Cell Biology
Published by TSRI Press®. © Copyright 2005,
The Scripps Research Institute. All rights reserved.
Lindsey MacPherson, Graduate Student, and
Ardem Patapoutian, Ph.D., Associate Professor,
Department of Cell Biology
Published by TSRI Press®. © Copyright 2005,
The Scripps Research Institute. All rights reserved.
CELL BIOLOGY 2005 19
DEPAR TMENT OF
CELL BIOLOGY
S TA F F
Sandra L. Schmid, Ph.D.*
Professor and Chairman
Francisco Asturias, Ph.D.**
Associate Professor
William E. Balch, Ph.D.*
Professor
Bridget Carragher, Ph.D.**
Associate Professor
Benjamin Cravatt, Ph.D.***
Professor
Gaudenz Danuser, Ph.D.**
Assistant Professor
Philip E. Dawson,
Ph.D.****
Associate Professor
Stephen P. Mayfield,
Ph.D.****
Associate Professor
Associate Dean, Kellogg
School of Science and
Technology
Mark Mayford, Ph.D.*****
Associate Professor
Lindsey Miles, Ph.D.
Associate Professor
Alan Bell, B.S.C.S.
Xerox Palo Alto Research
Center
Palo Alto, California
Ronald A. Milligan, Ph.D.**
Professor
Ulrich Müller, Ph.D.*****
Associate Professor
Ardem Patapoutian, Ph.D.†††
Associate Professor
Clinton Potter, B.S.**
Associate Professor
Martin Friedlander, M.D.,
Ph.D.
Professor
Lisa Stowers, Ph.D. ††††
Assistant Professor
Steve Kay, Ph.D. †
Professor
Natasha Kralli, Ph.D.
Assistant Professor
Peter Kuhn, Ph.D.**
Associate Professor
David Loskutoff, Ph.D.
Professor Emeritus
Mark J. Yeager, M.D., Ph.D.
Professor
ADJUNCT APPOINTMENTS
James Quigley, Ph.D.
Professor
Shelley Halpain, Ph.D.*****
Associate Professor
John R. Yates III, Ph.D.
Professor
Alan McLachlan, Ph.D. ††
University of Illinois
Chicago, Illinois
Velia Fowler, Ph.D.**
Professor
Larry R. Gerace, Ph.D.*
Professor
Elizabeth Winzeler, Ph.D. †††
Associate Professor
Heidi Stuhlmann, Ph.D.
Associate Professor
Kevin F. Sullivan, Ph.D.**
Associate Professor
Peter N.T. Unwin, Ph.D.**
Professor
Clare Waterman-Storer,
Ph.D.**
Associate Professor
Xiaohua Gong, Ph.D.
University of California
Berkeley, California
Klaus Hahn, Ph.D.
University of North Carolina
Chapel Hill, North Carolina
Eric Peeters, Ph.D.
Xerox Palo Alto Research
Center
Palo Alto, California
S TA F F S C I E N T I S T S
Michael Bracey, Ph.D.
Richard Bruce, Ph.D.
Xerox Palo Alto Research
Center
Palo Alto, California
Douglas Curry, B.S. (E.E.C.S.)
Xerox Palo Alto Research
Center
Palo Alto, California
Bertil Daneholt, M.D.
Karolinska Institutet
Stockholm, Sweden
Scott Elrod, Ph.D.
Xerox Palo Alto Research
Center
Palo Alto, California
Anchi Cheng, Ph.D.
Elena Deryugina, Ph.D.
Robert Fischer, Ph.D.
Thomas Schultz, Ph.D.
Elizabeth Wilson, Ph.D.
Yuanxin Zhu, Ph.D. ††
Fair Issac Corporation
San Diego, California
SENIOR RESEARCH
A S S O C I AT E S
Mark Ginsberg, M.D.
University of California
San Diego, California
Brian Adair, Ph.D.
David Goldberg, Ph.D.
Xerox Palo Alto Research
Center
Palo Alto, California
Sean Conner, Ph.D. ††
University of Minnesota,
Twin Cities
Minneapolis, Minnesota
Yuriy Chaban, Ph.D.
S E C T I O N C O V E R F O R T H E D E P A R T M E N T O F C E L L B I O L O G Y : Intraocular vascula-
ture of a mouse imaged in vivo. Visible in this image are the vessels of the iris, hyaloid, and retina.
Vessels of this 16-day-old mouse were visualized by using intravenous injection of fluorescent dye
and confocal microscopy. Approximately 150 images over a depth of about 3 mm were collected to
Mari Manchester, Ph.D.**
Associate Professor
construct the image shown. This approach allows visualization of functionally intact ocular blood vessels and serial imaging of the same blood vessels over time. Image by Matthew Ritter, Ph.D., in the
laboratory of Martin Friedlander, M.D., Ph.D.
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The Scripps Research Institute. All rights reserved.
20 CELL BIOLOGY 2005
Ralf-Peter Czekay, Ph.D. ††
Albany Medical College
Albany, New York
Aurelia Cassany, Ph.D.
Mark Daniels, Ph.D.
Ihsiung Chen, Ph.D.
Emily Chen, Ph.D.
Thomas Fath, Ph.D. ††
The Children’s Hospital at
Westmead
Sydney, Australia
Claire Kidgell, Ph.D.
Katsuhiro Kita, Ph.D.
Atanas Koulov, Ph.D.
Michael Fitch, Ph.D.
Scott E. Franklin, Ph.D. ††
Rincon Pharmaceuticals
La Jolla, California
Yei Hua Chen, Ph.D.
Paul LaPointe, Ph.D.
Maria Gonzalez, Ph.D.
Charmian Cher, Ph.D.
Nicole Lazarus, Ph.D.
Nicolas Grillet, Ph.D.
Jeremiah Joseph, Ph.D.
Smita Chitnis, Ph.D.
Donmienne Leung, Ph.D.
Cemal Gurkan, Ph.D.
Edward Korzus, Ph.D.
Parag Chowdhury, Ph.D.
John Lewis, Ph.D.
Frank Harmon, Ph.D.
Stephan Miller, Ph.D.
Jill Chrencik, Ph.D.
Lujian Liao, Ph.D.
Samuel Hazen, Ph.D.
Matthew Ritter, Ph.D.
Jessie Chu, Ph.D.
Maria Lillo, Ph.D.
Johannes Hewel, Ph.D.
Martin Schwander, Ph.D.
Michael Churchill, Ph.D.
Francesco Conti, Ph.D.
Edith Hintermann, Ph.D. ††
Institut für Allgemeine
Pharmakologie und
Toxikologie
Frankfurt, Germany
Judith Coppinger, Ph.D.
Hide Hyracoidea, Ph.D. †††††
Michael Hock, Ph.D.
Geza Ambrus-Aikelin, Ph.D.
Amy Cullinan, Ph.D. ††
Invitrogen Corporation
Carlsbad, California
Angelique Aschrafi, Ph.D.
Leif Dehmelt, Ph.D.
Eric Hwang, Ph.D.
Hongdong Bai, Ph.D.
Claudia Dellas, Ph.D. †††††
Sun Wook Hwang, Ph.D. ††
Korea University
Seoul, Korea
Juliana Conkright, Ph.D. ††
Scripps Florida
Gina Story, Ph.D.
Andries Zijlstra, Ph.D.
R E S E A R C H A S S O C I AT E S
Jessica Alexander, Ph.D.
Claudia Barros, Ph.D.
Ph.D. ††
Janna Bednenko,
University of Rochester
Rochester, New York
Maria Beligni, Ph.D.
Ph.D. ††
Johannes De Rooij,
Netherlands Cancer Institute
Amsterdam, the Netherlands
Ajay Dhaka, Ph.D.
Meng-Qui Dong, Ph.D.
K.E. Hua, Ph.D.
Takato Imaizumi, Ph.D.
Ph.D. ††
Miwako Ishido,
Trans-Science Inc.
Tokyo, Japan
Klara Limback-Stokin, Ph.D.††
University of Ljubljana
School of Medicine
Ljubljana, Slovenia
Jennifer Lin, Ph.D.
Ryan Littlefield, Ph.D.
Chuan-Yin Liu, Ph.D.
Songkai Liu, Ph.D. ††
University of California
San Francisco, California
Dinah Loerke, Ph.D.
Matthias Machacek, Ph.D.
Mark Madsen, Ph.D.
Claudio Masuda, Ph.D.
Naoki Matsuo, Ph.D.
Richard Belvindrah, Ph.D.
Michael Dorrell, Ph.D.
Khuloud Jaqaman, Ph.D.
Daniel McClatchy, Ph.D.
Kari Bradtke, Ph.D.
Kelly A. Dryden, Ph.D.
Anass Jawhari, Ph.D.
Ghislain Breton, Ph.D.
Anna Durrans, Ph.D.
Nadim Jessani, Ph.D. ††
Celera Genomics
San Francisco, California
Caroline McKeown, Ph.D.
Ph.D. ††
Anja Bubeck, Ph.D.
Andrei Efimov,
Vanderbilt University
Nashville, Tennessee
Barbara Calabrese, Ph.D.
Samer Eid, Ph.D.
Gregory Cantin, Ph.D.
Eva Farre, Ph.D.
Florence Brunel, Ph.D.
Published by TSRI Press®. © Copyright 2005,
The Scripps Research Institute. All rights reserved.
Lin Ji, Ph.D.
Kimon Kanelakis, Ph.D. ††
Johnson & Johnson
San Diego, California
Juncai Meng, Ph.D. ††
Merck Pharmaceuticals
Whitehorse Station, New
Jersey
Helena Mira, Ph.D.
Jennifer Mitchell, Ph.D.
CELL BIOLOGY 2005 21
Jacobus Neels, Ph.D.
Alan Saghatelian, Ph.D.
Ge Yang, Ph.D.
Sherry Niessen, Ph.D.
Kumar Saikatendu, Ph.D.
Jian Ming Yang, Ph.D. ††
Office of Environmental
Health Hazard Assessment
Oakland, California
John Offer, Ph.D. ††
University of Oxford
Glycobiology Institute
Oxford, England
Silvia Ortega-Gutierrez,
Ph.D.
Cleo Salisbury, Ph.D.
Trey Sato, Ph.D. ††
Scripps Florida
Defne Yarar, Ph.D.
Eric C. Schirmer, Ph.D. ††
University of Edinburgh
Edinburgh, Scotland.
Masahiro Yasuda, Ph.D.
Rie Yasuda, Ph.D.
Lesley Page, Ph.D.
Ning Pan, Ph.D. ††
University of Massachusetts
Medical School
Boston, Massachusetts
Thomas Schroeter,
Scripps Florida
Ph.D. ††
Stephan Sieber, Ph.D.
Erquan Zhang, Ph.D.
Wenhong Zhu, Ph.D. ††
Burnham Institute
La Jolla, California
* Joint appointment in the
Department of Molecular
Biology
** Joint appointment in the
Center for Integrative Molecular
Biosciences
*** Joint appointments in the
Department of Chemistry, the
Skaggs Institute for Chemical
Biology, and the Helen L.
Dorris Child and Adolescent
Neurological and Psychiatric
Disorder Institute
**** Joint appointment in the
Skaggs Institute for Chemical
Biology
***** Joint appointment in the
Institute for Childhood and
Neglected Diseases
†
Pratik Singh, Ph.D.
Zhongmin Zou, Ph.D.
Alessia Para, Ph.D.
Fabienne Soulet,
Ph.D. †††††
Brian Petrich, Ph.D. †††††
Scott Stagg, Ph.D.
Sarah Stanton,
Ph.D. †††††
Barbie Pornillos, Ph.D.
††
Appointment completed; new
location shown
†††
Joint appointments in the
Institute for Childhood and
Neglected Diseases and the
Genomics Institute of the
Novartis Research Foundation
S C I E N T I F I C A S S O C I AT E S
Aaron Ponti, Ph.D. †††††
Hilda Edith Aguilar de Diaz,
M.D.
Mark Surka, Ph.D.
Faith Barnett, Ph.D.
Judith Prieto, Ph.D.
††††
Claire Tiraby, Ph.D.
Alexei Brooun, Ph.D.
Natalie Prigozhina, Ph.D.
Ph.D. ††
Jose Pruneda-Paz, Ph.D.
Florian Topert,
Uexkull and Stolberg
Hamburg, Germany
Emily Burke, Ph.D.
Claire Delahunty, Ph.D.
Rajesh Ramachandran, Ph.D.
Tuija Uusitalo, Ph.D.
Tinglu Guan, Ph.D.
Vandana Ramachandran,
Ph.D.
John Venable, Ph.D.
Leon Reijmers, Ph.D.
Josep Villena, Ph.D.
Gang Ren, Ph.D. ††
Baylor College of Medicine
Houston, Texas
Xiaodong Wang, Ph.D.
Christopher Reyes, Ph.D.
Ann Wheeler, Ph.D.
Anna Reynolds, Ph.D.
Margaret Wilson, Ph.D.†††††
Olga Rodriguez, Ph.D.
Torsten Wittmann, Ph.D.
Benoit Roger, Ph.D. †††††
James Wohlschlegel, Ph.D.
Edwin Romijn, Ph.D.
Christine Wu, Ph.D. ††
University of Colorado
Denver, Colorado
Anand Kolatkar, Ph.D.
Fakhruddin Palida, Ph.D.
Cristian Ruse, Ph.D.
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The Scripps Research Institute. All rights reserved.
Kathryn Spencer, Ph.D.
BinQing Wei, Ph.D.
Joint appointments in the
Institute for Childhood and
Neglected Diseases and the
Department of Biochemistry
†††††
Joint appointment in the Helen
L. Dorris Child and Adolescent
Neuro-Psychiatric Disorder
Institute
Appointment completed
22 CELL BIOLOGY 2005
Sandra Schmid, Ph.D.
Chairman’s Overview
ou would think that with the complete list of parts
provided by the human genome sequence, the task
of deciphering the inner workings of cells and how
cells interact with each other and with their environment
would be easier. But in fact, having ready access to the
list of parts and the ability to discover their interconnectedness means that cell biologists must grapple with
greater complexity than before and with larger questions
of physiology and development. For the past several
decades, cell biologists have focused on enumerating
the parts of subcellular machines and individually assigning functions to the parts, usually in the context of specific pathways or cellular processes. Now, to have a
significant impact, the reductionists among us must
establish molecular mechanisms and unravel the structure-function relationships for each cellular machine.
Those who study specific cellular processes, such as
migration, metastasis, intracellular trafficking, and signal transduction, must unravel the complex regulation
and cross talk between cellular processes. For example,
signal transduction is controlled and modulated by the
spatial localization of signaling molecules and second
messengers. This spatial localization in turn is regulated
Y
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The Scripps Research Institute. All rights reserved.
by intracellular trafficking, by exchange of signaling molecules between subcellular compartments, and by dynamic
association with cytoskeletal elements and/or with structurally distinct membrane microdomains. The goal now is
to understand how the integration of each of these cellular
processes alters cell physiology in health and disease.
Those who study higher-order functions, such as
sensory perception, learning and memory, circadian
rhythms, and development, encounter even greater complexity and must use the powerful new tools of molecular
genetics to probe cause and effect and then to understand how anatomy and the organization of tissues affects
the higher-order functions. Finally, those who study specific diseases must identify all of the molecular changes
that occur and then determine the relevance of the
changes to the disease.
How are we tackling these complex biological problems to generate important and valuable new insight?
We collaborate! As I read through the reports that follow, I was struck by the extent and nature of ongoing
collaborations that are driving our discoveries in the
Department of Cell Biology. As the accompanying diagram (Fig. 1) illustrates, to tackle the ever-increasing
complexity of questions being addressed, members of
the department are taking advantage of the breadth and
depth of biomedical research ongoing at Scripps and are
weaving an increasingly complex network of collaborations.
Bill Balch, for example, is fulfilling his reductionist
roots by collaborating with Bridget Carragher and Clint
Potter, directors of the National Resource for Automated
Molecular Microscopy, to solve the structure of a novel
coat complex that drives vesicle formation from the
endoplasmic reticulum and with Ian Wilson to solve
high-resolution x-ray structures that reveal how individual components of the membrane trafficking machinery
carry out the cellular functions. Bill is also collaborating
with Jeff Kelly to elucidate how the cell’s biosynthetic
machinery distinguishes properly folded and misfolded
proteins and what properties of this sorting machinery
leave humans susceptible to protein misfolding diseases,
collectively known as amyloidoses. Finally, he is collaborating with John Yates to use innovative proteomics
methods to identify what components of the cell’s biosynthetic machinery might be limiting for controlling the
export of mutant proteins. In combination, these multidisciplinary approaches and collaborations are enabling
Dr. Balch and his colleagues to reveal potential new
avenues for therapeutic intervention in diseases, such as
cystic fibrosis, Gaucher’s disease, emphysema, diabetes,
CELL BIOLOGY 2005
23
Other transitions include the well-deserved promotions of Phil Dawson and Elizabeth Winzeler to associate
professor; Ardem Patapoutian and Clare Waterman-Storer
to associate professor with tenure; and Martin Friedlander to full professor. The success of these scientists
attests to the richness of the scientific environment at
Scripps Research.
F i g . 1 . Department of Cell Biology summary. Intradepartmental
and interdepartmental collaborations enrich research in all biology.
GNF indicates Genomics Institute of the Novartis Research Foundation; MB, Department of Molecular Biology; PARC, Palo Alto
Research Center.
and amyloidosis. Please explore the following reports to
see how interdisciplinary collaborations between Scripps
scientists are ensuring that members of the Department
of Cell Biology remain innovative leaders in the members’ respective areas of research.
Another report worth reading is Dave Loskutoff ’s.
Dave retired this year after nearly 25 years at Scripps,
where he served as chairman of the Department of
Vascular Biology from 1993 to 2003. During his distinguished career, Dave made numerous seminal contributions to our understanding of fibrinolysis and serum
proteases and their regulation. More recently, he focused
on the cell signaling role of the serine protease inhibitor
plasminogen activator inhibitor-1 in obesity and cardiovascular disease. His productivity and the importance
of his research efforts remained high throughout his
career. Nine articles describing his research findings
were published in excellent journals during the past
year. Our daily interactions with Dave will be missed,
but we look forward to informally enjoying his collegiality for some time.
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The Scripps Research Institute. All rights reserved.
24 CELL BIOLOGY 2005
INVESTIGATORS’ R EPORTS
Structural Characterization of
Macromolecular Machines
F.J. Asturias, Y. Chaban, J.Z. Chadick, A. Jawhari,
M. Sedova, D. Wepplo
e use state-of-the-art electron microscopy and
image analysis to calculate the 3-dimensional
structures of macromolecular complexes that
carry out a variety of cellular processes, including DNA
replication and transcription and fatty acid synthesis.
Our ultimate goal is to reveal the mechanism of action
of a macromolecular complex by determining the structures of the different conformations the complex adopts
as it carries out its function. Images of individual complexes are recorded under physiologically relevant conditions and then are computationally combined to obtain
structures of low-to-moderate resolution (10–25 Å).
Often, further information is obtained by docking atomicresolution structures of component subunits in the lower
resolution map of an entire complex. Macromolecular
electron microscopy is an ideal technique for these
studies because the technique requires only a small
amount of material and the conditions for preparing
samples are relatively gentle.
DNA transcription and its regulation are major areas
of interest. Previously, we characterized a number of
complexes that form part of the basal transcription
machinery in the yeast Saccharomyces cerevisiae,
arguably the most important model organism for studies of eukaryotic transcription. More recently, we extended
our studies to the human basal machinery. We are
characterizing complexes formed by human RNA polymerase II and general transcription factors to understand how these factors contribute to the initiation of
transcription. Also under study are complexes involved
in the regulation of transcription during initiation and
during previous steps in which the structure of chromatin is altered to make DNA accessible to the transcription machinery. By comparing and contrasting the
function and structure of homologous complexes in different eukaryotes, we hope to identify fundamental
features of the transcription regulation mechanism.
Our interest in the importance of conformational
changes for the function of macromolecular machines
prompted us to study the structure of mammalian
W
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fatty acid synthase (FAS), the enzyme responsible for
the synthesis of long-chain fatty acids. In animal cells,
the different enzymes involved in the synthesis of fatty
acids have fused into a single polypeptide chain with
7 different enzymatic activities. Moreover, the functional
form of FAS is a dimer in which 2 such chains cooperate to create 2 centers of fatty acid synthesis. FAS is
therefore a true “molecular assembly line.”
Despite the importance of FAS in metabolism, cellular development, and certain types of cancer, information on the structure of the enzyme is limited because
of the large number of conformations apparently adopted
by FAS. Even the organization of the 2 monomers in the
FAS dimer had remained uncertain, thereby precluding
an understanding of the functional complementation
between active sites required for fatty acid synthesis. In
addition, the results from the most recent functional
complementation studies conflict with the traditional
model for FAS organization in which the 2 component
monomers are depicted as fully extended and oriented
in an antiparallel arrangement.
We used a novel approach in which FAS point
mutants were imaged in the presence of substrates.
With this approach, the enzyme is effectively paused
at a given catalytic step and conformational variability
is reduced. Distinct but related FAS conformations
appear to be associated with specific catalytic steps
(Fig. 1). Calculating the structure of an FAS monomer
F i g . 1 . Structure in projection of different FAS paused mutants.
The active site compromised in each mutant and the specific mutations are indicated. Imaging mutants in the presence of substrates
considerably reduced the normal conformational variability of FAS.
CELL BIOLOGY 2005
and labeling the N termini of the 2 monomers in the
active form of the enzyme resulted in a revised understanding of FAS organization and in a new model in
which 2 coiled FAS monomers are intertwined to facilitate different functional interactions in the active FAS
dimer (Fig. 2). This revised model for FAS organization is a starting point for further characterization of
the enzyme and the related modular polyketide synthases, the molecules responsible for synthesis of a
number of antibiotics and other important physiologically active compounds.
F i g . 2 . A 16-Å resolution 3-dimensional structure of mammalian
FAS. A, Front view of the enzyme. B, Labeling of the 2 FAS N termini indicates that both are located near the center of the FAS
structure. C, Structure of an FAS monomer and the way in which 2
monomers might interact to form the active dimeric form of FAS.
Scale bar = 100 Å.
PUBLICATIONS
Asturias, F.J. Another piece in the transcription initiation puzzle. Nat. Struct. Mol.
Biol. 11:1031, 2004.
Asturias, F.J., Chadick, J.Z., Cheung, I.K., Stark, H., Witkowski, A., Joshi, A.K.,
Smith, S. Structure and molecular organization of mammalian fatty acid synthase.
Nat. Struct. Mol. Biol. 12:225, 2005.
Bourbon, H.M., Aguilera, A., Ansari, A.Z., Asturias, F.J., Berk, A.J., Bjorklund, S.,
et al. A unified nomenclature for protein subunits of Mediator complexes linking
transcriptional regulators to RNA polymerase II. Mol. Cell 14:553, 2004.
Chadick, J.Z., Asturias, F.J. Structure of eukaryotic Mediator complexes. Trends
Biochem. Sci. 30:264, 2005.
Witkowski, A., Ghosal, A., Joshi, A.K., Witkowska, H.E., Asturias, F.J., Smith, S.
Head-to-head coiled arrangement of the subunits of the animal fatty acid synthase.
Chem. Biol. 11:1667, 2004.
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25
Biological Chemistry and
Systems Biology of
Conformational Disease
W.E. Balch, Y. An, C. Chen, J. Conkright-Johnson,
D. Fowler, C. Gurkan, D. Hutt, A. Koulov, J. Matteson,
P. LaPointe, L. Page, H. Plutner, A. Pottekat, A. Sawkar,
S. Stagg, X. Wang
major challenge is to understand and treat the
many protein-misfolding diseases that affect
human health, including cystic fibrosis, emphysema, type 2 diabetes, and amyloidosis. These abnormalities are classified as membrane-trafficking conformational
diseases because a defect in protein folding at some
stage of the eukaryotic secretory pathway results in loss
of activity or protein aggregation. A key concern is to
determine the underlying defect in protein folding and
how that defect affects the ability of the protein to function normally within the context of the cell’s intracellular
transport machinery or in the extracellular environment
of the host.
Our broad objective is to define the molecular
basis for the trafficking of normal and misfolded proteins through the secretory pathway of eukaryotic
cells. We use chemical, structural, biological, and
bioinformatics approaches.
Eukaryotic cells are highly compartmentalized; each
compartment of the exocytic and endocytic pathways
provides a unique chemical landscape in which protein
function and folding may be modulated. Movement
between these compartments involves the activity of
both anterograde and retrograde transport tubules and
vesicles. Many conformational diseases are a consequence of dysfunction at different stages of this transport pathway or outside of the cell.
Transport through the secretory pathway involves
a selective mechanism in which cargo molecules are
concentrated into carrier vesicles. Vesicle-mediated
transport is regulated by a diverse group of small GTPases
belonging to the Ras superfamily. Each of these molecules acts as a “molecular sensor” to regulate different
steps in the reversible assembly of vesicle coats and
targeting-fusion complexes. During export from the first
compartment of the secretory pathway, the endoplasmic reticulum, coat recruitment to budding sites involves
activation of the GTPase Sar1. After activation, the
cytosolic coat components Sec23/24 and Sec13/31
A
26 CELL BIOLOGY 2005
form the coatomer complex II coat that polymerizes to
promote budding from the surface of the endoplasmic
reticulum. Recently, we showed that Sec 13/31 selfassembles to form a cage that drives vesicle budding.
In collaboration with C. Potter and B. Carragher, Department of Cell Biology, using single-particle analysis, we
solved the 2-dimensional electron cryomicroscopy structure of the Sec13/31 cage. We also found that cargo
selection by the assembling polymer involves “exit codes”
found on the cytoplasmic domains of cargo and cargo
receptors. These exit codes bind to a pocket in the Sec24
coat component. In studies with J.R. Yates, Department of Cell Biology, we are using shotgun proteomics
(multidimensional protein identification technology or
MudPIT) to identify unknown components involved in
cargo selection.
After vesicles separate from the endoplasmic reticulum, targeting and fusion of coatomer complex II transport vesicles to the next step of the secretory pathway,
the Golgi apparatus, require a different class of Raslike GTPases that belong to the Rab family. Members
of the large Rab family (>70 members) act as molecular switches that assemble complexes involved in
vesicle tethering and fusion. Using a bioinformatics
approach, in collaboration with J. Hogenesch, Genome
Technology, Scripps Florida, we found that each Rab
GTPase executes targeting and fusion decisions at a
distinct step in the exocytic or endocytic pathway. By
coordinating the function of multiple distinct effectors
at each step, Rab GTPases act as hubs to define the
function and highly distinctive membrane architecture
of eukaryotic cells found in different tissues. This systems biology approach provides for the first time a
global view of membrane traffic from the top down,
integrating form with function.
An important common effector for all Rab GTPases
is the protein GDP-dissociation inhibitor (GDI). GDI
extracts Rabs from membranes after vesicle fusion,
thereby forming a cytosolic complex that sequesters the
prenyl groups for redelivery to the membrane during
budding. Combined biochemical and structural studies
revealed the surprising importance of the Hsp90 chaperone system in GDI-dependent Rab recycling. In addition, Rab proteins coordinate the activity of tethers that
direct vesicle docking and fusion. Our recent structure
of the Rab1 tether p115, determined in collaboration
with I.A. Wilson, Department of Molecular Biology,
reveals a crucial role for coiled coil interactions in mediating membrane docking.
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Although traffic from the endoplasmic reticulum
can be disrupted by mutations that prevent proper protein folding during synthesis, other protein conformational diseases have mutations that disrupt function at
later steps of the secretory pathway and outside the
cell in new chemical environments that can alter the
protein fold. In collaboration with J. Kelly, Department
of Chemistry, we are studying the link between trafficking defects and the protein-folding energetics (Fig. 1)
of a number of conformational diseases, including cystic fibrosis, hereditary childhood emphysema, Gaucher
disease, familial amyloidosis of Finnish type, and transthyretin amyloidosis. The latter analyses have led to a
new understanding of the function of the endoplasmic
reticulum in normal physiology, suggesting that this
compartment functions as a capacitor for protein folding.
F i g . 1 . Export of cargo (ball-and-stick icons) from the endoplasmic
reticulum to the cell surface occurs through cargo-selective budding
regions (lower part of image). Cargo export is thought to be restricted
to fully folded structures by “quality control” pathways. Surprisingly,
analysis of protein-folding energetics and secretion indicated that
protein variants destabilized relative to the wild-type fold are secreted
with wild-type efficiency, triggering deposition of amyloid fibril aggregates in the extracellular space (upper part of image). Appreciation
of the importance of the global energetics of the protein fold in
secretion leads to a key shift in understanding the secretory pathway, from quality control to mechanistic pathways.
CELL BIOLOGY 2005
Through a multidisciplinary approach that combines
the tools of chemistry, biology, systems biology, and
structure, we hope to gain critical insight into the basis
for a variety of inherited transport diseases. Knowledge
of the function of these cargo selection pathways will
enable the development of small-molecule chemical
chaperones to encourage export and stability of misfolded proteins, leading to restoration of normal cellular function.
PUBLICATIONS
Bannykh, S.I., Plutner, H., Matteson, J., Balch, W.E. COPI-independent polarization of the Golgi stack. Traffic, in press.
Gurkan, C., Alory, C., Su, A.I., Lapp, H., Hogenesch, J., Balch, W.E. Large-scale profiling of Rab GTPase trafficking networks: the membrome. Mol. Biol. Cell, in press.
Page, L.J, Huff, M.E., Kelly, J.W., Balch, W.E. Ca2+ binding protects against gelsolin amyloidosis. Biochem. Biophys. Res. Commun. 322:1105, 2004.
Page, L.J., Suk, J.Y., Huff, M.E., Lim, H.J., Venable, J., Yates, J.R. III, Balch,
W.E., Kelly, J.W. Metalloprotease cleavage within the extracellular matrix triggers
the final stages of gelsolin amyloidogenesis. EMBO J., in press.
Sekijima, Y., Wiseman, R.L., Matteson, J., Hammarstrom, P., Miller, S.R., Sawkar,
A.R., Balch, W.E., Kelly, J.W. The biological and chemical basis for tissue-selective
amyloid disease. Cell 121:73, 2005.
Wang, X., Matteson, J., An, Y., Moyer, B., Yoo, J.S., Bannykh, S., Wilson, I.A.,
Riordan, J.R., Balch, W.E. COPII-dependent export of cystic fibrosis transmembrane conductance regulator from the ER uses a di-acidic exit code. J. Cell Biol.
167:65, 2004.
Wang, X., Riordan, J.R., Balch, W.E. Rescue of ∆508 CFTR by inducible folding
chaperones. J. Biol. Chem., in press.
Wiseman, L., Balch, W.E. Druging protein folding pathways through stress: a new
pharmacological adventure. Trends Biochem. Sci., in press.
Automated Molecular Imaging
B. Carragher, C.S. Potter, A. Cheng, D. Fellmann, F. Guerra,
G. Lander, S. Mallick, P. Mercurio, J. Pulokas, J. Quispe,
27
holds great promise for routinely and efficiently providing structural information at a resolution sufficient
to resolve the secondary structure in these large molecular machines. This method could then be used in
conjunction with high-resolution x-ray structures of
individual proteins to interpret very large complexes to
near-atomic resolution.
Unfortunately, the methods generally used in molecular microscopy are both time-consuming and labor
intensive. These include the preparation of suitable
specimens, the acquisition of the required very large
numbers of electron micrographs, and the supervision
of the sometimes-complex software needed for analysis and reconstruction of the 3-dimensional electron
density maps.
The challenge then is to transform structure determination via electron microscopy into a high-throughput method. Success in this endeavor will not only
facilitate the process of molecular microscopy but also
expand the scope of accessible problems and make
possible investigations that currently are deemed too
high risk because of the inordinate effort involved. To
this end, we are developing technologies to address
automation for specimen handling, image acquisition,
data processing, and integration of data information.
We have created an integrated software system, called
Leginon, that automatically collects electron micrographs
of macromolecular structures (Fig. 1). This system has
been integrated with automated particle-selection algorithms and analysis and processing software.
A major focus of our activities is the National
Resource for Automated Molecular Microscopy (NRAMM),
a biotechnology resource center funded by the National
S. Stagg, C. Suloway, C. Yoshioka, Y. Zhu
lucidating the structure and mechanism of action
of molecular machines is an emerging frontier in
understanding how the information in the genome
is transformed into cellular activities. Molecular machines
are associations of individual components (e.g., proteins,
nucleic acids, lipids) in the form of large complexes;
examples include ribosomes, transcription complexes,
track-motor complexes, and membrane-embedded pumps
and channels. These machines are large and may also
be conformationally and compositionally dynamic or
present in comparatively low numbers, factors that make
them extremely challenging (or impossible) objects for
study by x-ray crystallography and nuclear magnetic
resonance methods. Molecular microscopy, however,
E
Published by TSRI Press®. © Copyright 2005,
The Scripps Research Institute. All rights reserved.
F i g . 1 . Multiscale image collection from a transmission electron
microscope is controlled by using Leginon, an automated data collection system. Data are managed by using a relational database
and can be visualized by using a Web browser.
28 CELL BIOLOGY 2005
Center for Research Resources, National Institutes of
Health. The overall mission is to develop, test, and
apply technology to completely automate the processes
involved in using electron cryomicroscopy to solve macromolecular structures. The current focus of NRAMM is
the development of new approaches for specimen handling, automated acquisition, automated processing,
and information handling.
The activities of the NRAMM are closely coupled
to a number of collaborative and service projects in
which fundamental biological goals are incentives for
developing the new technology. During 2004, more than
20 of these projects were actively pursued. Specific
examples include structural studies of coatomer complex II–coated vesicles, which are responsible for transport of proteins from the endoplasmic reticulum to the
Golgi apparatus; characterization of viruslike particles
manufactured in recombinant expression systems; structural studies of the crustacean clotting protein; structural
studies of coronaviruses; and the structural characterization of the chloroplast ribosome. All of these collaborative projects guided the development of new approaches
in the 4 core technologies while simultaneously providing new structural information relevant to specific
biological problems.
An additional project, sponsored by the National
Science Foundation, is the development of automated
data collection techniques for imaging serial sections
obtained by using an electron microscope. Understanding the fine structure of cells and cellular components
contributes to a more profound understanding of cellular function and intracellular or intercellular interactions.
In order to visualize these large, complex structures in
3 dimensions at resolutions sufficient to observe structure on the nanoscale, the cells must be cut into sections
and then examined by using a transmission electron
microscope. Acquiring high-magnification images of a
long series of sections is difficult and extremely labor
intensive. The region of interest in each section must
be tracked across sections and across grids, a process
that requires examining the sections at a variety of scales
before acquiring high-magnification images of interesting areas. Multiscale imaging of this sort is not straightforward because the image formed by an electron
microscope shifts and rotates as the magnification is
changed. The overall task of reconstructing a 3-dimensional volume from a set of serial sections is challenging
and time-consuming, and the number of large-scale
reconstructions has been limited to a few spectacular
Published by TSRI Press®. © Copyright 2005,
The Scripps Research Institute. All rights reserved.
examples. Our objectives are to design, develop, and
implement a software application to automate the task of
acquiring high-magnification images of specific regions of
the cell across tens to hundreds of serial sections.
PUBLICATIONS
Dang, T.X., Farah, S.J., Gast, A., Robertson, C., Carragher, B., Egelman, E., WilsonKubalek, E.M. Helical crystallization on lipid nanotubes: streptavidin as a model protein. J. Struct. Biol. 150:90, 2005.
Mallick, S.P., Carragher, B., Potter, C.S., Kriegman, D.J. ACE: automated CTF
estimation. Ultramicroscopy 104:8, 2005.
Mallick, S.P., Zhu, Y., Kriegman, D. Detecting particles in cryo-EM micrographs
using learned features. J. Struct. Biol. 145:52, 2004.
O’Keefe, M.A., Turner, J.H., Musante, J.A., Hetherington, C.J.D., Cullis, A.G.,
Carragher, B., Jenkins, R., Milgrim, J., Milligan, R.A., Potter, C.S., Allard, L.F.,
Blom, D.A., Degenhardt, L., Sides, W.H. Laboratory design for high-performance
electron microscopy. Microsc. Today 12:8, 2004.
Suloway, C., Pulokas, J., Fellmann, D., Cheng, A., Guerra, F., Quispe, J., Stagg,
S., Potter, C.S., Carragher, B. Automated molecular microscopy: the new Leginon
system. J. Struct. Biol., in press.
Chemical Physiology
B.F. Cravatt, J. Alexander, K. Barglow, M.H. Bracey,
K. Chiang, M. Evans, H. Hoover, N. Jessani, D. Leung,
K. Matsuda, A. Mulder, S. Niessen, M. McKinney,
A. Saghatelian, S. Sieber, G. Simon, A. Speers, B. Wei
e are interested in understanding complex
physiology and behavior at the level of chemistry and molecules. At the center of cross
talk between different physiologic processes are endogenous compounds that provide a molecular mode for
intersystem communication. However, many of these
molecular messages remain unknown, and even in the
instances in which the participating molecules have
been defined, the mechanisms by which these compounds function are for the most part still a mystery.
We are investigating a family of chemical messengers termed the fatty acid amides, which affect many
physiologic functions, including sleep and pain. In particular, one member of this family, oleamide, accumulates selectively in the cerebrospinal fluid of tired
animals. This finding suggests that oleamide may function as a molecular indicator of an organism’s need for
sleep. Another fatty acid amide, anandamide, may be
an endogenous ligand for the cannabinoid receptor in
the brain.
The in vivo levels of chemical messengers such as
the fatty acid amides must be tightly regulated to maintain proper control over the influence of the messengers
W
CELL BIOLOGY 2005
on brain and body physiology. We are characterizing a
mechanism by which the level of fatty acid amides can
be regulated in vivo. Fatty acid amide hydrolase degrades
the fatty acid amides to inactive metabolites. Thus, the
hydrolase effectively terminates the signaling messages
conveyed by fatty acid amides, possibly ensuring that
these molecules do not generate physiologic responses
in excess of their intended purpose.
We are using transgenic and synthetic chemistry
techniques to study the role of the hydrolase in the
regulation of fatty acid amide levels in vivo. In collaboration with R.C. Stevens, Department of Molecular
Biology, we solved the first 3-dimensional structure of
fatty acid amide hydrolase. We are using this information to explore the molecular mechanism of action of
the enzyme and to design inhibitors of the hydrolase.
We are also interested in proteins responsible for the
biosynthesis of fatty acid amides.
Another area of interest is the design and use of
chemical probes for the global analysis of protein function. The evolving field of proteomics, defined as the
simultaneous analysis of the complete protein content
of given cell or tissue, encompasses considerable conceptual and technical challenges. We hope to enhance
the quality of information obtained from proteomics
experiments by using chemical probes that indicate the
collective catalytic activities of entire classes of enzymes.
These activity-based probes could be used to record
variations in protein function independent of alterations
in protein abundance and would be a powerful and complimentary set of tools for proteome analysis.
To date, we have generated activity-based probes
for more than a dozen enzyme classes, including serine
hydrolases, metalloproteases, glutathione S-transferases,
and several oxidoreductases. We are using the probes
to explore the roles that enzymes play in a variety of
physiologic and pathologic processes, especially the
progression of cancer. We are also developing complementary strategies for profiling the complete content of
metabolites in cells and tissues (the “metabolome”) to
facilitate the assignment of endogenous substrates to
enzymes of uncharacterized function.
PUBLICATIONS
Azad, S.C., Monory, K., Marsicano, G., Cravatt, B.F., Lutz, B., Zieglgansberger,
W., Rammes, G. Circuitry for associative plasticity in the amygdala involves endocannabinoid signaling. J. Neurosci. 24:9953, 2004.
Barglow, K.T., Cravatt, B.F. Discovering disease-associated enzymes by proteome
reactivity profiling. Chem. Biol. 11:1523, 2004.
Chiang, K.P., Gerger, A.L., Sipe, J.C., Cravatt, B.F. Reduced cellular expression
and activity of the P129T mutant of human fatty acid amide hydrolase: evidence
for a link between defects in the endocannabinoid system and problem drug use.
Hum. Mol. Genet. 13:2113, 2004.
Published by TSRI Press®. © Copyright 2005,
The Scripps Research Institute. All rights reserved.
29
Cravatt, B.F., Lichtman, A.H. The endogenous cannabinoid system and its role in
nociceptive behavior. J. Neurobiol. 61:149, 2004.
Cravatt, B.F., Saghatelian, A., Hawkins, E.G., Clement, A.B., Bracey, M.H., Lichtman, A.H. Functional disassociation of the central and peripheral fatty acid amide
signaling systems. Proc. Natl. Acad. Sci. U. S. A. 101:10821, 2004.
Jessani, N., Humphrey, M., McDonald, W.H., Niessen, S., Gangadharan, B., Yates,
J.R. III, Mueller, B.M., Cravatt, B.F. Carcinoma and stromal enzyme activity profiles associated with breast tumor growth in vivo. Proc. Natl. Acad. Sci. U. S. A.
101:13756, 2004.
Jessani, N., Niessen, S., Mueller, B.M., Cravatt, B.F. Breast cancer cell lines
grown in vivo: what goes in isn’t always the same as what comes out. Cell Cycle
4:253, 2005.
Jessani, N., Young, J.A., Diaz, S.L., Patricelli, M.P., Varki, A., Cravatt, B.F. Class
assignment of sequence-unrelated members of enzyme superfamilies by activitybased protein profiling. Angew. Chem. Int. Ed. 44:2400, 2005.
Leung, D., Wu, W., Hardouin, C., Cheng, H., Hwang, I., Cravatt, B.F., Boger,
D.L. Discovery of an exceptionally potent and selective class of fatty acid amide
hydrolase inhibitors enlisting proteome-wide selectivity screening: concurrent optimization of enzyme inhibitor potency and selectivity. Bioorg. Med. Chem. Lett.
15:1423, 2005.
Lichtman, A.H., Leung, D., Shelton, C.C., Saghatelian, A., Hardouin, C., Boger,
D.L., Cravatt, B.F. Reversible inhibitors of fatty acid amide hydrolase that promote
analgesia: evidence for an unprecedented combination of potency and selectivity. J.
Phamacol. Exp. Ther. 311:441, 2004.
Saghatelian, A., Cravatt, B.F. Global strategies to integrate the proteome and
metabolome. Curr. Opin. Chem. Biol. 9:62, 2005.
Saghatelian, A., Jessani, N., Joseph, A., Humphrey, M. Cravatt, B.F. Activitybased probes for the proteomic profiling of metalloproteases. Proc. Natl. Acad. Sci.
U. S. A. 101:10000, 2004.
Saghatelian, A., Trauger, S.A., Want, E.J., Hawkins, E.G., Siuzdak, G., Cravatt,
B.F. Assignment of endogenous substrates to enzymes by global metabolite profiling. Biochemistry 43:14332, 2004.
Sieber, S.A., Mondala, T., Head, S.R., Cravatt, B.F. Microarray platform for profiling enzyme activities in complex proteomes [published correction appears in J. Am.
Chem. Soc. 127:4114, 2005]. J. Am. Chem. Soc. 126:15640, 2004.
Mechanics of Cell Migration and
Chromosome Segregation
G. Danuser, J. Dorn, H. Jaqaman,* K. Jaqaman, L. Ji,
A. Kerstens, D. Loerke, M. Machacek, A. Matov, G. Yang,
Y. Ying
* Bethlehem University, Bethlehem, West Bank
ur goal is to understand how the action of thousands of force-generating molecular machines is
regulated to achieve complex outputs, such as
the directed movement of a cell or of its organelles.
Specifically, we investigate how assembly and contraction of the actin cytoskeleton and the structural interactions of actin filaments with other components of the
cytoskeleton mediate cell migration. In parallel, we
study how assembly and disassembly of microtubules
are orchestrated during cell division to symmetrically
distribute the replicated DNA from the mother cell into
2 daughter cells.
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30 CELL BIOLOGY 2005
To examine these molecular systems, we develop
computational models that predict how the convoluted
dynamics of many molecular machines could transform
into cell-level responses. Subsequently, we deduce
molecular-level mechanisms by fitting the models to
measurements of cell dynamics. The challenges arising
from such data-driven, multiscale modeling are 2-fold:
the precise measurement of single-cell dynamics and the
implementation of numerical tools for fitting large sets of
cell-level data to models with molecular resolution.
During the past year, we made important advances
in both directions. In collaboration with C.M. WatermanStorer, Department of Cell Biology, we used quantitative
fluorescent speckle microscopy to analyze the architectural dynamics of the actin cytoskeleton during cell
migration. We discovered that the initiating events of
cell protrusion are mediated by 2 materially distinct,
yet spatially overlapping, actin networks. This puzzling
find raises several questions that define the focus of
our current research: What are the molecular mechanisms by which cells assemble 2 networks of the same
polymer? What are the functional differences between
the networks? Do signals target the networks differentially, and if so, which signals coordinate the dynamics of the networks?
To study chromosome segregation, we have developed automated 3-dimensional light microscopy to probe
the changes in microtubule dynamics due to mutations in
kinetochore proteins in yeast. We will use this information to model the functional relationships between
kinetochore proteins and between the kinetochore and
the attached microtubules, which couple the chromosome to the force-generating machinery of the spindle.
We made an important step toward this goal by implementing a novel mathematical framework for matching
stochastic models of microtubule dynamics to singlecell data. Using this framework not only enables us to
test mechanistic models of the kinetochore-microtubule
interaction but also provides a generic tool for comparing
statistical models of molecular processes with measurements obtained by using light microscopy. We are also
using the framework to analyze the functional links
between actin cytoskeleton dynamics and clathrinmediated endocytosis.
PUBLICATIONS
Adams, M.C., Matov, A., Yarar, D., Gupton, S.L., Danuser, G., Waterman-Storer,
C.M. Signal analysis of total internal reflection fluorescent speckle microscopy (TIRFSM) and wide-field epi-fluorescence FSM of the actin cytoskeleton and focal adhesions in living cells. J. Microsc. 216(Pt. 2):138, 2004.
Published by TSRI Press®. © Copyright 2005,
The Scripps Research Institute. All rights reserved.
Gupton, S., Anderson, K.L.., Kole, T.P., Fischer, R.S., Ponti, A., Hitchcock-DeGregori, S., Danuser, G., Fowler, V.M., Wirtz, D., Hanein, D., Waterman-Storer, C.M.
Cell migration without a lamellipodium: translation of actin dynamics into cell
movement mediated by tropomyosins. J. Cell Biol. 168:619, 2005.
Künzi, P.A., Lussi, J., Aeschimann, L., Danuser, G., Textor, M., de Rooij, N.F.,
Staufer, U. Nanofabrication of protein-patterned substrates for future cell adhesion
experiments. Microelectron. Eng. 78-79:582, 2005.
Ponti, A., Machacek, M., Gupton, S.L., Waterman-Storer, C.M., Danuser G. Two distinct actin networks drive the protrusion of migrating cells. Science 305:1782, 2004.
Vallotton, P., Danuser, G., Bohnet, S., Meister, J.-J., Verkhovsky, A. Tracking retrograde flow in keratocytes: news from the front. Mol. Biol. Cell 16:1223, 2005.
Bioorganic Chemistry
of Proteins
P.E. Dawson, F. Brunel, M. Churchill, T. Tiefenbrunn,
N. Metanis
ur focus is the development and use of methods
to incorporate unnatural chemical groups into
proteins. We developed a chemical approach for
producing the large polypeptide chains that make up
protein molecules, enabling us to change the structure
of a protein in ways impossible by natural means. This
chemical ligation approach greatly facilitates the synthesis of proteins of moderate size and has opened the
world of proteins to the synthetic tools of organic chemistry. During the past year, we used these tools to study
the agouti protein, vitronectin, basic helix-loop-helix
DNA-binding domains, and the enzyme 4-oxalocrotonate
tautomerase. Our goal is to introduce noncoded amino
acids and other chemical groups into proteins to better understand the molecular basis of protein function.
O
E L E C T R O S TAT I C I N T E R A C T I O N S I N E N Z Y M E
C ATA LY S I S A N D D N A B I N D I N G
The relative contributions of electrostatic interactions
and hydrogen-bonding interactions of arginine residues in
proteins have been difficult to evaluate directly because
this amino acid side chain has no natural isosteric
replacement. To solve this problem, we used the unnatural amino acid citrulline to probe these interactions
in the small enzyme 4-oxalocrotonate tautomerase.
Citrulline can present 2 neutral hydrogen bonds from
the urea side chain in a manner superimposable to the
charged guanidinium group of arginine.
We found that neutralization of arginine at position 39, which binds to the ketoacid group of the substrate, resulted in a 1000-fold change in the catalytic
turnover rate, strongly supporting the key role of electrostatic interactions in this enzyme. Recently, we used
CELL BIOLOGY 2005
these citrulline analogs of 4-oxalocrotonate tautomerase
to design an enzyme with high specificity for an unnatural ketoamide substrate. These studies complement
our earlier work indicating that 4-oxalocrotonate tautomerase, which naturally uses an acid-base mechanism, could be converted, with a change in a single
amino acid, into a nucleophilic catalyst for the decarboxylation of oxaloacetate. We also used citrulline
analogs to probe interactions between transcription
factors and the phosphate backbone of DNA. As was
the case with 4-oxalocrotonate tautomerase, the citrulline mutation was destabilizing, although the dynamic
range of the assay was limiting.
H I V VA C C I N E D E S I G N
Eliciting broadly neutralizing antibodies is a major
goal of HIV vaccine design. This effort is complicated
by the poor accessibility of conserved regions of HIV
envelope proteins to antibodies. The membrane proximal region of the HIV envelope protein gp41 has generated marked research because of the discovery of 2
neutralizing antibodies that bind this sequence. We
are collaborating with D.R. Burton, Department of
Immunology, and I. Wilson, Department of Molecular
Biology, to design peptides that mimic the helical conformation of the peptide that binds to the neutralizing
antibody 4E10.
Immunization of simple peptides obtained from
gp41 elicited a nonneutralizing response, presumably
because of differences between the neutralizing conformation of the peptide and the most immunogenic
conformation of the peptide. We fully characterized
the 4E10 epitope and constrained the peptide to adopt
the primarily helical conformation recognized by the
4E10 antibody. These studies have yielded a series of
peptidomimetics that bind as tightly to the 4E10 antibody as the full-length gp41 glycoprotein does. Our goal
is to eliminate all surfaces of the peptide that are not
required for 4E10 binding or to mask them with soluble
polymers and carbohydrates. We plan to use this negative design approach in combination with binding studies,
immunization, and structural analysis by crystallography.
PUBLICATIONS
Beltran, A.C., Dawson, P.E., Gottesfeld, J.M. Role of DNA sequence in the binding
specificity of synthetic basic-helix-loop-helix domains. Chembiochem 6:104, 2004.
Brunel, F.M., Dawson, P.E. Synthesis of constrained helical peptides by thioether
ligation: application to analogs of gp41. Chem. Commun. (Camb.) 2552, 2005,
Issue 20.
Deechongkit, S., Dawson, P.E., Kelly, J.W. Toward assessing the position-dependent contributions of backbone hydrogen bonding to β-sheet folding thermodynamics employing amide-to-ester perturbations. J. Am. Chem. Soc. 126:16762, 2004.
Published by TSRI Press®. © Copyright 2005,
The Scripps Research Institute. All rights reserved.
31
Kamikubo, Y., De Guzman, R., Kroon, G., Curriden, S., Neels, J.G., Churchill,
M.J., Dawson, P., Oldziej, S., Jagielska, A., Scheraga, H.A., Loskutoff, D.J.,
Dyson, H.J. Disulfide bonding arrangements in active forms of the somatomedin B
domain of human vitronectin. Biochemistry 43:6519, 2004.
McNulty, J.C., Jackson, P.J., Thompson, D.A., Chai, B., Gantz, I., Barsh, G.S.,
Dawson, P.E., Millhauser, G.L. Structures of the agouti signaling protein. J. Mol.
Biol. 346:1059, 2005.
Metanis, N., Brik, A., Dawson, P.E., Keinan, E. Electrostatic interactions dominate
the catalytic contribution of Arg39 in 4-oxalocrotonate tautomerase. J. Am. Chem.
Soc. 126:12726, 2004.
Metanis, N., Keinan, E., Dawson P.E. A designed synthetic analog of 4-OT is specific for a non-natural substrate. J. Am. Chem. Soc. 127:5862, 2005.
Actin Dynamics in Cell
Morphogenesis and Function
V.M. Fowler, T. Fath, R.S. Fischer, C. McKeown, J. Moyer,
R. Nowak, J. Palomique
egulation of actin dynamics at the ends of filaments determines the organization and turnover
of actin cytoskeletal structures and is critical
for cell motility and cell architecture. For example, in
striated muscle and red blood cells (RBCs), actin filaments are organized into regular architectural arrays
that persist for the lifetime of the cell and are important
for maintenance of cell shape, mechanical properties,
and physiologic function. In contrast, in motile cells,
new actin filaments are rapidly assembled at the barbed
ends of the filaments and disassembled at the pointed
ends during extension of lamellipodia or filopodia.
Our goal is to elucidate the distinct regulatory mechanisms that control polymerization and dynamics of the
rapidly turning over actin filaments of motile cells as
compared with the stable, long-lived actin filaments of
nonmotile cells. Specifically, we focus on regulation by
the family of tropomodulin proteins that cap the pointed
ends of actin filaments and the roles of these proteins in
actin-based morphogenetic processes in development.
Tropomodulins are a conserved family of tropomyosin-regulated proteins of about 40 kD that cap the
pointed ends of actin filaments. Tropomodulins have
2 domains: an unstructured, flexible N-terminal domain
and a compact, folded C-terminal domain consisting
of 5 leucine-rich repeats. The N-terminal domain binds
tropomyosin and caps tropomyosin-actin pointed ends
with nanomolar affinity. The C-terminal domain caps
actin pointed ends with submicromolar affinity and is
unaffected by tropomyosin. In striated muscle myofibrils, tropomodulin 1 is associated with the pointed ends
of thin filaments and controls the length of filaments
by transiently capping the pointed ends and regulating
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32 CELL BIOLOGY 2005
actin association and dissociation. The N- and C-terminal
domains of tropomodulin 1 may have distinct functions
in stabilizing thin filaments and in controlling filament
length, respectively, but how the 2 domains regulate
dynamics of the pointed ends in vivo to control filament
assembly and disassembly is unclear.
To investigate the in vivo function of tropomodulin 1
in regulating assembly and disassembly of thin filaments
in muscle, we use mice that lack the gene for this tropomodulin. We showed previously that myofibril assembly
in the heart is grossly aberrant in the embryos of these
mutants, leading to aborted cardiac development and
the death of embryos between days 9 and 10 of development. To examine the primary defect in myofibril
assembly, we examined nascent myofibrils on myocyte
membranes in embryos at 7–8 days of development,
before the appearance of gross cardiac abnormalities.
In wild-type embryos, the earliest myofibrils contain 1–3 sarcomeres in tandem with regularly spaced
Z bodies and continuous F-actin, indicative of unregulated filament lengths. Such sarcomere structures are
never observed in the absence of tropomodulin 1; instead,
α-actinin and F-actin are present in rodlike, aberrant
Z disc structures on myocyte membranes. This finding
suggests that tropomodulin 1 has a novel early function
in the organization of Z discs into sarcomeres before the
stage of regulation of the length of thin filaments, a
later event of myofibril formation. In ongoing experiments, we are determining how the absence of tropomodulin 1 affects assembly of tropomyosin and other
components of thin or thick filaments and are examining how defective assembly of myofibrils in turn leads
to myocyte disorganization and disruption of cellular
contacts that occur in the absence of tropomodulin 1.
Tropomodulin 1 is also present in RBCs, where it
caps the pointed ends of the short actin filaments in
the spectrin membrane skeleton. To investigate the consequences in RBCs of deleting the gene for this tropomodulin, we prevented death in embryos of the mutant
mice by expressing a tropomodulin 1 transgene solely in
the heart. The result was viable mice with no tropomodulin 1 in their RBCs. Western blotting indicated
that levels of tropomodulin 3 were increased in tropomodulin 1–deficient RBCs, but the levels were considerably less than the normal amount of tropomodulin 1.
Hematologic analyses revealed that these mice had
a compensated mild hemolytic anemia, with increased
reticulocytosis and RBCs that were abnormally variable
in size in blood smears. Measurements of mechanical
stability and deformability indicated that tropomodulin
1–deficient RBCs were less deformable and more fragPublished by TSRI Press®. © Copyright 2005,
The Scripps Research Institute. All rights reserved.
ile than were normal RBCs. Using these mutant RBCs,
we can test our hypothesis that tropomodulin 1 regulates the lengths of the short actin filaments in RBCs,
thus influencing stability of the membrane skeleton and
cell survival and function in vivo.
The tropomodulin 3 isoform is expressed ubiquitously, including in motile endothelial cells, where it is
enriched in leading lamellipodia and acts as a negative
regulator of cell motility. Recently, we found that unlike
other members of the tropomodulin family, tropomodulin 3 can also bind to actin monomers in addition to
capping pointed ends,. Tropomodulin 3 can be chemically cross-linked to actin in a 1-to-1 complex, creating the expected 82-kD species and providing a powerful
tool for identifying the amino acids at the tropomodulin 3–actin binding interface. The results of coimmunoprecipitation and fluorescence staining assays for actin
monomers indicate that tropomodulin 3 also binds monomers in endothelial cells in vivo.
We propose that binding of the actin monomer to
tropomodulin 3 can reduce the amount of tropomodulin 3 available for binding to actin pointed ends, providing a mechanism to regulate tropomodulin 3 activity
and preferentially target tropomodulin 3 capping to
higher affinity tropomyosin-actin pointed ends.
PUBLICATIONS
Gupton, S.L., Anderson, K.L., Kole, T.P., Fischer, R.S., Ponti, A., HitchcockDeGregori, S.E., Danuser, G., Fowler, V.M., Wirtz, D., Hanein, D., WatermanStorer, C.M. Cell migration without a lamellipodium: translation of actin dynamics
into cell movement mediated by tropomyosin. J. Cell Biol. 168:619, 2005.
Schmid, M., Nanda, I., Hoehn, H., Schartl, M., Haaf, T., Buerstedde, J.M., et al.
Second report on chicken genes and chromosomes 2005. Cytogenet. Genome Res.
109:415, 2005.
Angiogenesis-Dependent
Disease and Membrane
Protein Topogenesis
M. Friedlander, E. Aguilar, E. Banin, F. Barnett, M. Dorrell,
S.F. Friedlander, R. Gariano, S. Moreno, A. Otani, M. Ritter,
L. Scheppke, H. Uusitalo-Jarvinen, W. Ruf, P.R. Schimmel,
D.A. Cheresh,* S.M. Simon,** K. Philipson***
* University of California, San Diego, California
** Rockefeller University, New York, New York
*** University of California, Los Angeles, California
ANGIOGENESIS-DEPENDENT DISEASE
M
ost diseases that cause catastrophic loss of
vision do so as a result of abnormal growth
of blood vessels. Similarly, tumors depend on
CELL BIOLOGY 2005
a blood supply for their growth and use these new vessels as an avenue for metastasis. Blood vessels themselves can generate tumors (e.g., hemangiomas) when
the growth and organization of vascular endothelial cells
is not properly controlled. Our goal is to understand the
mechanisms of ocular neovascularization in normal and
pathologic situations.
We use a neonatal mouse retina model to identify
regulators of developmental angiogenesis and understand endothelial guidance mechanisms. In addition, in
a long-standing collaboration with D.A. Cheresh, University of California, San Diego, we are using this system to
evaluate the role of integrins in these processes. In collaboration with P.R. Schimmel, Department of Molecular
Biology, we found that fragments of tryptophan tRNA
synthetase are potent angiostatics, and using a recombinant protein, we showed that a form of cell-based delivery caused significantly reduced retinal vascularization.
Most recently, we used combination therapy to show
that targeting multiple, distinct angiogenic pathways
with fragments of tryptophan tRNA synthetase and
antagonists of integrins and vascular endothelial cell
growth factor provides highly synergistic, potent angiostatic activity. Although this therapeutic approach should
be useful in the treatment of diseases in which complete inhibition of angiogenesis is desirable, it may not
be efficacious in the treatment of ischemic retinal disease. In ischemic retinal disease, relief of hypoxia by
vascular reconstruction, rather than destruction, may
be the desired outcome.
To examine possible therapy for diseases of retinal
ischemia, we explored the potential usefulness of lineage-negative hematopoietic stem cells derived from
the bone marrow of adult mice for cell-based delivery
of angiostatic and neurotrophic substances and for the
trophic actions of the cells themselves in vascular and
neuronal degenerative diseases. We found that the stem
cells contain a variety of progenitor (stem) cells, including endothelial precursor cells, and that the precursor
cells specifically target activated retinal astrocytes,
incorporate into new vessels, and, in a mouse model
of retinal degeneration, rescue and stabilize a degenerating retinal vasculature. We also showed that these
stem cells derived from the bone marrow of adults have
a profound neurotrophic effect when injected into the
eyes of mice with inherited retinal degeneration; not only
is the vasculature rescued in these mice but photoreceptors and visual function are also preserved. The stem
cells also can rescue retinal vasculature subject to
hypoxic stress and may be useful for the treatment of
Published by TSRI Press®. © Copyright 2005,
The Scripps Research Institute. All rights reserved.
33
ischemic retinopathies such as diabetes and retinopathy of prematurity.
Glioblastoma multiforme is an incurable brain tumor
that is usually fatal within 1 year after diagnosis. We
are using gene therapy and a rat model of this disease
to study the efficacy of an antiangiogenic approach in
treating these tumors. Hemangiomas are endothelial
tumors that proliferate rapidly and later involute spontaneously. We are using DNA microarrays to study
changes in gene expression as hemangiomas progress.
Our goal is to identify (1) new targets for therapy for
these tumors and (2) novel regulators of angiogenesis.
In collaboration with G.R. Nemerow, Department of
Immunology, we used pseudotyped adenovirus to selectively target specific cell types in the retina. By using
the appropriate fiber type, we can deliver transgenes
to cells, such as photoreceptors, that ordinarily are
not targeted by adenovirus.
MEMBRANE PROTEIN TOPOGENESIS
We are also studying the mechanism whereby proteins are asymmetrically integrated into cell membranes.
In addition to studies of membrane protein topogenesis
at the molecular level, we have been studying defects in
protein processing and insertion that occur in several
degenerative diseases of the eye. In collaboration with
K. Philipson, University of California, Los Angeles, we
are investigating the topology of the cardiac sodium-calcium exchanger. On the basis of hydropathy analysis of
the amino acid sequence, the exchanger is proposed to
contain 12 hydrophobic segments, the first of which is
a cleaved signal sequence. Using a variety of reporter
domains (glycosylation sites, epitopes, and proteolytic
cleavage sites), we analyzed the topology of the exchanger
both in vitro and in oocyte expression systems. Because
nearly all other polytopic eucaryotic membrane proteins
do not have cleaved signal sequences, we are investigating the putative role of such a sequence in the insertion and targeting of these exchangers.
Our results indicate that the native, cleaved N-terminal signal sequence is not necessary for insertion of a
functional exchanger into the cell membrane. In contrast, the photoreceptor exchanger does not have a
cleaved N-terminal signal sequence. If the N-terminal
65 amino acids are deleted, translocation of the N terminus of the protein is disrupted, but the remainder of
the exchanger is integrated into the membrane. We are
also using large-scale genomic analysis to study transgenic mice in which a mutated exchanger is expressed
and mice that lack the gene for the exchanger.
34 CELL BIOLOGY 2005
PUBLICATIONS
Barnett, F.H., Scharer-Schusz, M., Wood, M., Yu, X., Wagner, T.E., Friedlander, M.
Intra-arterial delivery of endostatin gene to brain tumors prolongs survival and
alters tumor vessel ultrastructure. Gene Ther. 11:1283, 2004.
Dorrell, M.I., Friedlander, M. Cell guidance in retinal angiogenesis. Prog. Retin.
Eye Res., in press.
Henderson, S.A., Goldhaber, J.I., So, J.M., Han, T., Motter, C., Ngo, A., Chantawansri, C., Ritter, M.R., Friedlander, M., Nicoll, D.A., Frank, J.S., Jordan, M.C., Roos,
K.P., Ross, R.S., Philipson, K.D. Functional adult myocardium in the absence of Na+Ca2+ exchange: cardiac-specific knockout of NCX1. Circ. Res. 95:604, 2004.
Otani, A., Dorrell, M.I., Kinder, K., Moreno, S.K., Nusinowitz, S., Banin, E.,
Heckenlively, J., Friedlander, M. Rescue of retinal degeneration by intravitreally
injected adult bone marrow-derived lineage-negative hematopoietic stem cells. J.
Clin. Invest. 114:765, 2004.
Otani, A., Friedlander, M. Retinal vascular regeneration. Semin. Ophthalmol.
20:43, 2005.
Ritter, M., Aguilar, E., Banin, E., Scheppke, L., Uusitalo-Jarvinen, H., Friedlander, M.
Three-dimensional in vivo imaging of the mouse ocular vasculature. Invest. Ophthal. Vis. Sci., in press.
Ritter, M., Friedlander, M. Integrins in ocular angiogenesis. In: Ocular Angiogenesis. Tobran-Tink, J., Barnstable, C. (Eds.). Humana Press, Totowa, NJ, in press.
Nucleocytoplasmic Transport
and Role of the Nuclear
Lamina in Higher Level
Nuclear Organization
L. Gerace, G. Ambrus-Aikelin, A. Aschrafi, J. Bednenko,
A. Bubeck, A. Cassany, B. Chen, T. Guan, K. Kanelakis
he nuclear envelope is a specialized domain of
the endoplasmic reticulum that forms the boundary of the nucleus in eukaryotic cells. The envelope consists of inner and outer nuclear membranes,
the nuclear lamina, and nuclear pore complexes (NPCs).
The nuclear lamina, a protein meshwork lining the
inner nuclear membrane, provides a structural scaffold
for the nuclear envelope and an anchoring site at the
nuclear periphery for chromatin. NPCs are large supramolecular assemblies that span the nuclear envelope
and serve as channels for molecular transport between
the nucleus and the cytoplasm. We are using a combination of biochemical, structural, and functional
approaches to investigate NPCs and the lamina.
T
NUCLEOCYTOPLASMIC TRANSPORT MECHANISMS
Transport of protein and RNA through NPCs is an
energy-dependent process mediated by nucleocytoplasmic shuttling receptors of the karyopherin β family.
Karyopherins bind to transport signals on protein or
RNA cargo molecules, and the receptor-cargo comPublished by TSRI Press®. © Copyright 2005,
The Scripps Research Institute. All rights reserved.
plexes are translocated through the NPC by binding of
the receptors to a group of NPC proteins (nucleoporins)
that contain phenylalanine-glycine amino acid motifs.
The directionality of nuclear transport is determined
largely by the small GTPase Ran, which directly interacts with karyopherins and thereby regulates cargo
binding. Conformational flexibility of karyopherins is
thought to be fundamental to their dynamic interactions
with cargo, Ran, and nucleoporins.
We are using in vitro assays with digitonin-permeabilized cells to analyze the molecular events that specify translocation of cargo-receptor complexes through
NPCs. Recently, using site-directed mutagenesis of
importin β, the prototypical nuclear import receptor,
we characterized 2 distinct binding sites in importin β
for nucleoporins containing the phenylalanine-glycine
motif and defined mutational hot spots for cargo binding. A major goal is to determine how the conformational dynamics of importin β are linked to discrete
transport steps. To this end, we are complementing
structure-function studies with analysis involving smallmolecule inhibitors.
We are also investigating movement of cargo-receptor
complexes though the central channels of NPCs, which
form the major permeability barrier of the pore. We are
focusing on the transport of large protein complexes,
which have more stringent Ran and energy requirements
for translocation than small proteins do.
In a related project, we are analyzing nuclear import
of the adenovirus genome, which consists of a 36-kb
double-stranded DNA molecule. Results from our in
vitro transport studies indicate that adenovirus DNA
transport is driven by import signals on DNA-associated
proteins. Our characterization of multiple import signals in adenovirus protein VII and the tight association
of the protein with the genome suggest that this viral
protein may be the protein adaptor involved in the DNA
import. Nuclear import of protein VII involves several
of the major cellular importins, suggesting that adenovirus has evolved to use redundant import pathways
to ensure efficient nuclear delivery of its genome.
We also are analyzing nuclear export of HIV type 1
mRNA mediated by the viral regulatory protein Rev.
Rev polymerizes on a cis-acting sequence of viral
mRNAs, providing a platform for assembly of nuclear
export factors. We are using proteomics combined with
a permeabilized cell assay for Rev-dependent HIV mRNA
export to functionally characterize the proteins assembled on the Rev platform. This project is part of a larger
CELL BIOLOGY 2005
collaboration with a research team at Scripps Research
to identify small-molecule inhibitors of Rev function;
the goal is to find compounds for developing new drugs
to inhibit HIV replication in humans.
Organization and Function of
the Neuronal Cytoskeleton
NUCLEAR LAMINA AND HIGHER LEVEL NUCLEAR
S. Halpain, J. Braga, B. Calabrese, L. Dehmelt, E. Hwang,
35
O R G A N I Z AT I O N
The nuclear lamina in vertebrates contains a polymer of 2–4 related intermediate filament proteins called
lamins, which are associated with a number of transmembrane proteins of the inner nuclear membrane. The
lamina plays essential roles in nuclear structure and
function, as indicated by the recent findings that more
than 15 inherited diseases in humans, including a range
of muscular dystrophies, are caused by mutations in
lamins or lamina-associated transmembrane proteins.
The involvement of the lamina in disease is thought to
be linked to its roles in nuclear integrity, chromatin
structure, and gene expression. Until recently, only about
12 transmembrane proteins specific to the nuclear envelope had been identified.
To survey the complete complement of proteins in
the nuclear envelope, we carried out a proteomics analysis of the nuclear envelope of rodent liver in collaboration with J.R. Yates, Department of Cell Biology. We
identified 67 novel putative nuclear envelope transmembrane proteins. Analysis of a subset of these proteins
indicated that most are authentic components of the
nuclear envelope.
We are now doing a systems-level analysis of the
lamina involved in muscle differentiation. For these
studies, we are using transcriptional microarray analysis, quantitative proteomics, and gene silencing by RNA
interference in cultured myoblasts. The goals are to
identify genes that may have a role in human muscular
dystrophies and to further elucidate how the protein
network consisting of lamins and associated transmembrane proteins acts in nuclear structure and function.
PUBLICATIONS
Gerace L. TorsinA and torsion dystonia: unraveling the architecture of the nuclear
envelope. Proc. Natl. Acad. Sci. U. S. A. 101:8839, 2004.
Ohba, T., Schirmer, E.C., Nishimoto, T., Gerace, L. Energy- and temperaturedependent transport of integral proteins to the inner nuclear membrane via the
nuclear pore. J. Cell Biol. 167:1051, 2004.
Schirmer, E.C., Florens, L., Guan, T., Yates, J.R. III, Gerace, L. Identification of
novel integral membrane proteins of the nuclear envelope with potential disease
links using subtractive proteomics. Novartis Found. Symp. 264:63, 2005.
Schirmer, E.C., Gerace, L. The stability of the nuclear lamina polymer changes
with the composition of lamin subtypes according to their individual binding
strengths. J. Biol. Chem. 279:42811, 2004.
Wiethoff, C.M., Wodrich, H., Gerace, L., Nemerow, G.R. Adenovirus protein VI mediates membrane disruption following capsid disassembly. J. Virol. 79:1992, 2005.
Published by TSRI Press®. © Copyright 2005,
The Scripps Research Institute. All rights reserved.
K. Spencer
uring the past year, we made significant progress
in research on the development and regeneration of neurons. In 2 main projects, we focused
on cytoskeletal proteins of nerve cells, key proteins
that underlie the structure and morphologic flexibility
required by neurons for transmitting, storing, and processing synaptic signals (Fig. 1). We used biochemical,
molecular biological, and microscopy-based approaches
to understand the function of these molecules. Fluorescence time-lapse imaging of living neurons is an
important tool that we use to uncover structure-function
relationships for cytoskeletal proteins and the consequences of dysfunction of the proteins.
D
F i g . 1 . Diagram of a brain cell axon illustrating the dense array
of protein fibers that make up the neuronal cytoskeleton. Actin filaments and microtubules interact to regulate the initiation, elongation, branching, and guidance of neuronal axons and dendrites in
the developing brain. Illustration courtesy of J. Lim, graduate student, Scripps Research.
One project concerns microtubule-associated proteins (MAPs). These proteins are important in regulating the assembly and stability of microtubules and the
interactions of microtubules with other components of
the cytoskeleton. We found that one microtubule-binding protein, MAP2, also directly binds actin filaments
and induces filament bundling. Using fluorescencebased time-lapse imaging and high-resolution confocal
microscopy, we tracked the behaviors of microtubules
36 CELL BIOLOGY 2005
and actin filaments in living neuronal cells with normal and mutant forms of MAP2.
Recently, we found that the microtubule-based
molecular motor dynein plays a key role in transporting microtubule bundles toward the cell periphery.
This dynein-dependent activity provides a key force
that pushes the cell membrane outward during neurite
initiation. Currently, we are identifying other cytoskeletal proteins and signal transduction pathways crucial
to the initiation of neurites.
A second project concerns the regulation of dendritic spines, specialized cellular protrusions that form
the receptive, postsynaptic element at glutamate synapses. Spines become lost or dysmorphic in many types
of mental retardation and in psychiatric conditions such
as chronic depression and schizophrenia. Furthermore,
spines are vulnerable to injury in diseases such as stroke
and epilepsy, in which excessive release of glutamate
can induce neuronal injury and subsequent cell death
(a condition termed excitotoxicity). Understanding how
spines form, what regulates their stability, and how they
recover from injury is therefore of therapeutic interest
for several neurologic conditions.
Our most recent results suggest a neuroprotective
role for spines, because preventing the collapse of dendritic spines attenuates neuronal cell death induced by a
subsequent lethal stimulus. The spine cytoskeleton is
composed mainly of actin filaments. We discovered
that actin filaments in spines are rapidly broken down
within minutes of an injury-inducing stimulus. However,
this damage to the spine can be rapidly reversed within
minutes under appropriate conditions, indicating for
the first time that spines can regrow after they collapse.
We identified 2 key molecules involved in regulating the shape and stability of spines. The first is the
serine protease cathepsin B, a classical lysosomal protease that functions in other subcellular domains. We
also discovered that myristoylated alanine-rich C kinase
substrate (MARCKS), a protein that regulates both
membrane trafficking and the actin cytoskeleton, is one
of the many potential substrates of cathepsin B. MARCKS
is a major downstream target of protein kinase C, and
our recent studies implicated a key role for protein
kinase C and MARCKS in the regulation of spine shape
and stability. Together these projects contribute to our
understanding of molecular events in normal brain development and in regeneration of neuronal structure after
injury and disease.
Published by TSRI Press®. © Copyright 2005,
The Scripps Research Institute. All rights reserved.
PUBLICATIONS
Calabrese, B., Halpain, S. Essential role for the PKC target MARCKS in maintaining dendritic spine morphology. Neuron 48:77, 2005.
Dehmelt, L., Halpain, S. The MAP2/tau family of microtubule-associated proteins.
Genome Biol. 6:204, 2005.
Graber, S., Maiti, S., Halpain, S. Cathepsin B-like proteolysis and MARCKS degradation in sub-lethal NMDA-induced collapse of dendritic spines. Neuropharmacology 47:706, 2004.
Graber, S., Morrison, M.E., Halpain, S. Dendritic spines in cognition and neurological
disease. Cyberounds [on-line review journal]. Available at: http://www.cyberounds.com/
conf/phsychiatryneuroscience/2004.
Halpain, S., Spencer, K., Graber, S. Dynamics and pathology of dendritic spines.
Prog. Brain Res. 147:29, 2005.
Genetics and Genomics of
Circadian Clocks
S.A. Kay, G. Breton, J. Chu, A. DeSchopke, E. Farre,
F.G. Harmon, S.P. Hazen, T. Imaizumi, B. Jenkins, C.Y. Liu,
C. Masuda, A. Para, J. Pruneda, T.F. Schultz, H.G. Tran,
D.K. Welsh,* E. Zhang
* University of California, San Diego, California
vast array of cellular processes fluctuate with a
24-hour periodicity, and an endogenous circadian
clock is responsible for generating these biological rhythms. Circadian rhythms are found in all kingdoms of life and control diverse events ranging from
sleep-wake cycles in mammals to the overall rate of
photosynthesis in plants. Many pathologic changes in
humans, such as sleep disorders, most likely are due
to a defect in circadian rhythms, so understanding how
the circadian clock operates within the cell will have
significance for human health. To study how circadian
clocks are built inside of cells, we use molecular, genetic,
and genomic approaches in 2 model systems: mouse
and Arabidopsis.
In mammals, the circadian clock plays an integral
role in timing many physiologic rhythms, such as blood
pressure, body temperature, and liver metabolism, in
anticipation of dusk and dawn. The master circadian
clock resides within a region of the brain that receives
light information from the eyes. However, this brain
region can keep time even in the absence of light, as
occurs in some who are visually blind. Mutations in
the genes that encode components of the circadian
clock are manifested as abnormal activity rhythms in
rodents and as sleeping disorders in humans, although
which photoreceptors set the clock is unclear. Thus,
although marked advancements have been made in
A
CELL BIOLOGY 2005
understanding how the mammalian clock itself runs, little is known about how light transduces synchronizing
signals to the clock.
To address this major question, we are using genetic
and genomic approaches to identify new gene functions
in circadian biology. We are generating a number of
mouse strains with mutations in known and potential
photoreceptors and are testing the mice for defects in
circadian rhythm. Thus far, we have determined that
one photoreceptor, melanopsin, is an important contributor in maintaining synchrony between the clock and
environmental light conditions. With the recently completed sequencing of the human and mouse genomes,
we now know the sequences of more than 30,000
genes that can be investigated for potential roles in
circadian function. We developed large-scale, in vitro,
cell-based assays that can be used to rapidly determine
if genes control clock activity. Combining this approach
with genetic analysis will enable us to further dissect
the connection between environmental stimuli, in the
form of light, and the behavioral and physiologic events
regulated by the circadian clock.
In recent years, researchers have found that intrinsic circadian clocks exist in various peripheral tissues
and cell types, directly controlling local physiology and
behavior. We are studying the circadian oscillators in
the liver and in the vasculature. As the first step, we
are investigating the heterogeneity and distinct functions
of the central and peripheral oscillators. In particular, we
are examining the distinct roles of the retinoid-related
orphan nuclear receptors in the clock mechanism. These
nuclear receptors were recently identified in our functional genomics studies.
Second, we are asking how environmental cues,
mainly light-dark cycles and feeding time, entrain or
synchronize peripheral oscillators. Peripheral oscillators most likely are synchronized by hormonal outputs
of the suprachiasmatic nucleus or by physiologic inputs
such as feeding-mediated metabolic changes. We are
using transgenic mice, mice lacking certain genes, and
immortalized hepatocytes and vascular smooth muscle
cells in these studies along with real-time bioluminescence imaging and biochemical and genetic approaches.
Furthermore, we are using high-resolution bioluminescence imaging to determine whether single neurons in
the suprachiasmatic nucleus and peripheral cells are
autonomous circadian clocks and to characterize the
precise nature of synchronization of the molecular clockwork of individual cells.
Published by TSRI Press®. © Copyright 2005,
The Scripps Research Institute. All rights reserved.
37
Flowering is a major event in the life cycle of higher
plants. Many plants use seasonal changes in the length
of days as a signal to flower, and higher plants use their
circadian clocks to perceive these changes. Recently, we
defined a molecular link between the circadian clock
and day length–dependent regulation of flowering. A
flowering time gene known as CONSTANS was identified a number of years ago and is regulated by the circadian clock. We showed that clock regulation of
CONSTANS expression is the key to seasonal control of
flowering in Arabidopsis. We are extending these studies
by comparing gene expression profiles under conditions
of long days and short days to identify other components involved in perception of day length.
By combining molecular, genetic, and genomic
approaches, we are beginning to define a number of
molecular links between the circadian clock and rhythmic regulation of behavior and development. Analysis
of circadian rhythms in multiple organisms provides a
unique opportunity to define molecular controls for the
behavior of whole organisms. These results will provide targets for clinical and agricultural applications
to improve the quality of life.
PUBLICATIONS
Flechner, S.M., Kurian, S.M., Head, S.R., Sharp, S.M., Whisenant, T.C., Zhang, J.,
Chismar, J.D., Horvath, S., Mondala, T., Gilmartin, T., Cook, D.J., Kay, S.A., Walker,
J.R., Salomon, D.R. Kidney transplant rejection and tissue injury by gene profiling of
biopsies and peripheral blood lymphocytes. Am. J. Transplant. 4:1475, 2004.
Sato, T.K., Panda, S., Miraglia, L.J., Reyes, T.M., Rudic, R.D., McNamara, P.,
Naik, K.A., FitzGerald, G.A., Kay, S.A., Hogenesch, J.B. A functional genomics
strategy reveals Rora as a component of the mammalian circadian clock. Neuron
43:527, 2004.
Ueda, H.R., Hayashi, S., Matsuyama, S., Yomo, T., Hashimoto, S., Kay, S.A.,
Hogenesch, J.B., Iino, M. Universality and flexibility in gene expression from bacteria to human. Proc. Natl. Acad. Sci. U. S. A. 101:3765, 2004.
Welsh, D.K., Yoo, S.H., Liu, A.C., Takahashi, J.A., Kay, S.A. Bioluminescence
imaging of individual fibroblasts reveals persistent, independently phased circadian
rhythms of clock gene expression. Curr. Biol. 14:2289, 2004.
Function of Nuclear Receptors
in Stress and Mitochondrial
Homeostasis
A. Kralli, J. Cardenas, M.B. Hock, C. Tiraby-Nguyen,
J. Villena
uclear receptors are ligand-regulated transcription factors with important roles in mammalian
development and physiology. Although many
nuclear receptors are activated by recognized small
N
38 CELL BIOLOGY 2005
lipophilic ligands, such as steroids and other lipids,
some have no known ligands and are called orphans.
We focus on nuclear receptors and receptor cofactors
that affect energy metabolism pathways.
GLUCOCORTICOIDS AND RESPONSE TO STRESS
The ability of organisms to respond and adapt to
stressors is fundamental for life. Response to stress
involves activation of the neuroendocrine system and
the secretion of adrenal glucocorticoids. Glucocorticoids
act via the glucocorticoid receptor to enable mobilization
of energy resources, recovery from the stress response,
and preparation for future stressors. The transcriptional
response mediated by receptors for glucocorticoids
integrates the hormonal signal with signals indicating
the type of stressor, the physiologic state of the organism, and the cellular environment. We are studying the
regulatory mechanisms that enable the integration of
such diverse signals in the activity of glucocorticoid
receptors. In particular, we address the function of transcriptional coactivators as integrators of such responses.
Peroxisome proliferator-activated receptor γ coactivator-1α (PGC-1α), an inducible coactivator that coordinates transcriptional programs important for energy
homeostasis, is a potent, tissue-specific modulator of glucocorticoid receptors. Our studies suggest that PGC-1α,
glucocorticoid receptors, and the orphan nuclear receptor
estrogen-related receptor α (ERRα) are parts of the transcriptional network that coordinates the stress response.
ERRα AND MITOCHONDRIAL FUNCTION
ERRα was the first orphan nuclear receptor identified, yet we are just starting to understand its physiologic function. We showed that ERRα is regulated by
PGC-1α, which binds ERRα and converts it from a factor with little or no transcriptional activity to a potent
activator of gene expression. This activation enables the
induction of ERRα target genes, including the ERRα
gene itself. As a result, levels of ERRα in vivo are highest in tissues that express PGC-1α, such as heart, kidney, brown adipose tissue, and muscle, and are induced
in response to signals that relay metabolic needs and
that upregulate PGC -1α, such as exposure to cold,
fasting, and exercise.
The similar spatial and temporal expression of
PGC-1α and ERRα suggests that the complex consisting of these 2 molecules mediates the induction of some
of the known PGC-1α–responsive pathways. Indeed, the
results of our gain-of- and loss-of-function studies support the notion that ERRα regulates facets of mitochondrial metabolism, including mitochondrial biogenesis,
Published by TSRI Press®. © Copyright 2005,
The Scripps Research Institute. All rights reserved.
fatty acid oxidation, the tricarboxylic acid cycle, and
oxidative phosphorylation. Mitochondrial dysfunction
and the reduced expression of genes associated with
oxidative phosphorylation have been implicated as
underlying factors in the development of insulin resistance. Our findings suggest that strategies aiming at
increasing ERRα activity could be beneficial in the
treatment of metabolic disease, such as type 2 diabetes.
PUBLICATIONS
Teyssier, C., Ma, H., Emter, R., Kralli, A., Stallcup, M.R. Activation of nuclear
receptor coactivator PGC-1α by arginine methylation. Genes Dev., in press.
Detection of Circulating Tumor
Cells in Peripheral Blood
P. Kuhn, R. Bruce,* K. Bethel, B. Hsieh,* M. Humphrey,*
R. Krivacic,* L. Kroener, N. Lazarus,* A. Ladanyi,*
D. Marrinucci, J. Nieva
* Palo Alto Research Center, Palo Alto, California
he spread of cancer from a primary site to secondary sites, known as metastasis, is correlated
with a high incidence of mortality. Research indicates that metastasis begins when primary tumor cells
disseminate, infiltrate, and invade the circulatory system. Although most disseminated cells are destroyed
in the circulatory system via apoptosis, a surviving cell
can invade a secondary organ, micrometastasize, and
form a secondary tumor. Detection of these circulating
tumor cells (CTCs), especially before metastasis occurs,
is critical for increasing survival rates.
CTCs exist in the peripheral blood in cancer patients
at ultralow concentrations, at an estimated rate of about
1 in 10 million leukocytes. Although existing technology
can be used to detect CTCs in patients with metastasis and to correlate the cells with overall disease survival, no method has sufficient sensitivity to reliably
measure a statistically significant number of cells at
early stages of the disease when treatment can be most
effective. Generally, optical scan technologies are used
to detect immunocytochemically labeled tumor cells,
and with some of the technologies, pathologic examination of the cells is possible. Without cell enrichment,
scan rates are far too slow for analysis of the sufficiently large population of cells needed to be statistically relevant.
Immunomagnetic enrichment relies on consistent and
substantial expression of the membrane adhesion recep-
T
CELL BIOLOGY 2005
tor EpCAM. Although this method has been approved by
the Food and Drug Administration for use in patients
with metastasis, the phenotypic heterogeneity of CTCs
suggests that the sensitivity of this approach is too low
to detect all of the CTCs present in peripheral blood.
We have developed an instrument that can be used to
directly scan and analyze 50 million nucleated cells
and detect rare cells in less than 2 minutes without
using antigen-dependent enrichment technology. This
method is 500 times faster than other cell-based scan
approaches because of the instrument’s exceptionally
wide field of view made possible by the use of fiberoptic array scanning technology (FAST). With the FAST
cytometer (Fig. 1), cells can be detected at a rate of
25 million cells per minute, with a sensitivity of 98%
and a specificity of 4 x 10 −6 . In addition, we have
developed a sample preparation method, the live-cell
attachment protocol, that is optimized for maximal
cell retention.
F i g . 1 . Major components of the FAST system. Reprinted with
permission from Curry, D.N., Krivacic, R.T., Hsieh, H.B., Ladanyi,
A., Bergsrud, D.E., Ho, M.Y., Chen, L.B., Kuhn, P., Bruce, R.H.
High-speed detection of occult tumor cells in peripheral blood.
Proc. IEEE Eng. Med. Biol. Soc. Vol. 26, 2004. © 2004 IEEE.
We are using the FAST instrument and the cellpreparation method to investigate the prevalence of
CTCs in patients with metastasis and in patients with
early-stage disease. Figure 2 shows a CTC in a background of leucocytes.
Peripheral blood from patients is processed and
labeled for detection of cytokeratin, a suitable biomarker for epithelial cancers. Because cytokeratin is
not expressed in normal hematopoietic cells, CTCs can
be identified by using a mixture of monoclonal antibodies against 9 different human cytokeratin proteins. The
samples are then scanned by using the FAST cytometer.
During the scan, the locations of detected objects are
collected. These locations are specified by a substrate
coordinate system determined by alignment marks on
Published by TSRI Press®. © Copyright 2005,
The Scripps Research Institute. All rights reserved.
39
F i g . 2 . A circulating tumor cell from a sample of cells obtained
from a patient with metastasis and labeled for cytokeratin is easily
distinguishable from a background of leukocytes.
the substrate. In order to relocate a FAST object with
a microscope, these coordinates are transformed to a
coordinate system on the microscope stage with the
same alignment marks. Via object relocation, the objects
detected by using FAST are scanned in the microscope
to detect true cancer cells.
In addition to determining numbers of CTCs, object
relocation will be useful in further characterizing these
rare cells. In a preliminary trial with cells from patients
with metastatic cancer and cells from healthy donors,
CTCs were located in all of the cell samples from cancer patients and in none of the samples from healthy
donors. These findings suggest that this method is
promising both for diagnosis of cancer and for monitoring responses to therapy.
PUBLICATIONS
Krivacic, R.T., Ladanyi, A., Curry, D.N., Hsieh, H.B., Kuhn, P., Bergsrud, D.E.,
Kepros, K.F., Barbera, T., Ho, M.Y., Chen, L.B., Lerner, R.A., Bruce, R.H. A rarecell detector for cancer. Proc. Natl. Acad. Sci. U. S. A. 101:10501, 2004.
Novel Mechanisms of
Cardiovascular Disease
D.J. Loskutoff, R.P. Czekay, C. Dellas, G. Giandomenico,
Y. Kamikubo, S. Konstantinides, S. Koschnick, J. Neels
P
lasminogen activator inhibitor-1 (PAI-1) is a serine protease inhibitor (serpin) that regulates proteases that remove pathologic fibrin deposits
40 CELL BIOLOGY 2005
from the vasculature. PAI-1 differs from most other serpins because it is the product of an immediate-early gene
and it binds to the adhesive glycoprotein vitronectin. Leptin is synthesized by adipocytes and acts on hypothalamic receptors to reduce food intake and to increase
energy expenditure. Juvenile-onset obesity develops in
mice that lack functional leptin (ob/ob mice) or its receptor (db/db mice). Our studies indicate that PAI-1 and leptin promote cardiovascular disease in mice.
D E TA C H M E N T O F C E L L S B Y PA I - 1
The binding of PAI-1 to cell-surface urokinase promotes the inactivation and internalization of adhesion
receptors (e.g., integrins, the urokinase receptor) and
leads to cell detachment from a variety of extracellular
matrices. Cell detachment is not a specific property of
PAI-1, because protease nexin-1 also detaches cells,
and it is not simply the result of the binding of macromolecules to urokinase and/or of the inactivation of
urokinase, because neither antibodies to urokinase nor
active-site inhibitors of urokinase can detach the cells.
The binding of PAI-1 and protease nexin-1 to urokinase leads to the specific inactivation of the matrixengaged integrins, a process that requires a direct
interaction between urokinase receptors and those integrins. These inhibitor-mediated changes in the subcellular distribution of the urokinase receptor and integrins
may influence a variety of cardiovascular disorders.
THROMBOTIC PHENOTYPE OF MICE WITH A
C O M B I N E D D E F I C I E N C Y I N PA I - 1 A N D V I T R O N E C T I N
The role of vitronectin in thrombosis is not fully
understood, primarily because this adhesive glycoprotein not only stabilizes PAI-1 and thus protects fibrin
from premature lysis but also binds to platelet integrins
and may influence platelet aggregation. Using novel
quantitative approaches, we examined the thrombotic
phenotype of mice with a combined deficiency in both
PAI-1 and vitronectin. Although unstable thrombi developed in all deficient mice, the thrombotic phenotype
of mice with the combined deficiency did not differ
significantly from the phenotype of mice with deficiencies in only PAI-1 or vitronectin. This observation suggests that vitronectin may influence thrombus stability
by regulating PAI-1 and not platelets.
L E P T I N A N D C A R D I O VA S C U L A R D I S E A S E
Obesity in humans is associated with elevated levels of leptin and an increased risk for thrombosis and
for cardiovascular diseases. Endogenous leptin appears
to regulate thrombosis in vivo, because administration
of leptin promoted arterial thrombosis in mice, and comPublished by TSRI Press®. © Copyright 2005,
The Scripps Research Institute. All rights reserved.
pared with lean mice, leptin-deficient ob/ob mice had
unstable thrombi and an attenuated thrombotic response
to injury. Moreover, when lean mice were pretreated
with a leptin-neutralizing antibody before injury of
the carotid artery, the mice had prolonged times to
thrombotic occlusion and the development of unstable, embolizing thrombi.
Thus, inhibiting leptin converted the thrombotic
phenotype of wild-type mice into one that closely resembled that of ob/ob mice. The antibody also protected
mice from experimentally induced venous thrombosis
and pulmonary embolism. Thus, inhibition of circulating leptin protects against arterial and venous thrombosis in mice, and possibly in hyperleptinemic humans
with obesity.
In other studies, compared with mice fed normal
chow, wild-type mice fed an atherogenic, high-fat diet
had elevated (9-fold) leptin levels and significantly
enhanced neointimal thickening after injury of the carotid
artery. Unexpectedly, the atherogenic diet had no effect
on injured vessels from leptin-deficient ob/ob mice
despite aggravating obesity, diabetes, and hyperlipidemia in these animals. Daily administration of leptin
to ob/ob mice during the 3-week period after injury
dramatically increased neointimal thickness and the
severity of luminal stenosis but had no effect on vessels
from db/db mice, which lack the receptor for leptin.
These results suggest that a direct, leptin receptor–mediated link exists between the hyperleptinemia in human
obesity and the increased risk for cardiovascular complications associated with this condition.
We also found that administration of leptin promoted
platelet activation and thrombosis in mice and potentiated the aggregation of human platelets induced by
low concentrations of ADP, collagen, and epinephrine.
However, the responses of human platelets varied significantly according to the donor of the cells. Platelets
from some donors (~40%) consistently responded to
leptin (responders), whereas platelets from other donors
(~60%) never responded (nonresponders). Although
platelets from both groups expressed only the signaling
form of the leptin receptor, the platelets from responders
expressed higher levels of this receptor than did those
of nonresponders. Ligand-binding assays indicated specific, saturable binding of leptin to platelets from both
groups. Thus, the decreased sensitivity to leptin of
platelets from nonresponders is not due to the absence
of the signaling form of the leptin receptor but may
reflect differences in its level of expression and/or affinity
CELL BIOLOGY 2005
for leptin. These studies indicate that platelets are a
major source of leptin receptors in the circulation and
suggest that leptin-responsive persons may have a higher
risk for obesity-associated thrombosis than do nonresponsive persons.
Preliminary studies indicate that the leptin receptor
is upregulated upon platelet activation and that platelets
do not store or release significant amounts of leptin.
These results suggest that activation of platelets leads to
increased binding of circulating leptin, further enhancing
platelet activation.
PUBLICATIONS
Campagnolo, L., Leahy, A., Chitnis, S., Koschnick, S., Fitch, M.J., Fallon, J.T., Loskutoff, D., Taubman, M.B., Stuhlmann, H. EGFL7 is a chemoattractant for endothelial cells
and is up-regulated in angiogenesis and arterial injury. Am. J. Pathol. 167:275, 2005.
Czekay, R.P., Loskutoff, D.J. Unexpected role of plasminogen activator inhibitor 1
in cell adhesion and detachment. Exp. Biol. Med. (Maywood) 229:1090, 2004.
Degryse, B., Resnati, M., Czekay, R.-P., Loskutoff, D.J., Blasi, F. Domain 2 of the
urokinase receptor contains an integrin-interacting epitope with intrinsic signaling
activity: generation of a new integrin inhibitor. J. Biol. Chem., in press.
Dellas, C., Loskutoff, D.J. Historical analysis of PAI-1 from its discovery to its
potential role in cell motility and disease. Thromb. Haemost. 93:631, 2005.
Giandomenico, G., Dellas, C., Czekay, R.-P., Koschnick, S., Loskutoff, D.J. The
leptin receptor system of human platelets. J. Thromb. Haemost. 3:1042, 2005.
Konstantinides, S., Schäfer, K., Neels, J.G., Dellas, C., Loskutoff, D.J. Inhibition
of endogenous leptin protects mice from arterial and venous thrombosis. Arterioscler. Thromb. Vasc. Biol. 24:2196, 2004.
Koschnick, S., Konstantinides, S., Schäfer, K., Crain, K., Loskutoff, D.J. Thrombotic phenotype of mice with a combined deficiency in plasminogen activator
inhibitor 1 and vitronectin. J. Thromb. Haemost., in press.
Pandey, M., Loskutoff, D.J., Samad, F. Molecular mechanisms of tumor necrosis factor-α-mediated plasminogen activator inhibitor-1 expression in adipocytes. FASEB J.,
in press.
Schäfer, K., Halle, M., Goeschen, C., Dellas, C., Pynn, M., Loskutoff, D.J., Konstantinides, S. Leptin promotes vascular remodeling and neointimal growth in
mice. Arterioscler. Thromb. Vasc. Biol. 24:112, 2004.
Viral Nanoparticles:
Pathogen Inhibitors and
Biomolecular Sensors
M. Manchester, E. Burke, G. Destito, M. Estrada,
41
Previously, researchers focused primarily on chemically derived materials such as dendrimers or polymers to develop synthetic nanosized particles (<100
nm in diameter) for biological applications such as
molecular therapeutics, tumor targeting, and in vivo
biomedical imaging.
We use cowpea mosaic virus as a nanoparticle platform for antivirals, antitoxins, vaccines, and tumor-targeting agents. The virus is an icosahedral, 31-nm particle
that can be produced easily and inexpensively in blackeyed pea plants. In contrast to the structure of most
other nanomaterials, the structure of the capsid of
cowpea mosaic virus is defined and can be engineered
to display peptides or proteins in controlled orientations
on particle surfaces via either genetic manipulation of
the viral genome or by chemical attachment to the particle surface.
V I R A L N A N O PA R T I C L E S A S VA C C I N E S A N D
ANTITOXINS
We have generated viral nanoparticles that display
T-cell epitopes on the surface in a multivalent fashion.
In vitro, these nanoparticles can be taken up into several types of antigen-presenting cells, including dendritic
cells and macrophages, and in vivo, particles given
orally or intravenously localize to antigen-presenting
cells. Viral nanoparticles pass through the gut epithelial lining via M cells that sample particulate matter in
the gastrointestinal tract. The uptake appears to be via a
slow endocytic pathway, with the nanoparticles localizing in perinuclear endosomes. Viral nanoparticles
displaying T-cell epitopes can induce pathogen-specific
T-lymphocyte responses in vivo that are protective
against pathogen challenge, indicating that the particles leave the endosomes to enter the antigen-presentation pathway associated with class I MHC molecules.
We are also developing combination inhibitor-vaccine approaches for other viral and bacterial pathogens. Finally, we have shown that soluble pathogen
receptors are protective against challenge with anthrax
lethal toxin in vivo, and we are studying the efficacy
of multivalent arrays of these receptor decoy domains
on viral nanoparticles.
M.J. Gonzalez, K. Koudelka, E. Powell, C. Rae, P. Singh,
V I R A L N A N O PA R T I C L E S F O R VA S C U L A R I M A G I N G
D. Thomas
A N D T U M O R TA R G E T I N G
he goal of nanotechnology in biomedical science
is the design of tiny nanomachines with multiple
functions that can be used to detect, target, and
treat human disease in vivo, thereby eliminating the
need for invasive diagnostic or therapeutic procedures.
Our goal is to develop viral nanoparticles that can
be delivered in a noninvasive manner, home to a tumor
in vivo, act as an image-contrast agent for detection by
magnetic resonance imaging or other noninvasive imaging techniques, and deliver an antitumor therapeutic
T
Published by TSRI Press®. © Copyright 2005,
The Scripps Research Institute. All rights reserved.
42 CELL BIOLOGY 2005
or tumoricidal gene. These studies are being done in collaboration with M.G. Finn, Department of Chemistry, J.
Johnson and A. Schneemann, Department of Molecular
Biology, and H. Stuhlmann, Department of Cell Biology.
Fluorescently labeled viral nanoparticles are very
bright, nontoxic materials that are excellent reagents
for imaging the vasculature in live animals. Working
with Dr. Stuhlmann and J. Lewis, Department of Cell
Biology, we showed that viral nanoparticles can be used
to effectively image the complete embryonic vasculature
in several species. These particles are also useful for
highlighting tumor vasculature in the chick chorioallantoic membrane tumor onplant model. Uptake of particles
into endothelial cells occurs, yielding a bright imaging
signal that can also be used to differentiate between
arterial and venous vessels.
We have also investigated the usefulness of viral
nanoparticles as biomolecular sensors to detect, image,
and treat tumors in vivo. We have now designed several tumor-specific nanoparticles that target various
types of tumor cells specifically and are taken up into
tumor cells both in vivo and in vitro. These studies
will allow us to further the design of antitumor agents
that can provide localized, specific imaging and therapy in order to visualize and eliminate cancer in its
earliest stages.
PUBLICATIONS
Naniche, D., Garenne, M., Rae, C., Manchester, M., Buchta, R., Brodine, S.K.,
Oldstone, M.B. Decrease in measles virus-specific CD4 T cell memory in vaccinated subjects. J. Infect. Dis. 190:1387, 2004.
Portney, N.G., Singh, K., Chaudhary, S., Destito, G., Schneemann, A., Manchester, M.,
Ozkan, M. Organic and inorganic nanoparticle hybrids. Langmuir 21:2098, 2005.
Scobie, H.M., Thomas, D., Marlett, J.M., Destito, G., Wigelsworth, D.J., Collier
R.J., Young J.A.T., Manchester, M. A soluble receptor decoy protects rats against
anthrax lethal toxin challenge. J. Infect. Dis., in press.
Singh, P., Gonzalez, M.J., Manchester, M. Viruses and their uses in nanotechnology. Drug Dev. Res., in press.
Translational Regulation in
Chloroplasts and Expression of
Human Monoclonal Antibodies
in Eukaryotic Algae
S.P. Mayfield, M. Beligni, D. Barnes, E. Brown, A. Coragliotti,
K. Espina, S. Franklin, R. Henry, A. Manuell, J. Schultz
G
ene expression in chloroplasts is primarily controlled during the translation of plastid mRNAs
into proteins, and understanding how this pro-
Published by TSRI Press®. © Copyright 2005,
The Scripps Research Institute. All rights reserved.
cess is regulated is key to understanding plant development and function. Controlling chloroplast translation is also an essential component of optimizing the
production of human therapeutic proteins in algae.
Using proteomic and bioinformatic analysis, we
identified the set of proteins that function in chloroplast translation. These studies indicated that the
translational apparatus of chloroplasts is related to
that of bacteria but that chloroplasts have incorporated additional proteins that allow more complex regulatory mechanisms. Chloroplasts also contain a number
of RNA elements that are not found in bacteria, and
even conserved RNA elements, such as ribosome-binding sequences, are positioned in chloroplast mRNAs far
upstream compared with their bacterial counterparts.
These unique components provide the opportunity for
regulation of chloroplast translation that cannot be
achieved in simpler bacterial systems.
To understand the unique, and conserved, aspects
of plant translation, we have begun a structural analysis of both the chloroplast and the cytoplasmic ribosomes of Chlamydomonas reinhardtii. Using electron
cryomicroscopy and single-particle reconstruction, we
determined the structure of cytoplasmic ribosomes and
found that they are almost identical to ribosomes from
animals, including ribosomes from mammals. We also
determined a preliminary structure of chloroplast ribosomes and found that they differ substantially from
ribosomes of bacteria, as predicted. These studies have
revealed the structural basis for identifying the molecular
and biochemical interactions of mRNA and ribosomes
that result in regulated translation of mRNAs.
In addition to these basic studies on translational
regulation, we have developed a system for the expression of recombinant proteins, including human therapeutic agents, in C reinhardtii. We constructed strains
of C reinhardtii that express variants of human monoclonal antibodies against herpes simplex virus. These
antibodies assemble in the algal cells to form fully
functional antibodies that bind herpes simplex proteins.
We also showed that this alga-based system can be
used to produce a number of other proteins with potential human therapeutic value. These studies indicate
that eukaryotic algae have tremendous potential for
the expression of recombinant human therapeutic proteins, because algae can be grown economically on a
very large scale. Our continued genetic, biochemical,
and structural studies should lead to a greater understanding of the mechanism of chloroplast translation
and to higher levels of expression of therapeutic proteins.
CELL BIOLOGY 2005
PUBLICATIONS
Barnes, D., Cohen, A., Bruick, R.K., Kantardjieff, K., Fowler, S., Efuet, E., Mayfield, S.P. Identification and characterization of a novel RNA binding protein that
associates with the 5′-untranslated region of the chloroplast psbA mRNA. Biochemistry 43:8541, 2004.
Beligni, M.V., Yamaguchi, K., Mayfield, S.P. Chloroplast elongation factor Ts proprotein is an evolutionarily conserved fusion with the S1 domain-containing plastidspecific ribosomal protein-7. Plant Cell 16:3357, 2004.
Beligni, M.V., Yamaguchi, K., Mayfield, S.P. The translational apparatus of Chlamydomonas reinhardtii chloroplast. Photosyn. Res. 82:315, 2004.
Franklin, S.E., Mayfield, S.P. Recent developments in the production of human
therapeutic proteins in eukaryotic algae. Expert Opin. Biol. Ther. 5:225, 2005.
Manuell, A., Beligni, M.V., Yamaguchi, K., Mayfield, S.P. Regulation of chloroplast
translation: interactions of RNA elements, RNA-binding proteins, and the plastid
ribosome. Biochem. Soc. Trans. 32(Pt. 4):601, 2004.
Mayfield, S.P., Franklin, S.E. Expression of human antibodies in eukaryotic microalgae. Vaccine 23:1828, 2005.
Somanchi, A., Barnes, D., Mayfield, S.P. A nuclear gene of Chlamydomonas reinhardtii, Tba1, encodes a putative oxidoreductase required for translation of the
chloroplast psbA mRNA. Plant J. 42:341, 2005.
Molecular Basis of Cognitive
Function and Dysfunction
M. Mayford, E. Korzus, K. Limbaeck-Stokin, G.J. Reijmers,
M. Yasuda, R. Yasuda, S. Miller, N. Matsuo
he ability to remember is perhaps the most significant and distinctive feature of our cognitive
life. We are who we are in large part because of
what we have learned and what we remember. Impairments in learning and memory are a component of disorders that affect human beings throughout life, from
childhood forms of mental retardation to psychiatric disorders such as schizophrenia with onsets in late adolescence and early adulthood to diseases of aging such
as Alzheimer’s disease.
T
CALCIUM SIGNALING AND MEMORY
We know relatively little at a molecular level about
how the brain stores new information. One hypothesis,
which we tested, is that calcium-regulated changes in
the strength of synaptic connections between nerve cells
can store information. The enzyme calcium/calmodulin-dependent protein kinase is abundant at synapses
and when activated by calcium can strengthen synaptic
connections. We used genetic manipulations in mice
to indiscriminately activate this kinase at all synapses
in the entorhinal cortex, a part of the brain that is important for memory and is affected in the earliest stages
of Alzheimer’s disease in humans.
Published by TSRI Press®. © Copyright 2005,
The Scripps Research Institute. All rights reserved.
43
We found not only that the formation of new memories was impaired but also that previously established
memories could be erased. If memories are stored as
precise patterns of synaptic weights, then the indiscriminate strengthening of synapses might be expected
to erase memories in a manner similar to the way
writing all 1’s in computer memory will erase previously stored information.
GENETIC MODELS OF DISEASE
The recent determination of the complete sequence
of the mouse and human genomes indicates that humans
are highly similar to mice at the genetic level. One
approach to understanding genetic diseases in humans
is to introduce the same mutations into mice to produce
a model of the disease for better understanding of the
molecular pathology and for testing possible treatments.
Rubenstein-Taybi syndrome is a developmental and
cognitive disorder that results from mutation in the
gene CBP. We produced a strain of mice with a defect
in CBP and found that the mice were impaired in several learning and memory tasks. More important, we
showed that these impairments were not due to problems in development of the brain because they could be
reversed by providing a normally functioning CBP gene
to adult mice. The protein encoded by CBP chemically
modifies histones to allow the expression of a large variety of other genes. We found that the memory deficits in
the mice could be reversed by treatment with a drug that
targets this histone-modifying function, suggesting a
treatment for this and possibly other cognitive disorders.
M O L E C U L A R A N AT O M Y O F M E M O R Y
When humans learn new information, they use only
a tiny fraction of the neurons in brain. One of the difficulties in studying memory is an inability to identify
and specifically manipulate those neurons that participate in a particular memory trace. We recently developed a genetic technique for use in mice that enables
us to specifically introduce genetic changes into neurons that are activated by behavioral stimuli. We are
using this approach to introduce marker proteins that
enable us to see the connections between neurons that
have been activated during learning. We can follow
these neurons over long periods to detect changes in
their structure and function as the particular memory
trace decays or strengthens with repeated training.
PUBLICATIONS
Limback-Stokin, K., Korzus, E., Nagaoka-Yasuda, R., Mayford, M. Nuclear calcium/calmodulin regulates memory consolidation. J. Neurosci. 24:10858, 2004.
44 CELL BIOLOGY 2005
Regulation of the Plasminogen
Activation System
A. Baik, F. Garcia-Bannach,* N. Gingles, J. Mitchell,
R.J. Parmer,* L. Teyton,** L.A. Miles
* University of California, San Diego, California
** Department of Immunology, Scripps Research
ssembly of plasminogen and plasminogen activators on cell surfaces is a key control point for
positive regulation of cell-surface proteolytic activity necessary in physiologic and pathologic processes.
Plasminogen-binding sites are markedly upregulated
when monocytoid cells undergo apoptosis. Monocytes
are major mediators of inflammation, and apoptosis is
a mechanism for regulating the inflammatory response
by eliminating activated macrophages. Therefore, we
are investigating the ability of plasminogen to modulate
apoptosis in monocytes.
We cultured monocytoid cells (freshly isolated human
monocytes and U937 cells) in plasminogen-deficient
serum and induced the cells to undergo apoptosis by
using either TNF-α or cycloheximide. When induced in
the presence of increasing concentrations of plasminogen,
apoptosis was inhibited in a dose-dependent manner; full
inhibition occurred at a concentration of plasminogen
equal to its physiologic concentration. Treatment with
plasminogen also markedly reduced intranucleosomal
DNA fragmentation and the active caspase-3, caspase-8,
and caspase-9 induced by TNF-α or by cycloheximide.
We also examined the requirement for plasmin proteolytic activity in the cytoprotective function of plasminogen. A plasminogen active-site mutant did not
recapitulate the cytoprotective effect of wild-type plasminogen. Furthermore, antibodies against protease-activated receptor 1 blocked the antiapoptotic effects of
plasminogen. The cytoprotective effect of plasminogen
required plasmin proteolytic activity and was mediated
by protease-activated receptor 1. Because apoptosis of
monocytes plays a key role in inflammation and atherosclerosis, our studies provide insight into a novel role of
plasminogen in these processes.
A
PUBLICATIONS
Garcia-Bannach, F.G., Gutierrez-Fernandes, A., Parmer, R.J., Miles, L.A. Interleukin-6- induced plasminogen gene expression in murine hepatocytes is mediated
by transcription factor CCAAT/enhancer binding protein β (C/EBPβ). J. Thromb.
Haemost. 2:2205, 2004.
Griffin, M.O, Jinno, M., Miles, L.A., Villarreal, F.J. Reduction of myocardial infarct size
by doxycycline: a role for plasmin inhibition. Mol. Cell. Biochem. 270:1, 2005.
Published by TSRI Press®. © Copyright 2005,
The Scripps Research Institute. All rights reserved.
Miles, L.A., Hawley, S.B., Baik, N., Andronicos, N.M., Castellino, F.J. Parmer,
R.J. Plasminogen receptors: the sine qua non of cell surface plasminogen activation. Front. Biosci. 10:1754, 2005.
Structure and Action of
Molecular Machines
R.A. Milligan, J. Chappie, P. Chowdhury, T. Dang, A. Efimov,
A. Mulder, G. Orca, M. Reedy,* M.K. Reedy,* C. Reyes,
B. Sheehan, K. Thompson, A.B. Ward,
E.M. Wilson-Kubalek, C. Yoshioka
* Duke University Medical Center, Durham, North Carolina
acromolecular assemblies may be composed of
from 2 to perhaps scores of proteins and are
the functional units—the molecular machines—
of the cell. We use electron cryomicroscopy and image
analysis to study the structure and mechanism of action
of several of these machines. We combine the 3-dimensional maps calculated from electron images of the
machines with biochemical data and high-resolution
x-ray structures of the individual components to provide insight into the operation of the machines. During
the past year, we continued our work on members of
the myosin and kinesin superfamilies, microtubule-stabilizing proteins, and membrane proteins.
Although the mechanism of plus end–directed, processive motion by conventional kinesins is now well
understood, the mechanism by which members of the
kinesin 14 class move toward the minus ends of microtubules is not. Likewise, in the myosin superfamily, how
nucleotide-mediated conformational changes in the motor
domain of class VI myosins result in “backward” motility
is not known. We are elucidating the molecular mechanisms of these more unusual members of the myosin
and kinesin superfamilies. (Movies showing the motions
of conventional myosin and kinesin can be viewed at
www.scripps.edu/milligan/projects.html.)
The kinesin Ncd belongs to the kinesin 14 class
of motor proteins. Compared with the situation with
plus end–directed kinesins, the nature and timing of
the structural changes that underlie the motility of
kinesin 14 motors are poorly understood. We used
electron cryomicroscopy and image analysis to calculate 3-dimensional maps of Ncd bound to microtubules
in various stages in its mechanochemical cycle. The
maps revealed a minus end–directed rotation of approximately 70° of a coiled coil mechanical element of
microtubule-bound Ncd upon ATP binding. In parallel
M
CELL BIOLOGY 2005
with these structural studies, our collaborators, N. Endres
and R. Vale at the University of California, San Francisco,
showed that extending or shortening this mechanical
element respectively increases or decreases movement
velocity without affecting ATPase activity. These results
indicate that as with other kinesins, the force-producing conformational change of Ncd occurs upon ATP
binding but, unlike the situation with other kinesins,
involves the swing of a rigid, lever arm–like mechanical element similar to that described for myosins.
Whereas most kinesins move along intact microtubules, members of the kinesin 13 class destabilize
and depolymerize microtubules and do not appear to
have motile properties. We found that a KinI fragment
consisting of only the conserved motor core is necessary and sufficient for ATP-dependent depolymerization. The motor core binds along microtubules in all
nucleotide states, but in the presence of a nonhydrolyzable ATP analog, depolymerization also occurs. Structural
characterization of the analog-induced depolymerization products provided a snapshot of the disassembly
machine at the microtubule ends.
Our data indicate that whereas conventional kinesins
use the energy of ATP binding to execute a power stroke
that results in unidirectional motion along the microtubule surface, KinIs at the ends of microtubules use the
energy to bend the underlying protofilament, thereby
destabilizing the microtubule lattice and leading to
microtubule depolymerization. Furthermore, when the
motor core is associated with the microtubule wall, the
core is stalled in a weakly bound, nucleotide-free state.
Progression to the strongly bound, ATP-containing state
is possible only when the KinI encounters a microtubule
end, where it can catalyze deformation of protofilaments and disassembly of microtubules. The unusual
mechanochemical coupling of this kinesin provides an
elegant mechanistic basis for its microtubule-depolymerizing activity.
The protein doublecortin is expressed in migrating
and differentiating neurons. In humans, mutations in
this protein disrupt brain development, causing lissencephaly. Although doublecortin is associated with and
stabilizes the microtubule cytoskeleton, it has no homology with other microtubule-binding proteins such as
MAP2 or tau. We found that doublecortin preferentially
nucleates and binds to 13-protofilament microtubules.
This specificity was explained when we discovered that
the protein binds in the valleys between the protofilaments of the microtubule wall. This binding site is
Published by TSRI Press®. © Copyright 2005,
The Scripps Research Institute. All rights reserved.
45
unique and appears to be ideally located for microtubule stabilization. In this location, doublecortin most
likely contributes to both the longitudinal and the lateral interactions that stabilize the microtubule wall.
In collaboration with G. Chang, Department of
Molecular Biology, we have grown well-ordered arrays
of several membrane proteins that are involved in multidrug resistance. These arrays, helical tubes and 2-dimensional crystals of membrane-embedded proteins, are
suitable for structural studies via electron microscopy.
In one instance, we trapped a drug transporter in various
stages of its mechanistic cycle and with substrates bound.
We anticipate that 3-dimensional electron microscopy maps of membrane-embedded transporters in
various states, together with the high-resolution x-ray
structures of the detergent-solubilized protein, will provide insights into the mechanisms used to transport
metabolites and drugs across membranes.
In other studies, we developed a general method
for helical crystallization of proteins on lipid tubules that
we are using to study the virulence factor PFO from
Clostridium perfringens. PFO is a cytolysin, an important class of proteins that oligomerize and embed within
membranes as part of their lytic function. We obtained
helical crystals of wild-type and several mutant forms of
PFO on nickel-lipid tubules. Three-dimensional maps of
these proteins derived from images of the helical crystals
will be used to complement our studies of PFO pore
formation on lipid layers. These studies will provide a
better understanding of the pathogenic function of
cytolysins. Additional studies involving tubular crystallization of membrane proteins and other bacterial toxins
are opening up promising new areas for future research.
PUBLICATIONS
Dang, T.X., Farah, S.J., Gast, A., Robertson, C., Carragher, B., Egelman, E., Wilson-Kubalek, E.M. Helical crystallization on lipid nanotubes: streptavidin as a
model protein. J. Struct. Biol. 150:90, 2005.
Dang, T.X., Hotze, E.M., Rouiller, I., Tweten, R.K., Wilson-Kubalek, E.M. Prepore to
pore transition of a cholesterol-dependent cytolysin visualized by electron microscopy.
J. Struct. Biol. 150:100, 2005.
Neuman, B., Adair, B.D., Burns, J.W., Milligan, R.A., Buchmeier, M.J., Yeager, M.
Complementarity in the supramolecular design of arenaviruses and retroviruses
revealed by electron cryomicroscopy and image analysis. J. Virol. 79:3822, 2005.
O’Keefe, M.A., Turner, J.H., Musante, J.A., Hetherington, C.J.D., Cullis, A.G.,
Carragher, B., Junkins, R., Milgrim, J., Milligan, R.A., Potter, C.S., Allard, L.F.,
Blom, D.A., Degenhardt, L., Sides, W.H.. Laboratory design for high-performance
electron microscopy. Microsc. Today 12:8, 2004.
46 CELL BIOLOGY 2005
Molecular Mechanisms of
CNS Development and
Mechanosensory Perception
U. Müller, R. Banan, C. Barros, R. Belvindrah, F. Conti,
S. Hankel, E. Hintermann, P. Kazmierczak, R. Radakovits,
C. Ramos, A. Reynolds, A. Sczaniecka, M. Schwander,
M. Senften, S. Webb
disproportionately large number of genes in the
genomes of vertebrates encode cell recognition
molecules that mediate cell-cell interactions and
interactions between cells and the extracellular matrix.
This finding most likely reflects an evolutionary trend
toward increasingly more complex cellular interactions in
higher metazoans. The highest diversity of such interactions occurs in the CNS, where thousands of different
neuronal subtypes are connected into defined neuronal
circuits. We use mouse genetics, genomics, cell biology,
biochemistry, and imaging technology to analyze the function of cell recognition molecules during the development
of neuronal circuits in the CNS. In another project, we are
elucidating the mechanisms by which cell recognition
molecules contribute to mechanosensory perception.
A
F O R M AT I O N O F C O R T I C A L S T R U C T U R E S I N T H E C N S
The establishment of the 3-dimensional cytoarchitecture of the nervous system depends on interactions
of receptors on neuronal cells with molecules presented
within the extracellular matrix and by neighboring cells.
Integrins are a class of neuronal receptors that mediate
interactions with glycoproteins secreted by the extracellular matrix and with membrane-anchored counterreceptors.
Recently, we found that integrins cooperate with
secreted signaling molecules such as sonic hedgehog and
Reelin to regulate important steps during CNS development, such as cell proliferation and formation of neuronal
layers during the development of the cerebral and cerebellar cortex. We are identifying the downstream signaling pathways activated by integrins during cortical
development. We are also studying signaling interactions
between integrins and other receptors such as receptor
tyrosine kinases. Finally, we have extended our studies to
the analysis of integrin functions in the CNS in adults.
CELL RECOGNITION MOLECULES, MECHANOSENSORY
PERCEPTION, AND DEAFNESS
Mechanosensation, the transduction of mechanical
force into an electrochemical signal, allows living organisms to detect touch, hear, register movement and gravity,
Published by TSRI Press®. © Copyright 2005,
The Scripps Research Institute. All rights reserved.
and sense changes in cell volume and shape. In mammals, the hair cells of the inner ear are the principle
mechanosensors for the detection of sound and movement. Hair cells elaborate stereocilia that contain mechanosensitive ion channels. The stereocilia of a hair cell
are interconnected by extracellular bridges into a bundle
and are situated next to specialized extracellular matrix
assemblies. Sound waves or head movements lead to
deflection of the stereocilia bundle, changes in the ion
permeability of the mechanosensitive channels, and depolarization of the hair cells. The molecules that regulate
development and function of hair cells are poorly defined.
Because defects in hair cells cause inherited forms
of deafness, we use human and mouse genetics as a
guideline to identify and study molecules that regulate
the development and function of mechanosensory hair
cells. Currently, about 70 genes have been identified
in which mutations lead to deafness. Many of these
genes encode membrane-anchored cell adhesion molecules and molecules secreted by the extracellular matrix.
Mutations in the gene for the cell adhesion molecule
cadherin 23 cause deafness in mice and humans. Our
findings provide strong evidence that cadherin 23 is a
component of the so-called tip-link, which has been
predicted to transmit force onto mechanically gated ion
channels in the stereocilia of hair cells. We are analyzing the function of cadherin 23, proteins that interact with this cell adhesion molecule, and proteins
encoded by additional “deafness” genes for mechanotransduction. We are also doing genetic screens in
mice to identify novel recessive deafness traits. Using
this strategy, we have already identified several novel
genes that may be associated with deafness.
PUBLICATIONS
Barros, C., Müller, U. Cell adhesion in nervous system development. In: Integrins
in Development. Danen, E. (Ed.). Landes Bioscience, Georgetown, TX, in press.
Belvindrah, R., Müller, U. Integrin signaling and central nervous system development. In: Extracellular Matrix and Disease. Miner, J. (Ed.). Elsevier, St. Louis.
Advances in Developmental Biology and Biochemistry, Vol. 15, in press.
Escher, P., Lacazette, E., Courtet, M., Blindenbacher, A., Landmann, L., Bezakova, G., Lloyd, K., Müller, U., Brenner H.R. Synapse formation in skeletal muscle
lacking neuregulin receptors. Science, in press.
Li, N., Zhang, Y., Naylor, M.J., Schatzmann, F., Maurer, F., Wintermantel, T., Schuetz,
G., Müller, U., Streuli, C.H., Hynes, N.E. β1 Integrins regulate mammary gland proliferation and maintain the integrity of mammary alveoli. EMBO J. 24:1942, 2005.
Müller, U. Integrins and extracellular matrix in animal models. In: Cell Adhesion.
Behrens, J., Nelson, W.J. (Eds.). Springer Verlag, New York, 2004, p. 217. Handbook of Experimental Pharmacology, Vol. 165.
White, D.E., Kurpios, N.A., Zuo, D., Hassell, J.A., Blaess, S., Müller, U., Muller, W.J.
Targeted disruption of β1 integrins in a transgenic mouse model of human breast cancer reveals an essential role in mammary tumor induction. Cancer Cell 6:159, 2004.
CELL BIOLOGY 2005
Molecular Mechanisms of
Thermosensation
A. Patapoutian, A. Dhaka, T. Earley, S. Eid, S.W. Hwang,
L. Macpherson, T. Miyamoto, A. Murray, G. Story
e are interested in the molecular description
of the function of sensory neurons. Of the 5
popularly characterized senses—sight, hearing, taste, smell, and touch—touch is among the most
varied and least understood. Within this sense is the
ability to sense mechanical forces, chemical stimuli, and
temperature, and the molecules that mediate this ability have been a long-standing mystery. Temperature sensation in particular has received relatively little attention
from biologists and yet is critical for interactions with
the environment.
We recently discovered proteins that may enable sensory neurons to convey information about temperature.
These proteins are ion channels activated by specific
changes in temperature; thus they act as the molecular
thermometers of the body. Specifically, our results have
led to the identification and characterization of a novel
warm-activated transient-receptor-potential (TRP) channel, TRPV3 (33°C threshold) and 2 novel cold-activated
TRP channels, TRPM8 (25°C threshold) and TRPA1
(ANKTM1, 17°C threshold). We found that TRPM8 is
also the receptor for the compound menthol, providing
a molecular explanation of why mint flavors are typically perceived as cooling. Furthermore, we discovered
that TRPA1 is activated by cinnamaldehyde and other
compounds with a burning sensory quality, consistent
with a role of TRPA1 in the detection of noxious cold
sensations. Together these temperature-activated channels represent a new subfamily of TRP channels that
we have dubbed thermoTRPs.
In agreement with a role in initiating temperature
sensation, most of the thermoTRPs are normally found
in subsets of neurons in dorsal root ganglia. A surprisingly
distinct expression pattern was observed for TRPV3, the
warm receptor. High levels of TRPV3 are observed solely
in skin keratinocytes in mice, suggesting that skin cells
might be able to “sense” temperature and then communicate this information to dorsal root ganglia neurons. How temperature information is coded from the
skin to the spinal cord is not well understood, and we
are using a variety of approaches to answer this question. For example, recent data from mice lacking the
gene for TRPV3 suggest that TRPV3 is indeed required
W
Published by TSRI Press®. © Copyright 2005,
The Scripps Research Institute. All rights reserved.
47
for proper heat sensation in vivo, reinforcing a role of
skin in thermosensation.
All organisms have a need for thermosensation.
Because some invertebrate species are more amenable
to genetic studies than mammals are, we asked whether
nonvertebrates also use thermoTRPs to sense temperature. We recently showed that the Drosophila ortholog
of TRPA1 is an ion channel activated by warm temperatures, suggesting an evolutionary conserved role of TRP
channels in temperature sensing. In collaborative efforts
with P. Garrity, Massachusetts Institute of Technology,
Cambridge, Massachusetts, and W. Shafer, University
of California, San Diego, we are using genetic studies
to examine the role of TRPA family members in invertebrate species.
Another key question is what makes thermoTRPs
temperature sensitive whereas other TRPs are not?
Answering this question requires insight into the fundamental biophysical mechanism of how temperature
activates ion channels. Our ongoing structure-function
experiments, including mutagenesis and chimeric protein analysis of the thermoTRPs, will provide us with
important clues about how cold or heat activates these
ion channels.
Our long-term goal is to synthesize an integrated
picture of sensory neuron function. By identifying the
proteins that most likely initiate the molecular cascade
leading to temperature perception, we have provided
the basis for probing the foundation of the sense of
temperature. We now have the opportunity to extend
these insights into important areas of human health,
such as pain pathophysiology. For example, TRPA1 is a
potential target for treating pain, and we are identifying
small-molecule inhibitors of TRPA1 in collaboration
with scientists at the Genomics Institute of the Novartis
Research Foundation, San Diego, California. Therefore,
the approaches we are using will yield insights into the
basic biology of the peripheral nervous system and may
also have an effect on novel treatments for pain.
PUBLICATIONS
Moqrich, A., Earley, T., Watson, J., Andahazy, M., Backus, C., Martin-Zanca, D., Wright,
D.E., Reichardt, L.F., Patapoutian, A. Expressing TrkC from the TrkA locus causes a subset of dorsal root ganglia neurons to switch fate. Nat. Neurosci. 7:812, 2004.
Moqrich, A., Hwang, S.W., Earley, T.J., Petrus, M.J., Murray, A.N., Spencer, K.S.R.,
Andahazy, M., Story, G., Patapoutian, A. Impaired thermosensation in mice lacking
TRPV3, a heat and camphor sensor in skin. Science 307:1468, 2005.
Patapoutian A., Wood, J.N. Mechanisms of nociception: molecules to behaviour. J.
Neurobiol. 61:1, 2004.
Rosenzweig, M., Brennan, K.M., Tayler, T.D., Phelps, P.O., Patapoutian, A., Garrity, P.A. The Drosophila ortholog of vertebrate TRPA1 regulates thermotaxis. Genes
Dev. 19:419, 2005.
48 CELL BIOLOGY 2005
Functional Proteins in Tumor
Metastasis and Angiogenesis
A. Zijlstra, J.P. Partridge, T. Kupriyanova, M. Madsen,
T. Papagiannakopoulas, M.C. Subauste, E.I. Deryugina,
J.P. Quigley
e have established a number of in vivo model
systems that can recapitulate the major cellular and tissue events that occur during tumor
metastasis and angiogenesis. The model systems allow
quantitative measurements, microscopic analysis in
real time, biochemical and immunologic probing, and
direct molecular and therapeutic interventions. Recently,
use of short interfering RNA molecules directed against
specific expressed genes provided insights into the contributory role of the gene products in tumor dissemination
and neovascularization. In addition, use of subtractive
immunization, which is used to generate unique neutralizing monoclonal antibodies, in combination with
immunoproteomics enables us to identify specific antigenic molecules that are functionally active in metastasis and angiogenesis.
W
M E TA S TA S I S
Selected human tumor cells inoculated onto the
chorioallantoic membrane of developing chick embryos
form primary tumors on the membrane in 4–7 days. A
small percentage of the cells in the primary tumor disseminate through the vasculature and within 3–4 days,
arrest and proliferate in secondary organs of the embryo.
Measuring a small number of early-arriving metastatic
cells (<200) growing and expanding in the secondary
organ has always been technically difficult. We now
use an approach in which unique regions of human DNA,
known as Alu repeat sequences, are amplified by polymerase chain reaction from the total DNA extracted
from various organs of the tumor-bearing chick embryo.
Chicken DNA contains no Alu sequences, so any product generated by the polymerase chain reaction indicates that human tumor cells are present in the chick
embryo organ and would have arrived there via the
known sequential steps in metastasis. We can now
detect as few as 25–50 human tumor cells present in
the entire chick embryo lung, liver, or brain and can
measure the expansion of these metastatic cells by
using the real-time polymerase chain reaction.
We are using various screening procedures in this
model system to identify molecules that enhance, or
conversely inhibit, the appearance of metastatic human
Published by TSRI Press®. © Copyright 2005,
The Scripps Research Institute. All rights reserved.
tumor cells in organs of chick embryos. The screening
procedures include direct inoculation of primary tumor
cells that have been transfected with various short interfering RNA constructs to delete specific genes that might
contribute to metastatic dissemination. Inoculating monoclonal antibodies directly into the tumor-bearing embryos
and monitoring the influence of the antibodies on metastasis are also part of our screening procedures.
We are also using a more conventional method of
monitoring human tumor metastasis in specific immunodeficient mice. However, compared with our chick
embryo metastasis assay, this method is less quantitative, requires more time (3–5 weeks), and is more difficult to use for inhibitor screening and molecular
intervention. We are using the mouse metastasis assay to
take advantage of mouse genetics and to confirm the efficacy of various effector molecules and inhibitors that initially are identified in the chick embryo metastasis assay.
ANGIOGENESIS
One of the most commonly used in vivo assays for
angiogenesis is the chick embryo chorioallantoic membrane assay. We developed a quantitative variation of
this assay that allows detection and measurement of the
newly sprouting blood vessels responding to an angiogenic stimulus such as a specific growth factor or a
growing tumor (Fig. 1). A highly specific metalloproteinase, MMP-13, has been implicated in the tissue
remodeling that occurs during the formation of the new
blood vessels. We characterized this proteolytic event
and found that collagen-cleaving metalloproteinases are
implicated directly in the outgrowth of new vessels.
We also discovered that another metalloproteinase,
MMP-9 (gelatinase B), most likely is involved in angiogenic tissue remodeling. The proteolytic activity of this
enzyme, which is quite distinct from that of MMP-13,
also appears to be necessary for a full angiogenic
response. Interestingly, these 2 critical enzymes are
actively imported into the vascular/stromal tissue by
distinct inflammatory cells responding to the angiogenic
stimulation. Neutrophil-like heterophils rapidly and
almost immediately import MMP-9 into the tissue,
whereas monocyte/macrophages actively deliver MMP-13
1–2 days later, possibly in response to specific secreted
products of the early arriving heterophils. Thus, normal angiogenesis and tumor angiogenesis are closely
linked to an accompanying host inflammatory response
that contributes critical functional molecules to the
angiogenic process.
We are dissecting out and identifying the molecules
and cells that link the inflammatory response to the
CELL BIOLOGY 2005
49
PUBLICATIONS
Blancafort, P., Chen, E.I., Gonzalez, B., Bergquist, S., Zijlstra, A., Guthy, D.,
Brachat, A., Brakenhoff, R.H., Quigley, J.P., Erdmann, D., Barbas, C.F. III.
Genetic reprogramming of tumor cells by zinc finger transcription factors. Proc.
Natl. Acad. Sci. U. S. A. 102:11716, 2005.
Deryugina, E.I., Zijlstra, A., Partridge, J.J., Kupriyanova, T.A., Madsen, M., Papagiannakopoulos, T., Quigley, J.P. Unexpected effect of matrix metalloproteinase
downregulation on vascular intravasation and metastasis of human fibrosarcoma cells
selected in vivo for high rates of dissemination. Cancer Res., in press.
Wilson, S., Greer, B., Hooper, J., Zijlstra, A., Walker, B., Quigley, J.P., Hawthorne S.
The membrane-anchored serine protease, TMPRSS2, activates PAR-2 in prostate
cancer cells. Biochem. J. 388(Pt. 3):967, 2005.
Zijlstra, A., Seandel, M., Kupriyanova, T.A., Partridge, J.J., Madsen, M., HahnDantona, E.A., Quigley, J.P., Deryugina, E.I. Pro-angiogenic role of neutrophil-like
inflammatory heterophils during neovascularization induced by growth factors and
human tumor cells Blood, in press.
Regulators of Clathrin-Mediated
Endocytosis
S.L. Schmid J. Chappie, S.D. Conner, M. Ishido,
M. Leonard, R. Ramachandran, F. Soulet, B.D. Song,
M.C. Surka, D. Yarar
lathrin-mediated endocytosis is essential for the
efficient uptake of nutrients and other macromolecules into cells and for the regulation of
signaling by cell-surface receptors. The process occurs
at clathrin-coated pits, which concentrate receptor-ligand
complexes, deform the membrane, invaginate, and
eventually pinch off, forming clathrin-coated vesicles
(CCVs). The major components involved in the formation
of CCVs are clathrin, adaptor proteins, and dynamin.
Clathrin self-assembles into a polygonal lattice and
serves as a scaffold for the formation of coated pits.
Adaptor protein-2 (AP2) is a heterotetrameric protein
that triggers clathrin assembly at the plasma membrane
and interacts directly with the cytoplasmic tails of surface receptors to concentrate the receptors into the
assembling coated pit. Dynamin is a multidomain
GTPase that regulates endocytosis. Each of these proteins also interacts with a myriad of accessory proteins,
whose function in the formation of CCVs is poorly understood. In the past year, we focused on factors that
regulate clathrin-mediated endocytosis.
Dynamin has an unusually low affinity for GTP and
a relatively high basal rate of GTP hydrolysis. Moreover, dynamin self-assembles into rings and helical
stacks of rings that are localized to the necks of deeply
invaginated coated pits. Dynamin self-assembly stimulates its GTPase activity approximately 100-fold; this
stimulation is mediated by dynamin’s GTPase effector
C
F i g . 1 . In vivo model to quantitate angiogenesis induced by
growth factors and tumor cells. Collagen onplants are placed on
the chorioallantoic membrane (CAM) of day 10 chick embryos developing ex ovo (white arrows, left panel, top row). In 3 days, new
vessels sprouting from preexisting vessels in the membrane grow up
through the collagen into the gridded nylon mesh (right panel, top
row) and with a dissecting microscope are clearly visible within
the grids (black arrowheads, middle row) and easily distinguished
from the background preexisting vessels (open circles, middle row).
The number of new vessels in the grids can be easily scored. Differential angiogenesis can be quantitated as indicated in the bar graph.
Aggressive human tumor cells, such as HT1080 cells, placed into
the onplants induce elevated levels of angiogenesis similar to those
induced by purified growth factors (VEGF/FGF) and blocked by specific metalloproteinase inhibitors (MMP inhibitor and TIMP-2). Nonaggressive human tumor cells, such as HeLa cells, do not induce
significant angiogenesis. If onplants containing HT1080 cells are
allowed to incubate on the embryo for 6 days, large vascularized
primary tumors develop (right panel, bottom row), resulting in dissemination of metastatic cells into secondary organs of the embryo.
angiogenic process and to the progression of malignant
neoplasms. We are also trying to decipher whether the
relevant functional molecules are derived from host cells
or from tumor cells.
Published by TSRI Press®. © Copyright 2005,
The Scripps Research Institute. All rights reserved.
50 CELL BIOLOGY 2005
domain. Although dynamin clearly is essential for
clathrin-mediated endocytosis, the role of GTP binding
and/or hydrolysis in endocytosis remains unclear.
Insight into the function of dynamin in endocytosis was derived from collaborative studies with
M. Ramaswami, University of Arizona, Tucson, Arizona.
Point mutations in shibire, the gene for dynamin in
Drosophila, cause temperature-sensitive defects in
endocytosis. We showed that the ts2 mutation, which
occurs in the switch 2 region of the dynamin GTPase
domain, compromises GTP-binding affinity. Three second-site suppressor mutations, 1 in the switch 1 region
of the GTPase domain and 2 in the GTPase effector
domain, fully rescued the ts2 defects in synaptic vesicle recycling. The functional rescue in vivo correlated
with a reduction in both the basal and assembly-stimulated GTPase activity in vitro. These findings indicated
that the GTPase effector domain is indeed an internal
dynamin GTPase-activating protein and establish that,
as for other GTPases, the function of dynamin in vivo
is negatively regulated by its activity as a GTPase-activating protein.
On the basis of these findings, we proposed a 2-step
model (Fig. 1) for dynamin during the formation of vesicles. In this model, an early regulatory GTPase-like
function precedes late, assembly-dependent steps during which GTP hydrolysis is required for vesicle release.
A 2-step model for dynamin function in clathrin-mediated
endocytosis. In stage 1, dynamin (ovals) plays a regulatory role
and functions as a kinetic timer controlling the recruitment and/or
activity of SH3 domain–containing molecules. In stage 2, dynamin
assembly and assembly-stimulated GTPase activity are required as
Fig. 1.
a fission apparatus, a curvature sensor, and/or a structural scaffold.
In this model, dynamin self-assembly marks the transition between early and late functions of dynamin. What
might control this transition? We identified a new protein
partner of dynamin, sorting nexin 9 (SNX9). SNX9 binds
directly to dynamin and potentiates dynamin assembly
and assembly-stimulated GTPase activity on liposomes
and in solution. Using total internal reflection fluorescence microscopy in living cells, we detected a transient
burst of recruitment of SNX9 labeled with green fluorescent protein to clathrin-coated pits that occurs during the
Published by TSRI Press®. © Copyright 2005,
The Scripps Research Institute. All rights reserved.
late stages of vesicle formation and coincides spatially
and temporally with a burst of fluorescence of dynamin
labeled with red fluorescent protein. These data suggest
that dynamin effectors, like SNX9, might coordinate
dynamin self-assembly with coat assembly, cargo
recruitment, or membrane invagination.
Clathrin-mediated endocytosis is also regulated by
phosphorylation events, but the kinases responsible,
some of which cofractionate with coat proteins, have
not been identified. Through mass spectroscopic analysis of preparations of adaptor proteins, we identified a
novel serine/threonine kinase, the coated vesicle–associated kinase of 104 kD (CVAK104), that belongs to
the SCY1-like family of protein kinases, previously
thought to be catalytically inactive. CVAK104 cofractionates with adaptor proteins extracted from CCVs and
directly binds to both clathrin and the plasma membrane
adaptor AP2. CVAK104 binds ATP, and kinase assays
indicate that it functions as a poly-L-lysine–stimulated
kinase that can autophosphorylate and phosphorylate
the β 2 -adaptin subunit of AP2. Further studies are
needed to determine the functional consequences of
this phosphorylation reaction.
Actin assembly is spatially and temporally coordinated with endocytosis, and numerous endocytic
accessory proteins directly or indirectly regulate actin
dynamics. However, functional evidence for a role of
actin during the formation of CCVs has been lacking.
In collaboration with C. Waterman-Storer, Department
of Cell Biology, using parallel biochemical and microscopic approaches, we reexamined the role of cortical
actin dynamics of clathrin-mediated endocytosis.
Using total internal reflection fluorescence microscopy, we found that disruption of the F-actin assembly
and disassembly cycle with latrunculin A or jasplakinolide resulted in nearly complete cessation of all aspects
of the dynamics of clathrin-coated structures labeled
with red fluorescent protein. Stage-specific biochemical assays and quantitative fluorescence and electron
microscopic analyses established that F-actin dynamics are required for multiple distinct stages of the formation of CCVs, including the formation, constriction,
and internalization of coated pits. In future studies,
we will address the molecular mechanisms that link
actin dynamics to clathrin-mediated endocytosis.
PUBLICATIONS
Conner, S.D., Schmid, S.L. CVAK104 is a novel poly-L-lysine-stimulated kinase
that targets the β2 subunit of AP2. J. Biol. Chem. 280:21539, 2005.
Leonard, M., Song, B.D., Ramachandran, R., Schmid, S.L. Robust colorimetric assays
for dynamin’s basal and stimulated GTPase activities. Methods Enzymol., in press.
CELL BIOLOGY 2005
Miwako, I., Schmid, S.L. Coated vesicle formation from isolated plasma membranes. Methods Enzymol., in press.
Narayanan, R., Leonard, M., Song, B.D., Schmid, S.L. Ramiswami, M. An internal GAP domain negatively regulates presynaptic dynamin in vivo: a two-step
model for dynamin function. J. Cell Biol. 169:117, 2005.
Song, B.D., Leonard, M., Schmid, S.L. Dynamin GTPase domain mutants that differentially affect GTP binding, GTP hydrolysis, and clathrin-mediated endocytosis.
J. Biol. Chem. 279:40431, 2004.
Soulet, F., Yarar, D., Leonard, M., Schmid, S.L. SNX9 regulates dynamin assembly and
is required for efficient clathrin-mediated endocytosis. Mol. Biol. Cell 16:2058, 2005.
Yarar, D., Waterman-Storer, C., Schmid, S.L. A dynamic actin cytoskeleton functions
at multiple stages of clathrin-mediated endocytosis. Mol. Biol. Cell 16:964, 2005.
Molecular Biology of Olfaction
L. Stowers, K. Flanagan, M. Gubernator, J. Lin, T. Marton,
C. Ramos, J. Riceberg
very breath samples the environment for olfactory chemical information, determining the quality of food, warning of danger, and confirming
safety. The sense of olfaction is composed of 2 types
of neurons: those that mediate an evocative perception
that varies with each individual’s experience and those
that regulate stereotyped innate social behaviors such
as aggression and mating. Neurons that elicit odorant
perception reside in the olfactory epithelium and relay
chemical information through activation of cAMP-responsive channels. Recently, we showed that behavior-generating neurons are located in the vomeronasal organ
and respond to pheromones through a cascade that
ultimately activates C-type transient-receptor-potential
2 (TrpC2) channels. We are using a molecular genetic
approach to characterize the function of these pheromoneresponsive neurons.
Through electrophysiologic recordings, we showed
that neurons in mutant mice lacking TrpC2 do not depolarize in response to natural sources of pheromones.
Behavioral assays with these animals revealed that this
pheromone response is necessary for both intermale
aggression and gender recognition. We are identifying
other unique molecular subpopulations of pheromoneresponsive neurons, and through genetic ablation, biochemistry, and electrophysiology, we are assigning a
biological function to each neuron type.
A full characterization of the repertoire of chemosensory neurons will be essential in understanding the
logic of olfactory information coding. To this end, we
are investigating a novel class of olfactory neurons that
lack both the cAMP and TrpC2 signaling components.
E
Published by TSRI Press®. © Copyright 2005,
The Scripps Research Institute. All rights reserved.
51
Analysis of these neurons by transcriptional profiling
and then molecular genetics and biochemistry is being
used to identify their role in olfactory function.
Elucidation of the function of specific neural circuits
that regulate mammalian behavior has been hindered
because the pheromone compounds that signal each
behavior have not been identified from the complex natural sources of the compounds. To obtain these important molecules, we are establishing a high-throughput
screening assay of chemical compounds that we expect
will provide identification of both agonists and antagonists specific to each of the 400 pheromone receptors.
These data will enable us both to activate specific neural
circuits and analyze the natural production and regulation of the signaling ligands. In total, we expect to
define the pheromone response pathway of mice and
to reveal general principles of neurons that govern
complex social behavior.
PUBLICATIONS
Stowers, L., Marton, T.F. What is a pheromone? Mammalian pheromones reconsidered. Neuron 46:699, 2005.
Molecular Regulation of Vascular
System Development in Mammals
M.J. Fitch, Z. Zou, S. Chitnis, J.D. Lewis, A. Durrans,
M. Schroeter,* L. Campagnolo,** W. LeVine, H. Stuhlmann
* University of Göttingen, Göttingen, Germany
** Università degli Studi di Roma Tor Vergata, Rome, Italy
he cardiovascular system is the first organ system to develop in mammalian embryos. Establishment of a functional circulatory system is crucial
for delivery of nutrients and oxygen to the embryos,
and defects result in death before birth or in congenital cardiovascular abnormalities. Our major goal is to
dissect the molecular pathways that regulate the 2
principal processes of vascular development: vasculogenesis and angiogenesis. We focus on the mouse
model because of the ready availability of genetic information on mice and experimental tools and because of
similarities between mice and humans. Previously, we
used a “gene trap” screen in mouse embryonic stem
cells and embryos to detect novel genes involved in
these processes.
T
A ZINC FINGER GENE ESSENTIAL FOR NORMAL
VA S C U L A R A N D LY M P H AT I C D E V E L O P M E N T
One endothelial-expressed gene identified in the
screen, Vezf1, encodes a 56-kD nuclear transcription
52 CELL BIOLOGY 2005
factor with 6 putative zinc finger domains. The gene
and its family members are highly conserved in vertebrate species. Using transgenic mice and mice deficient
in Vezf1, we determined that this gene plays an essential and dosage-dependent role in the proliferation,
remodeling, and integrity of the developing vasculature.
Homozygous and a fraction of heterozygous mutant
embryos had vascular and lymphatic endothelial abnormalities and died during midgestation. Electron and
confocal microscopy indicated that the primary phenotype involves defects in endothelial cell junctions and
basal membrane. These findings are supported by recent
studies in the embryoid body in vitro differentiation
model and in teratocarcinomas derived from mutant
embryonic stem cells. Of interest, the lethal phenotype in mice lacking Vezf1 can be at least partially
rescued by endothelial overexpression of the gene.
How does Vezf1 control vascular development?
We are beginning to explore the molecular pathways
of Vezf1 function. We have identified the regulatory
regions in the Vezf1 promoter in vitro and in vivo. To
identify target genes of Vezf1, we are using subtractive hybridization and cDNA/expressed sequence tag
microarray analysis. In addition, we are analyzing the
properties of primary cells from Vezf1 mutant embryos.
A N E A R LY M A R K E R F O R E N D O T H E L I A L C E L L S A N D
thelial progenitor cells in mouse embryos and during
differentiation of embryonic stem cells and to study
the potential of the cells to contribute to or regenerate
vascular tissues in embryos and adults.
D E V E L O P M E N T O F M U LT I VA L E N T V I R A L
N A N O PA R T I C L E S F O R I N V I V O VA S C U L A R
TA R G E T I N G A N D I M A G I N G
We are developing, in collaboration with M. Manchester, Department of Cell Biology, viral nanoparticles
for noninvasive imaging and targeting of the cardiovascular system in mammals. For these studies, fluorescent
dyes and peptides are chemically attached to lysine residues of the capsid of cowpea mosaic virus (CPMV). In
initial studies, we showed that fluorescent CPMV produces a bright and stable signal that allows visualization
of blood flow in developing mouse and chick embryos
and in tumor angiogenesis models. We are extending
these studies to target CPMV with multivalent attached
peptides to the vasculature both during development
and in disease models such as tumor angiogenesis.
PUBLICATIONS
Campagnolo, L., Leahy, A., Chitnis, S., Koschnick, S., Fitch, M.J., Fallon, J.T.,
Loskutoff, D., Taubman, M.B., Stuhlmann, H. EGFL7 is a chemoattractant for
endothelial cells and is up-regulated in angiogenesis and arterial injury. Am. J.
Pathol. 167:275, 2005.
Feral, C.C., Nishiya, N., Fenczik, C.A., Stuhlmann, H., Slepak, M., Ginsberg, M.H.
CD98hc (SLC3A2) mediates integrin signaling. Proc. Natl. Acad. Sci. U. S. A.
102:355, 2005.
THEIR PROGENITORS
A second endothelial gene identified in our screen,
Egfl7, encodes a 30-kD secreted protein with 2 internal epidermal growth factor–like domains. Expression
of Egfl7 is restricted to the vascular endothelium and
its progenitors in the yolk sac mesoderm. Expression
is downregulated in most of the quiescent vasculature
in adults but is transiently upregulated during vascular
injury and endothelial regeneration and during physiologic angiogenesis in the uterus during pregnancy. Clues
about a possible role of Egfl7 come from our studies in
which we found that EGFL7, the protein encoded by the
gene, acts as a chemoattractant for endothelial cells.
What is the function of Egfl7 during development
and adult angiogenesis? We are using gain- and lossof-function approaches in the mouse model to test our
hypothesis that Egfl7 plays a crucial role during vascular development in embryos and in angiogenesis in
adults. We are also determining if EGFL7 acts as a
growth factor or a cytokine through direct binding to
receptors or through interaction with proteins in the
extracellular matrix. Finally, we are using Egfl7 as a
molecular marker to isolate early populations of endoPublished by TSRI Press®. © Copyright 2005,
The Scripps Research Institute. All rights reserved.
Kuhnert, F., Campagnolo, L., Xiong, J.-W., Lemons, D., Fitch, M.J., Zou, Z.,
Kiosses, W.B., Gardner, H., Stuhlmann, H. Dosage-dependent requirement for
mouse Vezf1 in vascular system develpment. Dev. Biol. 283:140, 2005.
Kuhnert, F., Stuhlmann, H. Identifying early vascular genes through gene trapping
in mouse embryonic stem cells. Curr. Top. Dev. Biol. 62:261, 2004.
Ion Channels and
Fast Synaptic Transmission
N. Unwin
on channels play a central role in the rapid transmission of electrical signals throughout the nervous system. To determine how these membrane proteins
work, my colleagues and I are using electron microscopy to analyze their structures trapped in different
physiologic states. Current studies center on the nicotinic acetylcholine receptor at the nerve-muscle synapse.
We wish to find out how this ion channel achieves its
ion selectivity and high transport rate and how it opens
and desensitizes in response to acetylcholine released
into the synaptic cleft. For our studies, we use post-
I
CELL BIOLOGY 2005
synaptic membranes isolated from the (muscle-derived)
electric organ of a Torpedo ray, which form tubular
crystals of acetylcholine receptors.
The acetylcholine receptor is a member of a superfamily of transmitter-gated ion channels, which includes
the serotonin 5-HT3, γ-aminobutyric acids A and C,
and glycine receptors. It has a cation-selective pore,
delineated by a ring of 5 similar subunits, that opens
upon binding of acetylcholine to the 2 ligand-binding
(α) subunits at the subunit interfaces.
Recently, we obtained a refined atomic model of
the acetylcholine receptor in the closed-channel form.
We found that the individual subunits in the N-terminal
ligand-binding domain are organized around 2 sets of
β-sheets packed in a curled β-sandwich, as in the
related soluble pentameric acetylcholine-binding protein. Each of the subunits in the membrane- spanning
domain is made from 4 α-helical segments. The helical
segments arrange symmetrically, forming an inner ring
of helices that shape a water-filled pore and an outer
shell of helices that coil around each other and shield
the inner ring from the lipids. In the closed channel, the
helices in the inner ring come together near the middle of the membrane and make a constricting hydrophobic girdle. This girdle, which is about 50 Å from the
acetylcholine-binding sites, constitutes an energetic
barrier to ion permeation and functions as the gate of
the channel.
These details, together with those obtained earlier
from studies of the receptor trapped in the open-channel form, have enabled us to understand in outline the
allosteric mechanism by which acetylcholine opens the
pore. In the absence of acetylcholine, the pore is normally closed. When acetylcholine enters the binding
sites, localized rearrangements in the α-subunits occur
that stabilize an alternative extended conformation of
the channel in which the inner sets of β-sheets are
rotated by about 10° about axes perpendicular to the
membrane plane, relative to their orientations in the
closed channel. These rotations are communicated
through the inner membrane-spanning helices and open
the pore by breaking the hydrophobic girdle apart.
Improvements in resolution of the 3-dimensional
structure, in both the closed- and the open-channel
forms, are now being attempted so that the structural
mechanism of gating of the channel can be described
in greater detail. The knowledge gained from the refined
structure of the locations of amino acid residues, in
relation to the ion pathway, is also being used to develop
Published by TSRI Press®. © Copyright 2005,
The Scripps Research Institute. All rights reserved.
53
quantitative explanations of how the high cation selectivity and high conduction rates of this channel are
achieved. These studies are yielding crucial insight
into the nature of a number of neuromuscular disorders, including several well-characterized congenital
myasthenic syndromes. They are also providing important 3-dimensional information about the binding sites
for drugs that affect the brain by modulating the function
of the related γ-aminobutyric acid, serotonin, glycine,
and neuronal acetylcholine receptors.
PUBLICATIONS
Unwin, N. Refined structure of the nicotinic acetylcholine receptor at 4Å resolution.
J. Mol. Biol. 346:967, 2005.
Microscopes and Motility:
Systems Integration in
Cell Migration
C.M. Waterman-Storer, T. Wittmann, N. Prigozhina,
O. Rodriguez, S.L. Gupton, K. Kita, R. Littlefield, K. Hu,
A. Wheeler, W. Shin, M.L. Gardel
ell migration is critical to development, the
immune response, and wound healing. In cancer cells, loss of regulation of cell motility results
in deadly metastasis. The locomotion of vertebrate tissue cells is thought to require complex and dynamic
interactions between the microtubules and actin cytoskeletal polymers, the endomembrane trafficking system, and focal adhesions to the substrate. We develop
quantitative light microscopy methods to analyze the
dynamic interactions between these complex macromolecular systems in living cells to understand how
the systems are spatiotemporally coordinated to drive
directed cell movement. We then use these microscopic
assays to analyze cells with specific perturbations of
cytoskeletal, membrane, or adhesive proteins to dissect
the molecular mechanisms of the regulation of the
proteins and their contribution to cell morphogenesis
and migration.
We pioneered fluorescent speckle microscopy, a
powerful method that allows quantitative analysis of
the dynamics of macromolecular assemblies in living
cells. In the past year, we enhanced the sensitivity of
this technique by extending the technology to multispectral total internal fluorescence reflection fluorescence microscopy, allowing the first-ever analysis of
C
54 CELL BIOLOGY 2005
the integration of proteins within focal adhesion complexes with the actin cytoskeleton during cell migration.
The microtubule and actin cytoskeletons may interact in cells via coregulation by the same signaling
cascades to promote a coordinated effort that drives
polarized cell migration. In support of this notion, we
showed that a signaling cascade downstream of Rac1
GTPase coordinates the regulation of both actin and
microtubules at the protruding edge of migrating cells.
We found that Rac1 interacts directly with and activates
p21-activated kinase 1, whose activity is required for
the assembly of both microtubules and actin filaments
in cell protrusion. Microtubule growth is promoted when
the kinase directly phosphorylates and inactivates the
microtubule-destabilizing oncoprotein Op18/stathmin.
We also identified a second Rac1 effector, CLASP, that
regulates polarized microtubule behavior in migrating
cells. CLASP interaction with microtubules specifically
in the leading edge of migrating cells is promoted by
Rac1 activity via glycogen synthase kinase 3, the wellknown regulator of cell polarity.
It has been thought for some time that localized,
microtubule-dependent delivery of endomembrane components to the leading-edge plasma membrane is
required for continued cell motility. We used PKD, a
mutant kinase of the trans-Golgi network, to specifically
inhibit the transport of membrane from the trans-Golgi
network to the plasma membrane in cells and monitored
cell motile functions by using time-lapse microscopy. The
results indicated that PKD-mediated transport across
the trans-Golgi network to the plasma membrane along
microtubules is required for fibroblast locomotion and
localized Rac1-dependent leading-edge activity.
The actin cytoskeleton is locally regulated for functional specializations for cell motility. In collaboration
with G. Danuser, Department of Cell Biology, using
computational analysis of quantitative fluorescent
speckle microscopy of the actin cytoskeleton, we defined
2 spatially colocalized but functionally distinct actinbased machines in migrating epithelial cells. The lamellipodium consists of a treadmilling F-actin array with
rapid retrograde flow in which Arp2/3, a complex containing the actin-related proteins 2 and 3, and the actin
depolymerization factor cofilin concentrate, whereas the
lamella has spatially random punctae of F-actin assembly and disassembly with slow retrograde flow and contains myosin II and tropomyosin.
We sought to specifically inhibit the formation of
the lamellipodium to test its requirement in cell migraPublished by TSRI Press®. © Copyright 2005,
The Scripps Research Institute. All rights reserved.
tion. We increased cellular tropomyosin levels in epithelial cells, and using quantitative fluorescent speckle
microscopy, electron microscopy, and immunolocalization, showed that this increase blocked formation of
functional lamellipodia. Cells lacking a lamellipodium
had more persistent leading-edge protrusion and rapid
cell migration than did cells with a lamellipodium. Inhibition of endogenous tropomyosin reduced the persistence of lamellipodial protrusion. Thus, in stark contrast
to the dogma in cell migration, cells can migrate in the
absence of a lamellipodium and tropomyosin is a major
regulator of functional specialization of the F-actin
cytoskeleton in migrating cells.
PUBLICATIONS
Adams, M.C., Matov, A., Yarar, D., Gupton, S.L., Danuser, G., Waterman-Storer,
C.M. Signal analysis of total internal reflection fluorescent speckle microscopy (TIRFSM) and wide-field epi-fluorescence FSM of the actin cytoskeleton and focal adhesions in living cells. J. Microsc. 216(Pt. 2):138, 2004.
Gupton, S.L., Anderson, K.L., Kole, T.P., Fischer, R.S., Ponti, A., HitchcockDeGregori, S.E., Danuser, G., Fowler, V.M., Wirtz, D. Hanein, D., WatermanStorer, C.M. Cell migration without a lamellipodium: translation of actin dynamics
into cell movement mediated by tropomyosin. J. Cell Biol. 168:619, 2005.
Gupton, S.L., Waterman-Storer, C.M. Live-cell fluorescent speckle microscopy
(FSM) of actin cytoskeletal dynamics and their perturbation by drug perfusion. In:
Cell Biology: A Laboratory Handbook, 3rd ed. Celis, J., Small, J.V. (Eds.). Elsevier,
St. Louis, in press.
Ponti, A., Machachek, M., Gupton, S.L., Waterman-Storer, C.M., Danuser, G. Two distinct actin networks drive the protrusion of migrating cells. Science 305:1782, 2004.
Ponti, A., Matov, A., Waterman-Storer, C.M., Danuser, G. Computational fluorescent speckle microscopy, II: high-resolution comapping of F-actin flow and turnover
in migrating cells. Biophys. J., in press.
Torreano, P.A., Waterman-Storer, C.M., Cohan, C.S. The effects of collapsing factors on F-actin content and microtubule distribution of Helisoma growth cones. Cell
Motil. Cytoskeleton 60:166, 2005.
Wittmann, T., Littlefield, R., Waterman-Storer, C.M. Fluorescent speckle microscopy of cytoskeletal dynamics living cells. In: Live Cell Imaging: A Laboratory Manual. Spector, D.L., Goldman, R.D. (Eds.). Cold Spring Harbor Press, Cold Spring
Harbor, NY, 2004, p. 187.
Wittmann, T., Waterman-Storer, C.M. Spatial regulation of CLASP affinity for
microtubules by Rac1 and GSK3β in migrating epithelial cells. J. Cell Biol.
169:929, 2005.
Yarar, D., Waterman-Storer, C.M., Schmid, S.L. A dynamic actin cytoskeleton functions
at multiple stages of clathrin-mediated endocytosis. Mol. Biol. Cell 16:964, 2005.
Systems Biology of Malaria
E. Winzeler, C. Kidgell, J. Young, J. Johnson
s the causative agent of human malaria, parasites
of the genus Plasmodium are major contributors
to global morbidity and mortality. Approximately
300 million to 500 million cases of malaria occur each
year. Individuals who are repeatedly exposed to the par-
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CELL BIOLOGY 2005
asite become immune but not completely. The immunity is incomplete because either the parasites alter
the complement of antigens on the surface of the red
blood cells or on the surface of the merozoites during
the merozoites’ brief extracellular phase in the blood
or because a great deal of genetic diversity exists in
the parasite population.
Because no licensed malaria vaccine is available,
drugs are the best therapy for the disease. However,
resistance to inexpensive drugs such as chloroquine
has emerged and spread quickly. Multidrug-resistant
strains are now common. Global warming and human
migration may bring the disease back to areas where
it was once eliminated. The economic cost of malaria
to the developing world is enormous.
Despite the impact of malaria parasites on global
health and productivity, little is known about the proteins encoded by the Plasmodium genome. Many proteins do not have recognizable homologs in other
species, either because the proteins are involved in
parasite-specific processes or because of the parasite’s
preference for amino acids encoded by codons rich in
adenine and thymine. Thus, we also do not understand
how protein expression is regulated or know the identity of any key regulators of development.
Because traditional methods for discovering gene
function are laborious, we are using a systematic
approach to begin to predict the function of proteins
encoded by the genome of Plasmodium falciparum
and to discover networks and protein-protein interactions. We are interested in describing how the transcript and protein levels change for all the genes in
the genome under as many natural life-stage and environmental conditions as possible. Some of our current
studies involve globally comparing transcript and protein levels to systematically identify proteins whose
levels are controlled posttranscriptionally and extending our transcriptional analysis of the parasite’s genome
to the sexual development phase, which is responsible
for the transmission of malaria. This work has led to
the identification of sequence motifs in the parasite
genome that most likely are responsible for controlling
transcription during development. We are also interested in developing novel algorithms that can be used
to create probabilistic models of gene function based
on gene expression and proteomic and homology data.
PUBLICATIONS
Daily, J.P., Le Roch, K.G., Sarr, O., Ndiaye, D., Lukens, A., Zhou, Y., Ndir, O.,
Mboup, S., Sultan, A., Winzeler, E.A., Wirth, D.F. In vivo transcriptome of Plasmodium falciparum reveals overexpression of transcripts that encode surface proteins. J. Infect. Dis. 191:1196, 2005.
Published by TSRI Press®. © Copyright 2005,
The Scripps Research Institute. All rights reserved.
55
Kidgell, C., Winzeler, E.A. Elucidating genetic diversity with oligonucleotide arrays.
Chromosome Res. 13:225, 2005.
Le Roch, K., Johnson, J.R., Florens, L., Zhou, Y., Santrosyan, A., Grainger, M.,
Yan, S.F., Williamson, K.C., Holder, A.A., Carucci, D.J., Yates, J.R. III, Winzeler,
E.A. Global analysis of transcript and protein levels across the Plasmodium falciparum life cycle. Genome Res. 14:2308, 2004.
O’Neill, B.M., Hanway, D., Winzeler, E.A. Coordinated functions of WSS1, PSY2
and TOF1 in the DNA damage response. Nucleic Acids Res. 32:6519, 2004.
Simpson, K.M., Baum, J., Good, R.T., Winzeler, E.A., Cowman, A.F., Speed, T.P.
A comparison of match-only algorithms for the analysis of Plasmodium falciparum
oligonucleotide arrays. Int. J. Parasitol. 35:523, 2005.
Stubbs, J., Simpson, K.M., Triglia, T., Plouffe, D., Tonkin, C.J., Duraisingh, M.T.,
Maier, A.G., Winzeler, E.A., Cowman, A.F. Molecular mechanism for switching of
P falciparum invasion pathways into human erythrocytes. Science 309:1384, 2005.
Young, J.A., Fivelman, Q.L., Blair, P.L., de la Vega, P., Le Roch, K.G., Zhou, Y.,
Carucci, D.J., Baker, D.A., Winzeler, E.A. The Plasmodium falciparum sexual
development transcriptome: a microarray analysis using ontology-based pattern
identification. Mol. Biochem. Parasitol. 143:67, 2005.
Young, J.A., Winzeler, E.A. Using expression information to discover new drug and
vaccine targets in the malaria parasite Plasmodium falciparum. Pharmacogenomics 6:17, 2005.
Zhou, Y., Young, J.A., Santrosyan, A., Chen, K., Yan, F., Winzeler, E.A. In silico gene function prediction using ontology-based pattern identification. Bioinformatics 21:1237, 2005.
Advancing Applications in Mass
Spectrometry–Based Proteomics
J.R. Yates III, A.O. Bailey, G.T. Cantin, E. Chen, D. Cociorva,
J. Coppinger, C. Delahunty, M.Q. Dong, J. Hewel,
J.R. Johnson, L. Liao, I. MacLeod, D. McClatchy, J. Meng,
S. Niessen, R. Park, J.H. Prieto, E. Romijn, C.I. Ruse,
R. Sadygov, J. Venable, J. Wohlschlegel, C. Wu, T. Xu,
W.H. Zhu
ass spectrometry has emerged as a powerful
technique for cellular proteomics, complementing traditional gene-by-gene approaches with
a comprehensive description of the molecular factors
that contribute to a biologically relevant system. We
remain at the forefront of this field, developing new
strategies to address more sophisticated scientific questions through proteomics, such as how to quantify global
changes in protein abundance for mammalian systems,
how to characterize complex posttranslational modifications, and what information can be gained by comparing the proteome with the transcriptome.
Proteomics methods based on quantitative mass
spectrometry rely on strategies that introduce an internal
isotopic standard for every protein to be characterized.
The preferred method for introducing these standards
is metabolic labeling (i.e., providing an isotope-labeled
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56 CELL BIOLOGY 2005
amino acid in culture medium); however, this approach
has been limited to microorganisms and simple model
organisms. Recently, we developed a method to generate proteins and peptides labeled with nitrogen 15 in
Rattus norvegicus tissues; the method resulted in an
atomic enrichment greater than 90% in liver and plasma.
This labeling strategy has countless possibilities for use
in comparative proteomic analyses of mammalian tissues, particularly studies of animal models of disease.
In addition, we developed a novel technique for comparing the abundance of peptides with internal isotopic
standards from tandem mass spectrometry spectra.
Whereas in most quantitative proteomics approaches,
these differences are measured at the level of the mass
spectra of intact peptides, by quantifying the fragmented
peptide instead, we dramatically improved the signalto-noise ratio and dynamic range of these measurements.
The characterization of posttranslational modifications is also an emerging application of mass spectrometry–based proteomics. The modification of cellular
factors by small ubiquitin-like modifiers (SUMOs) is
an essential process in budding yeast, but the identities of the substrates remain largely unknown. Using
proteomics techniques, we identified 271 new SUMO
targets, illustrating for the first time the diverse roles
that SUMO plays in regulating eukaryotic cells. This
research also revealed coordinated SUMO modification
of multiple proteins in well-defined macromolecular
complexes. This intriguing result suggests that sumoylation may target protein complexes rather than individual proteins. In other studies, we are characterizing
the mechanism that underlies this observation.
Finally, in collaboration with E.A. Winzeler, Department of Cell Biology, we did a genome-wide comparison of the abundance of global protein and mRNA
transcripts for several stages throughout the life cycle
of Plasmodium falciparum, the parasite that causes
malaria. Previous work in other laboratories suggested
that little correlation exists between mRNA and protein
abundance. However, our comparison showed a significant correlation in P falciparum, particularly when a
delay between the synthesis of mRNA transcripts and
proteins was considered. Interestingly, correlating mRNA
and protein expression profiles for individual genes
revealed particular families of functionally related genes
that appeared to have similar patterns of mRNA and
protein accumulation. Using this gene-by-gene correlation analysis, we were able to identify those genes that
most likely are regulated by posttranscriptional controls
Published by TSRI Press®. © Copyright 2005,
The Scripps Research Institute. All rights reserved.
and will be useful for future studies to unravel the molecular mechanisms that underlie this type of regulation.
PUBLICATIONS
Le Roch, K.G., Johnson, J.R., Florens, L., Zhou, Y., Santrosyan, A., Grainger, M.,
Yan, S.F., Williamson, K.C., Holder, A.A., Carucci, D.J., Yates, J.R. III, Winzeler,
E.A. Global analysis of transcript and protein levels across the Plasmodium falciparum life cycle. Genome Res. 14:2308, 2004.
Venable, J.D., Dong, M.Q., Wohlschlegel, J., Dillin, A., Yates, J.R. Automated
approach for quantitative analysis of complex peptide mixtures from tandem mass
spectra. Nat. Methods 1:39-45, 2004.
Wohlschlegel, J.A., Johnson, E.S., Reed, S.I., Yates, J.R. III. Global analysis of protein sumoylation in Saccharomyces cerevisiae. J. Biol. Chem. 279:45662, 2004.
Wu, C.C., MacCoss, M.J., Howell, K.E., Matthews, D.E., Yates, J.R. III. Metabolic labeling of mammalian organisms with stable isotopes for quantitative proteomic analysis. Anal. Chem. 76:4951, 2004.
Macromolecular Assemblies
Visualized by Electron
Cryomicroscopy and Image
Analysis: Membrane Proteins
and Viruses
M. Yeager, B.D. Adair, K. Altieri, A. Cheng, M.J. Daniels,
K.A. Dryden, B. Ganser, J. Harless, Y. Hua, R. Nunn,
F.A. Palida, M.A. Arnaout,* A.R. Bellamy,** N. Ben-Tal,***
M.J. Buchmeier,**** F.V. Chisari,**** K. Coombs,*****
H.B. Greenberg,† J.E. Johnson,**** S. Matsui,† L.H. Philipson,††
T.D. Pollard,††† A. Rein†††† A. Schneeman,**** J.A. Tainer,****
J.A. Taylor,** V.M. Unger†††
* Harvard Medical School, Boston, Massachusetts
** University of Auckland, Auckland, New Zealand
*** Tel-Aviv University, Tel-Aviv, Israel
**** Scripps Research
***** University of Manitoba, Winnipeg, Manitoba
†
Stanford University, Stanford, California
††
University of Chicago, Chicago, Illinois
†††
Yale University, New Haven, Connecticut
††††
National Cancer Institute, Frederick, Maryland
he ultimate goal of our studies is to gain a deeper
understanding of the molecular basis of important human diseases, such as sudden death, heart
attacks, and HIV infection, that cause substantial mortality and suffering. The structural details revealed by
our research may provide clues for the design of more
effective and safer medicines.
At the basic science level, we are intrigued by questions at the interface between cell biology and struc-
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CELL BIOLOGY 2005
tural biology: How do membrane proteins fold? How do
membrane channels open and close? How are signals
transmitted across a cellular membrane when an extracellular ligand binds to a membrane receptor? How do
viruses attach to and enter host cells, replicate, and
assemble infectious particles? To explore such problems,
we use high-resolution electron cryomicroscopy and
computer image processing. With this approach, we
can examine the molecular architecture of supramolecular
assemblies such as membrane proteins and viruses.
In electron cryomicroscopy, biological specimens
are quick frozen in a physiologic state to preserve their
native structure and functional properties. A special
advantage of this method is that we can capture dynamic
states of functioning macromolecular assemblies, such
as open and closed states of membrane channels and
viruses actively transcribing RNA. Three-dimensional
density maps are obtained by digital image processing
of the high-resolution electron micrographs. The rich
detail in the density maps indicates the power of this
approach to reveal the structural organization of complex biological systems that can be related to the functional properties of such assemblies.
Research projects under way include the structure
analysis of (1) membrane proteins involved in cell-tocell communication (gap junctions), water transport
(aquaporins), ion transport (potassium channels), transmembrane signaling (integrins), and viral recognition
(rotavirus NSP4); (2) viruses responsible for significant
human diseases (retroviruses, hepatitis B virus, rotavirus,
astrovirus); and (3) viruses used as model systems to
understand mechanisms of pathogenesis (arenaviruses,
reoviruses, nodaviruses, tetraviruses, and sobemoviruses). The following sections summarize selected projects that exemplify the themes of our research program.
57
We have now extended this analysis to 5.7-Å in-plane
and 19.8-Å vertical resolution, a step that enables us to
identify the positions and tilt angles for the 24 α-helices
within each hemichannel (Fig. 1). The 4 hydrophobic
segments in connexin sequences were assigned to the
α-helices in the map on the basis of biochemical and
phylogenetic data. Evolutionary conservation and an
analysis of compensatory mutations in connexin evolution were used to identify the packing interfaces between
the helices. The final model, which specifies the coordinates of Cα atoms in the transmembrane domain,
provides a structural basis for understanding the different physiologic effects of almost 30 mutations and
polymorphisms in terms of structural deformations at
the interfaces between helices, revealing an intimate
connection between molecular structure and disease.
INTEGRINS
Integrins are a large family of heterodimeric transmembrane receptor proteins that modulate important
GAP JUNCTION MEMBRANE CHANNELS
Gap junction channels connect the cytoplasms of
adjacent cells by means of an intercellular conduit formed
by the end-to-end docking of 2 hexameric hemichannels
called connexons. Gap junctions play an essential
functional role by mediating metabolic and electrical
communication within tissues. For instance, in the heart,
gap junction channels organize the pattern of current
flow to allow a coordinated contraction of the muscle.
We expressed a recombinant cardiac gap junction
protein, termed connexin 43, and produced 2-dimensional crystals suitable for electron cryocrystallography.
Our previous findings indicated that each hexameric
connexon is formed by 24 closely packed α-helices.
Published by TSRI Press®. © Copyright 2005,
The Scripps Research Institute. All rights reserved.
F i g . 1 . Intercellular gap junction channels have a diameter of
about 65 Å and are formed by the end-to-end docking of 2 hemichannels, each composed of a hexamer of connexin subunits. A Cα
model (ribbons) for the membrane-spanning domain of the hemichannels was derived by combining the information from a computational analysis of connexin sequences, the results of more than
a decade of biochemical studies, and the constraints provided
by a 3-dimensional map derived by electron cryocrystallography.
Although individually none of these approaches provided high-resolution information, their sum yielded an atomic model that predicts
how connexin mutations (spheres) may interfere with formation of
functional channels by disrupting helix-helix packing.
58 CELL BIOLOGY 2005
biological processes such as development, cell adhesion,
angiogenesis, wound healing, and neoplastic transformation. The ectodomain of the integrin α v β 3 crystallizes in a bent, genuflexed conformation, which is
considered to be inactive (i.e., unable to bind physiologic ligands in solution) unless it is fully extended by
activating stimuli. To assess whether the bent integrin
can bind physiologic ligands, we collaborated with
M.A. Arnaout, Harvard Medical School, Boston, Massachusetts, to generate a stable, soluble complex of the
manganese-bound αvβ3 ectodomain with a fragment of
fibronectin containing type III domains 7–10 and the EDB
domain. Electron microscopy and single-particle image
analysis were used to determine the 3-dimensional
structure of this complex (Fig. 2).
F i g . 2 . The 3-dimensional density map (gray-scale transparency)
of the integrin αvβ3 in a complex with fibronectin was determined by
using electron microscopy and image analysis. The x-ray structures of
the αv and β3 chains have been docked into the electron microscopy
density envelope. Additional density (lower right) can accommodate
fibronectin domain 10 adjacent to the ligand-binding site as well as
domain 9 at the synergy site. The complex is shown adjacent to the
white box, which represents the 30-Å-thick hydrophobic part of the
cellular membrane across which signals are transmitted.
Most αvβ3 particles, whether unliganded or bound
to fibronectin, had compact, triangular shapes. A difference map comparing ligand-free and fibronectinbound integrin revealed density that could accommodate
the fibronectin type III domain 10 containing arginine–
glycine–aspartic acid in proximity to the ligand-binding
site of β3, with domain 9 just adjacent to the synergy
site binding region of αv.
We conclude that the ectodomain of αvβ3 has a
bent conformation that can stably bind a physiologic
Published by TSRI Press®. © Copyright 2005,
The Scripps Research Institute. All rights reserved.
ligand in solution. These results are relevant for understanding how binding of ligands to the extracellular
domain leads to conformational changes that transmit
signals across the plasma membranes of cells, culminating in changes in gene transcription in the nucleus.
N O D AV I R U S E S
The nodavirus Flock House virus (FHV) has a
genome consisting of 2 strands of RNA that are packaged in an icosahedral capsid formed by 180 protein
subunits. FHV is an exceedingly useful system for
understanding mechanisms of viral assembly. We used
electron cryomicroscopy and image reconstruction to
determine the structure of 4 types of FHV particles that
differed in RNA and protein content (Fig. 3).
3 . Electron cryomicroscopy and image reconstruction of 4
types of FHV particles that differed in RNA and protein content
revealed almost identical capsid shells. Despite differences in the
encapsidated RNA (electrophoretic profiles, a–d), a substantial fraction of the packaged nucleic acid, either viral or heterologous, was
organized as a dodecahedral cage of duplex RNA. A, Native FHV
particles purified from infected Drosophila melanogaster cells contain both RNA1 and RNA2. B, Particles obtained by expression of
Fig.
the FHV coat protein in Sf21 cells contain primarily cellular RNAs.
C, FHV particles assembled in Drosophila cells from coat protein
subunits lacking basic residues at the N terminus primarily encapsidate RNA1. D, FHV particles assembled in Sf21 cells from coat protein subunits lacking the N terminus contain primarily cellular RNAs.
RNA-capsid interactions were primarily mediated
via the N and C termini, which are essential for RNA
recognition and particle assembly. A substantial fraction
of the packaged nucleic acid, either viral or heterologous, was organized as a dodecahedral cage of duplex
RNA. The similarity in RNA tertiary structure suggests
CELL BIOLOGY 2005
that RNA folding is independent of sequence and length.
Computational modeling indicated that RNA duplex
formation involves both short- and long-range interactions. These studies suggest that the capsid protein
can exploit the plasticity of the RNA secondary structures, capturing those that are compatible with the
geometry of the dodecahedral cage. Further analysis
of capsid protein mutants and designed RNA molecules is under way.
PUBLICATIONS
Adair, B.D., Xiong, J.-P., Maddock, C., Goodman, S.L., Arnaout, M.A., Yeager, M.
Three-dimensional EM structure of the ectodomain of integrin αvβ3 in complex with
fibronectin. J. Cell Biol. 168:1109, 2005.
Becker, C.F.W., Strop, P., Bass, R.B., Hansen, K.C., Locher, K.P., Ren, G., Yeager,
M., Rees, D.C., Kochendoerfer, G.G. Conversion of a mechanosensitive channel to
a water-soluble form by covalent modification with amphiphiles. J. Mol. Biol.
343:747, 2004.
Fleishman, S.J., Unger, V.M., Yeager, M., Ben-Tal, N. A Cα model for the transmembrane α-helices of gap-junction intercellular channels. Mol. Cell 15: 879, 2004.
Gollapudi, R.R., Yeager, M., Johnson, A.D. Left ventricular cardiac tamponade in
the setting of cor pulmonale and circumferential pericardial effusion: case report
and review of the literature. Cardiol. Rev. 13:214, 2005.
Neuman, B.W., Adair, B.D., Burns, J.W., Milligan, R.A., Buchmeier, M.J., Yeager, M.
Complementarity in the supramolecular design of arenaviruses and retroviruses
revealed by electron cryomicroscopy and image analysis. J. Virol. 79:3822, 2005.
Turbedsky, K.T., Pollard, T.D., Yeager, M. Assembly of Acanthamoeba myosin-II
minifilaments: model of anti-parallel dimers based on EM and x-ray diffraction of
2D and 3D crystals. J. Mol. Biol. 345:363, 2005.
Published by TSRI Press®. © Copyright 2005,
The Scripps Research Institute. All rights reserved.
59