Cell Biology
Electron cryomicroscopy and single-particle analysis were
used to determine the 30-Å structure of the self-assembling
coat protein complex-II cage nanoparticle. Shown are the
2-fold (bottom), 3-fold (middle), and 4-fold (top) fold symmetry axes of the biologically unprecedented cuboctahedron
structure responsible for directing cargo selection and membrane curvature during endoplasmic reticulum vesicle budding. Reprinted from Stagg, S.M., Gurkan, C., Fowler, D.M.,
LaPointe, P., Foss, T.R., Potter, C.S., Carragher, B., Balch,
W.E. Structure of the Sec13/31 COPII coat cage. Nature
439:234, 2006. This work is a collaboration between the
laboratories of William Balch, Ph.D., Bridget Carragher, Ph.D,
and Clinton Potter, B.S., at the National Resource for
Automated Molecular Microscopy.
Gaudenz Danuser, Ph.D., Associate Professor
Dinah Leorke, Ph.D., Research Associate
James Lim, Graduate Student
Department of Cell Biology
CELL BIOLOGY
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
Kristin Baldwin, Ph.D.***
Assistant Professor
Bridget Carragher, Ph.D.**
Associate Professor
Benjamin Cravatt, Ph.D.****
Professor
Director, Helen L. Dorris
Child & Adolescent NeuroPsychiatric Disorder
Institute
2006
Stephen P. Mayfield,
Ph.D.*****
Professor
Associate Dean of Graduate
Studies
Mark Mayford, Ph.D.***
Associate Professor
Lindsey Miles, Ph.D.
Associate Professor
Ronald A. Milligan, Ph.D.**
Professor
Director, Center for
Integrative Biosciences
Ulrich Müller***
Professor
Clinton Potter , B.S.**
Associate Professor
Philip E. Dawson, Ph.D.*****
Associate Professor
Lisa Stowers, Ph.D. ††
Assistant Professor
Velia Fowler, Ph.D.**
Professor
Heidi Stuhlmann, Ph.D.
Associate Professor
Shelley Halpain, Ph.D.***
Associate Professor
Natasha Kralli, Ph.D.
Associate Professor
Peter Kuhn, Ph.D.**
Associate Professor
David Loskutoff, Ph.D.
Professor Emeritus
Mari Manchester, Ph.D.**
Associate Professor
Robert Fischer, Ph.D.
Alan Bell, B.S.C.S.
Xerox Palo Alto Research
Center
Palo Alto, California
Elizabeth Wilson, Ph.D.
19
SENIOR RESEARCH
A S S O C I AT E S
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
Brian Adair, Ph.D.
Barbara Calabrese, Ph.D.
Mark Daniels, Ph.D.
Jeremiah Joseph, Ph.D.
Edward Korzus, Ph.D. †††
University of California
Riverside, California
Matthew Ritter, Ph.D.
Ardem Patapoutian, Ph.D. †
Associate Professor
James Quigley, Ph.D.
Professor
Larry R. Gerace, Ph.D.*
Professor
ADJUNCT APPOINTMENTS
Bertil Daneholt, M.D.
Karolinska Institutet
Stockholm, Sweden
Gaudenz Danuser , Ph.D.**
Associate Professor
Martin Friedlander, M.D.,
Ph.D.
Professor
THE SCRIPPS RESEARCH INSTITUTE
Ph.D. †††
Kevin F. Sullivan,
University of Ireland
Galway, Ireland
Peter N.T. Unwin, Ph.D.**
Professor
Clare Waterman-Storer,
Ph.D.**
Associate Professor
Elizabeth Winzeler, Ph.D. †
Associate Professor
John R. Yates III, Ph.D.
Professor
Mark J. Yeager, M.D., Ph.D.
Professor
Scott Elrod, Ph.D.
Xerox Palo Alto Research
Center
Palo Alto, California
Mark Ginsberg, M.D.
University of California
San Diego, California
David Goldberg, Ph.D.
Xerox Palo Alto Research
Center
Palo Alto, California
Martin Schwander, Ph.D.
Gina Story, Ph.D. †††
Washington University
St. Louis, Missouri
Defne Yarar, Ph.D.
Andries Zijlstra, Ph.D.
R E S E A R C H A S S O C I AT E S
Xiaohua Gong, Ph.D.
University of California
Berkeley, California
Jessica Alexander, Ph.D.
Klaus Hahn, Ph.D.
University of North Carolina
Chapel Hill, North Carolina
Veronica Ardi, Ph.D.
Eric Peeters, Ph.D.
Xerox Palo Alto Research
Center
Palo Alto, California
Andrea Bacconi, Ph.D.
Geza Ambrus-Aikelin, Ph.D.
Angelique Aschrafi, Ph.D.††††
Hongdong Bai, Ph.D.
Kent Baker, Ph.D.
S TA F F S C I E N T I S T S
Claudia Barros, Ph.D.
Michael Bracey, Ph.D.
Maria Beligni, Ph.D.
Anchi Cheng, Ph.D.
Richard Belvindrah, Ph.D.
Elena Deryugina, Ph.D.
Edward Brignole III, Ph.D.
20 CELL BIOLOGY
2006
THE SCRIPPS RESEARCH INSTITUTE
Florence Brunel, Ph.D.
Anna Durrans, Ph.D.
Paul LaPointe, Ph.D.
Jacobus Neels, Ph.D.
Anja Bubeck, Ph.D. †††
Gen-Probe, Inc.
San Diego, California
Samer Eid, Ph.D. †††
Merck Research
Laboratories, Neuroscience
Drug Discovery
West Point, Pennsylvania
Nicole Lazarus, Ph.D.
Sherry Niessen, Ph.D.
Donmienne Leung, Ph.D. †††
Applied Molecular Evolution
San Diego, California
Silvia Ortega-Gutierrez,
Ph.D.
Gang Cai, Ph.D.
Lesley Page, Ph.D.
Gregory Cantin, Ph.D.
Eric Carlson, Ph.D.
Ph.D. †††
Michael Fitch,
Tanabe Research
Laboratories U.S.A.
San Diego, California
Lujian Liao, Ph.D.
Aurelia Cassany, Ph.D.
Santos Franco, Ph.D.
Yuriy Chaban, Ph.D.
Margaret Gardel, Ph.D.
Pablo Chamero, Ph.D.
Emily Chen, Ph.D.
Ihsiung Chen, Ph.D. †††
CODA Genomics
Laguna Hills, California
Yei Hua Chen, Ph.D.
Charmian Cher, Ph.D.
Smita Chitnis, Ph.D.
Esther Choi, Ph.D.
Parag Chowdhury, Ph.D.
John Lewis, Ph.D. †††
Dalhousie University
Halifax, Nova Scotia, Canada
Ph.D. †††
Maria Gonzalez,
Rincon Pharmaceuticals
La Jolla, California
Jorg Grandl, Ph.D.
Nicolas Grillet, Ph.D.
Cemal Gurkan, Ph.D. ††††
Johannes Hewel, Ph.D.
Michael Hock, Ph.D.
Ke Hu, Ph.D.
Maria Lillo, Ph.D. †††
University of Salamanca
Salamanca, Spain
Ana Maria Pasaperi-Limon,
Ph.D.
Olivier Pertz, Ph.D. †††
University of California
San Diego, California
Barbie Pornillos, Ph.D.
Jennifer Lin, Ph.D.
Anita Pottekat, Ph.D.
Ryan Littlefield, Ph.D. †††
University of Washington
Seattle, Washington
Judith Prieto, Ph.D.
Dinah Loerke, Ph.D.
Natalie Prigozhina, Ph.D. †††
Vala Sciences, Inc.
La Jolla, California
Darren Logan, Ph.D.
Thomas Pucadyil, Ph.D.
Bingen Lu, Ph.D.
Rajesh Ramachandran,
Ph.D.
Matthias Machacek, Ph.D.
Kalotina Machini, Ph.D.
Vandana Ramachandran,
Ph.D.
Jill Chrencik, Ph.D.
Michael Huber, Ph.D.
Mark Madsen, Ph.D.
Abbas Razvi, Ph.D.
Michael Churchill, Ph.D.
Darren Hutt, Ph.D.
Valentina Marchetti, Ph.D.
Leon Reijmers, Ph.D.
Francesco Conti, Ph.D.
Eric Hwang, Ph.D.
Julia Marin-Navarro, Ph.D.
Anna Reynolds, Ph.D.
Judith Coppinger, Ph.D.
Khuloud Jaqaman, Ph.D.
Michael Matho, Ph.D.
Kaustuv Datta, Ph.D.
Anass Jawhari, Ph.D. ††††
Naoki Matsuo, Ph.D.
Edwin Romijn, Ph.D. †††
Philips Scientific Equipment
Division
Eindhoven, the Netherlands
Leif Dehmelt, Ph.D.
Lin Ji, Ph.D.
Daniel McClatchy, Ph.D.
Ajay Dhaka, Ph.D.
Nobutaka Kato, Ph.D.
Caroline McKeown, Ph.D.
Anouk Dirksen, Ph.D.
Claire Kidgell, Ph.D. ††††
Marcel Mettlen, Ph.D.
Meng-Qui Dong, Ph.D.
Katsuhiro Kita, Ph.D.
Helena Mira, Ph.D.
Michael Dorrell, Ph.D.
Kevin Koehntop, Ph.D.
Jennifer Mitchell, Ph.D.
Alan Saghatelian, Ph.D. †††
Harvard University
Cambridge, Massachusetts
Kelly A. Dryden, Ph.D.
Jenny Kohler, Ph.D.
Machiko Muto, Ph.D.
Kumar Saikatendu, Ph.D.
Jerome Dupuy, Ph.D.
Atanas Koulov, Ph.D.
Andromeda Nauli, Ph.D.
Tomoyo Sakata, Ph.D.
Cristian Ruse, Ph.D.
Mohsen Sabouri-Ghomi,
Ph.D.
CELL BIOLOGY
2006
Cleo Salisbury, Ph.D.
Ge Yang, Ph.D.
Ian Schneider, Ph.D.
Christina Schroeder, Ph.D.
Masahiro Yasuda, Ph.D. †††
University of Michigan
Ann Arbor, Michigan
Stephan Sieber, Ph.D. †††
Ludwig-Maximilians University
Munich, Germany
Rie Yasuda, Ph.D. †††
Osaka University
Osaka, Japan
Pratik Singh, Ph.D.
Zhongmin Zou, Ph.D. †††
Institute of Combined Injury
of PLA, Third Military
Medical University
Chongqing, P.R. China
THE SCRIPPS RESEARCH INSTITUTE
* Joint appointment in the
Department of Molecular Biology
** Joint appointment in the Center
for Integrative Molecular
Biosciences
*** Joint appointment in the
Institute for Childhood and
Neglected Diseases
**** Joint appointments in the
Department of Chemistry, the
Scott Stagg, Ph.D.
Mark Surka, Ph.D.
Skaggs Institute for Chemical
Biology, and the Helen L. Dorris
Child and Adolescent NeuroPsychiatric Disorder Institute
***** Joint appointment in the Skaggs
Institute for Chemical Biology
†
Patricia Szainer, Ph.D.
Joint appointments in the
Institute for Childhood and
S C I E N T I F I C A S S O C I AT E S
Neglected Diseases and the
Claire Tiraby Nguyen, Ph.D.
Tuija Uusitalo, Ph.D. †††
University of Helsinki
Helsinki, Finland
Genomics Institute of the Novartis
Hilda Edith Aguilar de Diaz,
M.D.
Research Foundation
††
Joint appointments in the Helen
L. Dorris Child and Adolescent
Alexei Brooun, Ph.D.
Neuro-Psychiatric Disorder
Institute
Valerie Uzzell, Ph.D.
Claire Delahunty, Ph.D.
Mohammed El-Kalay, Ph.D.
Josep Villena, Ph.D.
Tinglu Guan, Ph.D.
Xiaodong Wang, Ph.D. †††
Medical University of Ohio
Toledo, Ohio
Anand Kolatkar, Ph.D.
Eranthie Weerapana, Ph.D.
BinQing Wei, Ph.D.
Scott Westenberger, Ph.D.
Ann Wheeler, Ph.D.
Torsten Wittmann, Ph.D. †††
University of San Francisco
San Francisco, California
James Wohlschlegel, Ph.D.
Catherine Wong, Ph.D.
Aaron Wright, Ph.D.
Lihua Wu, Ph.D. †††
Department of Immunology
Scripps Research
Appointment completed; new
location shown
John Venable, Ph.D.
Kari Bradtke Weber, Ph.D.
†††
††††
Appointment completed
21
22 CELL BIOLOGY
2006
Sandra Schmid, Ph.D.
Cell Biology Overview
embers of the Department of Cell Biology continue to excel in the rich environment of Scripps
Research, and their successes are being recognized by others. Clare Waterman-Storer, who launched
her independent career at Scripps Research, received the
National Institutes of Health Director’s Pioneer Award
this year, along with a 5-year grant to support her research
program. Dr. Waterman-Storer was 1 of 13 scientists
chosen from more than 800 applicants for this extremely
prestigious award; the Director’s Pioneer Award recognizes leading scientists in the United States and invests
in their potential to make important advances in biomedical research. Also this year John Yates received
the prestigious Christian B. Anfinsen Award from the
Protein Society, which recognizes significant technical
achievements in the field of protein science. Gaudenz
Danuser, Martin Friedlander, Dr. Waterman-Storer, and I
have given plenary addresses recognizing notable scientific achievements and leadership in our respective fields.
Finally, Dr. Danuser and Natasha Kralli were promoted
to associate professor, and Stephen Mayfield, who also
serves as associate dean of Graduate Studies, was promoted to full professor—positions befitting their academic and scientific accomplishments.
M
THE SCRIPPS RESEARCH INSTITUTE
A unique attribute of Scripps Research that distinguishes it from academic institutions whose faculty must
meet diverse educational obligations is its ability to build
research efforts around areas of strength, ensuring critical mass and leadership in important areas of biomedical research. Although the department is proud of the
success of its individual scientists, we believe that this
success reflects in part the strong synergies that have
developed as we have built on areas of strength within
cell biology. Synergy is defined as 2 or more groups
working together in such a way that the result is greater
than the sum of their individual capabilities. Although
there are numerous foci of synergy driving innovation
and research in the department, I highlight here the
3 areas that represent the strengths on which we will
continue to build.
An early and unique strength of our department, which
synergizes with the structural efforts in the Department
of Molecular Biology, is the use of electron cryomicroscopy to provide structural insights into the workings of
complex, multisubunit cellular machines. Francisco
Asturias, Bridget Carragher, Clint Potter, Ron Milligan,
Nigel Unwin, Mark Yeager, and their colleagues have
built an internationally preeminent center for electron
cryomicroscopy. Together, these groups are developing
new methodologies, solving important structures, and
training the next generation of electron cryomicroscopy
structural biologists, not only among students and fellows
at Scripps Research, but through popular intensive summer courses offered to students from around the world.
These synergistic interactions have driven unprecedented
productivity. Many important structures have been solved
in the past year, including (1) the chloroplast ribosome
to reveal functionally important differences between it
and its bacterial progenitor (Dr. Milligan in collaboration
with members of Dr. Mayfield’s laboratory), (2) the intact
infectious P22 bacteriophage to reveal the mechanisms
of viral DNA transfer into the host cell (Drs. Carragher
and Potter in collaboration with Jack Johnson in the
Department of Molecular Biology), (3) the coat protein
complex II cage to reveal a unique architecture for deforming a membrane and collecting cargo molecules into transport vesicles (Drs. Carragher and Potter in collaboration
with members of Bill Balch’s laboratory), (4) the structure of DNA polymerase epsilon, providing new insight
into its interaction with its DNA template (Dr. Asturias),
(5) the structure of a minus-end directed kinesin to reveal
the mechanism of its unusual directionality (Dr. Milligan
in collaboration with Ron Vale at the University of Cali-
CELL BIOLOGY
2006
fornia, San Francisco), and (6) the structure of an integrin complexed to its substrate revealing the activated
form of this cell adhesion molecule (Dr. Yeager). These
remarkable accomplishments reveal the accelerated pace
of electron cryomicroscopy structural biology made possible through the relatively recent expansion of these
efforts within the Center for Integrative Molecular Biosciences, and the resulting innovations in technology
and efforts to automate this historically slow and tedious
technology. The importance of these efforts, under the
leadership of Drs. Carragher and Potter, has been recognized with funding from the National Center for Research
Resources and designated as a National Resource for
Automated Molecular Microscopy.
A second area of synergy, which has been built in
association with the Institute for Childhood Diseases, is
in cellular and molecular neurobiology. Kristen Baldwin
recently joined these efforts from Columbia University
after completing her postdoctoral training with Richard
Axel, the recipient of the 2004 Nobel Prize in Physiology and Medicine for his work on olfaction. Using the
olfactory system as a model, Dr. Baldwin plans to dissect the mechanisms governing neuronal diversity and
the establishment of neural connectivities that enable us
to process and respond to complex sensory input. She
joins a group of investigators at the Institute for Childhood Diseases that includes Shelley Halpain, Mark Mayford, Uli Mueller, Ardem Patapoutian, and Lisa Stowers,
who work on diverse but complementary aspects of
neuronal development, sensory perception (especially
touch, smell, and hearing), the establishment of neuronal circuitry, and higher-order functions of learning
and memory. Their combined expertise allows them to
tackle the complexities of how the brain is wired for
higher-order functioning. The outcomes of their studies
have important implications for childhood diseases such
as autism, as well as for mental retardation, schizophrenia, and neurodegenerative diseases such as Alzheimer’s
and Parkinson’s.
A third area of synergy on which we plan to build is
in the quantitative spatial and temporal analyses of higherorder cellular processes, such as cell migration, signal
transduction, the establishment of polarity, and intracellular trafficking. These efforts, also being carried out
within the Center for Molecular Biosciences, are spearheaded by Dr. Danuser, Velia Fowler, and Dr. WatermanStorer. Traditionally, cell biologists have focused their
efforts on dissecting a single cellular process or a single
piece of the cellular machinery, often in isolation from
THE SCRIPPS RESEARCH INSTITUTE
23
the cell. The work of Drs. Danuser and Waterman-Storer
is revealing the complex molecular and physical interactions between multiple moving parts of the cell that
are required for directed cell locomotion. Dynamic interactions and interdependencies between the actin cytoskeleton and the endocytic machinery are being revealed
in collaboration with scientists in my laboratory. Spatial
and temporal regulation of signaling events that direct
cellular behavior are being analyzed in collaboration with
Gary Bokoch in the Department of Immunology. The
sophisticated microscopy, image analysis, and mathematical modeling techniques being developed in the
Center for Molecular Biosciences, combined with innovations in molecular biology, are enabling cell biologists to
manipulate and study the complex behavior of whole cells,
rather than the traditional and more limited “divide and
conquer” approaches of the past.
Although the Department of Cell Biology at Scripps
Research is clearly leading in these important areas of
research and innovation, in this fast-paced and competitive arena if you’re not moving forward, you’ll quickly
slip backward. Therefore, we hope to continue to build
on these areas of strength with the recruitment of talented and creative faculty who bring new and complementary expertise to our efforts and who will contribute
to and benefit from the synergistic environments we
have established.
24 CELL BIOLOGY
2006
INVESTIGATORS’ R EPORTS
Structural Characterization of
Macromolecular Machines
F.J. Asturias, Y. Chaban, J. Brown, E. Brignole, G. Cai
e use state-of-the-art electron microscopy and
image analysis techniques to determine the
3-dimensional structures of macromolecular
complexes involved in a variety of cellular processes,
including DNA transcription, DNA replication, chromatin
modification and remodeling, and fatty acid synthesis.
Macromolecular electron microscopy is an ideal technique for these studies because it requires only a small
amount of material and the conditions for preparing
samples are physiologically relevant. Images of individual macromolecules are recorded and then computationally combined to obtain structures of low to moderate
(25–10 Å) resolution. These structures are often interpreted by docking atomic resolution structures of component subunits in the lower resolution map of an entire
complex. Our ultimate goal is to use a combination of
biochemical and structural information to reveal the
mechanism by which a macromolecular complex carries out its function.
In our current research on DNA transcription and
its regulation, we are analyzing the basal machinery
and assembly of the RNA polymerase II preinitiation
complex. We are also studying complexes involved in
the regulation of transcription during initiation and in
previous steps in which the structure of chromatin is
altered to control access to DNA. We are particularly
interested in the structure and function of Mediator, a
complex that plays a central role in regulating transcription in eukaryotes at the time transcription begins.
We have developed a reproducible protocol for purifying Mediator that will enable us to pursue biochemical
and structural studies to get to the heart of the mechanism of regulation by Mediator. We are also gearing
up to use fluorescence microscopy to validate the in
vivo relevance of our in vitro studies.
In the past year we also made progress in analyzing the structure and mechanism of DNA polymerase ε
(Pol ε). We used electron microscopy and single-particle image analysis to calculate a 16-Å resolution of the
polymerase, the first of a multisubunit eukaryotic DNA
polymerase. We were able to determine the location of
W
THE SCRIPPS RESEARCH INSTITUTE
2 of the 4 subunit components of Pol ε and to document and measure changes in the relative orientation
of the 2 large domains that constitute the structure.
Although no atomic resolution structures of the component subunits were available to help in interpreting
the electron microscopy map, we used the structural
information to design template elongation assays that
resulted in a model for interaction of Pol ε with DNA
that explains the intrinsic processivity and capacity of
Pol ε to interact with a variety of templates (Fig. 1).
F i g . 1 . Structure of Pol ε and a model for its interaction with DNA.
Electron microscopy and single-particle image analysis were used
to calculate the structure of the polymerase at a resolution of 16 Å.
The structure of the catalytic Pol2 subunit was calculated independently, as was the structure of the Pol2–Dpb2 subunit complex.
Image analysis also revealed changes in the relative orientation of the
Pol2 and Dpb2-Dpb3-Dpb4 domains made possible by a flexible
connection. These changes in relative orientation might result in a
conformation that would allow access of the DNA to the active-site
cleft (top). A return of the tail to its normal conformation after entry
of double-stranded DNA into the active-site cleft would result in close
interaction of the nucleic acid with the extended tail domain (bottom).
This mode of interaction with DNA would explain the intrinsic processivity of Pol ε and the involvement of the Dpb3–Dpb4 subunit complex in double-stranded DNA binding. The processivity dependence
of the length of the double-stranded primer region revealed by elongation assays would be explained by the requirement for a minimal
length of double-stranded DNA to ensure proper interaction with the
full length of the extended tail domain in the Pol ε structure.
CELL BIOLOGY 2006
Finally, we continue to investigate the role that conformational changes play in the function of mammalian
fatty acid synthase (FAS), the enzyme responsible for
the synthesis of long-chain fatty acids. In this true
macromolecular assembly line, the different enzymes
involved in the synthesis of fatty acids have fused into
a single polypeptide chain that includes 6 catalytic and
1 acyl carrier protein domains. Using a novel approach
in which FAS point mutants were imaged in the presence of substrates (effectively pausing the enzyme at a
given catalytic step), we were able to determine an
FAS structure that led to a revised model for FAS
organization. That model has now been confirmed by
a recently published partial x-ray structure of FAS. As
a result of the molecular flexibility that appears to be
essential for the function of FAS, 2 of the FAS domains
were not observed in the x-ray structure. Further electron microscopy analysis of FAS will reveal the location
of all domains and provide information about the variety of conformational states that make possible the
multitude of interdomain interactions required for the
function of the enzyme.
PUBLICATIONS
Asturias, F.J., Cheung, I., Sabouri, N., Chilkova, O., Wepplo, D., Johansson, E.
Structure of Saccharomyces cerevisiae DNA polymerase epsilon by cryo-electron
microscopy. Nat. Struct. Mol. Biol. 13:35, 2006.
Takagi, Y., Chadick, J.Z., Davis, J.A., Asturias, F.J. Preponderance of free Mediator
in the yeast Saccharomyces cerevisiae. J. Biol. Chem. 280:31200, 2005.
Chemical Biology of
Conformational Disease
and Membrane Traffic
W.E. Balch, Y. An, C. Chen, J. Conkright-Johnson, D. Fowler,
C. Gurkan, D. Hutt, A. Koulov, P. LaPointe, J. Matteson,
A. Nauli, L. Page, H. Plutner, A. Pottekat, A. Razvi, S. Stagg,
P. Szajner, I. Yonemoto
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
A
25
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 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
form the coatomer complex II coat (COPII) that polymerizes to promote budding from the surface of the
endoplasmic reticulum. This machinery directs exit
from the endoplasmic reticulum of proteins encoded
by nearly one third of the genome in eukaryotes.
Recently, in collaboration with C. Potter and B. Carragher, Department of Cell Biology, we solved the
2-dimensional electron cryomicroscopy structure of the
Sec13/31 cage (Fig. 1). This cage is a self-assembling
nanoparticle that collects cargo by assembling into a
polymer scaffold that interacts with an adaptor protein
complex bound to “exit codes” found on the cytoplasmic domains of cargo and cargo receptors. These exit
codes bind to a multivalent adaptor platform found on
the surface of Sec24 facing the lipid layer. With J.R.
Yates, Department of Cell Biology, we are using state-ofthe-art proteomics (multidimensional protein identification
technology or MudPIT) to identify unknown components
involved in cargo selection.
26 CELL BIOLOGY
2006
F i g . 1 . Structure of the self-assembling COPII cage. Illustrated are
the 3 different symmetry-related views of the Sec13/31 complex that
self-assembles to form unprecedented cuboctahedron geometry on
the surface of the endoplasmic reticulum to form a molecular scaf-
fold (cage) that collects cargo for export. By coordinating cargo concentration with membrane curvature and fission, the cage can
generate a transit vesicle that mobilizes cargo to the cell surface.
Reprinted from Stagg, S.M., Gurkan, C., Fowler, D.M., LaPointe,
P., Foss, T.R., Potter, C.S., Carragher, B., Balch, W.E. Structure of
the Sec13/31 COPII coat cage. Nature 439:234, 2006.
After budding and fusion of COPII transport vesicles
from the endoplasmic reticulum, targeting and fusion of
the vesicles to generate the next compartment of the
secretory pathway, the Golgi apparatus, require a different class of Ras-like 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 involving hierarchial clustering and mRNA expression profiling (microarray), we
found that each Rab GTPase executes targeting and
fusion decisions at a distinct step in the exocytic or
endocytic pathway. By integrating the interactions of
multiple distinct effectors at each step, Rab GTPases
act as hubs to define the highly distinctive membrane
THE SCRIPPS RESEARCH INSTITUTE
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.
Of particular importance is our characterization of
the structure of the Rab1 tether p115, done in collaboration with I.A. Wilson, Department of Molecular Biology.
The structure reveals a superhelical coiled coil multivalent assembly platform that facilitates Rab-dependent
maturation of tethering-fusion complexes. In addition,
Rab proteins are recycled for use in multiple rounds of
tether assembly. We recently showed the surprising
importance of the Hsp90 chaperone system in Rab
recycling after vesicle fusion.
Many mutation disrupt cargo traffic from the endoplasmic reticulum by preventing proper protein folding
during synthesis, resulting in loss of recognition by the
COPII selection machinery. 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 of a
number of conformational diseases, including cystic
fibrosis, hereditary childhood emphysema, Gaucher
disease, familial amyloidosis of Finnish type, Parkinson’s disease, and transthyretin amyloidosis. These
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 and human evolution. Our
analysis of cystic fibrosis has revealed that system-wide
modification of the chaperone folding pathways (the
chaperone) can alter the steady-state energetic pools
of unfolded and folded macrostates (conformational
populations) that allow for rescue of the trafficking
defect and restore the function of chloride channels at
the cell surface.
Through a multidisciplinary approach that combines the tools of chemistry, biology, systems biology,
bioinformatics, and structure, we hope to gain critical
insight into the fundamental principles of cargo trafficking and 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.
CELL BIOLOGY
2006
PUBLICATIONS
Bannykh, S.I., Plutner, H., Matteson, J., Balch, W.E. The role of ARF1 and Rab
GTPases in polarization of the Golgi stack. Traffic 6:803, 2005.
Chen, C.Y., Balch, W.E. The Hsp90 chaperone complex regulates GDI-dependent
Rab recycling. Mol. Biol. Cell 17:3494, 2006.
Chen, C.Y., Sakisaka, T., Balch, W.E. Use of Hsp90 inhibitors to disrupt GDIdependent Rab recycling. Methods Enzymol. 403:339, 2005.
Fowler, D.M., Koulov, A.V., Alory-Jost, C., Marks, M.S., Balch, W.E., Kelly, J.W.
Functional amyloid formation within mammalian tissue. PLoS Biol. 4:e6, 2006.
Gurkan, C., Balch, W.E. Recombinant production in baculovirus-infected insect
cells and purification of the mammalian Sec13/Sec31 complex. Methods Enzymol.
404:58, 2005.
Gurkan, C., Lapp, H., Alory, C., Su, A.I., Hogenesch, J.B., Balch, W.E. Large-scale
profiling of Rab GTPase trafficking networks: the membrome. Mol. Biol. Cell
16:3847, 2005.
Gurkan, C., Lapp, H., Hogenesch, J.B., Balch, W.E. Exploring trafficking GTPase
function by mRNA expression profiling: use of the SymAtlas Web-application and
the Membrome datasets. Methods Enzymol. 403:1, 2005.
Kelly, J.W., Balch, W.E. The integration of cell and chemical biology in protein
folding. Nat. Chem. Biol. 2:224, 2006.
Page, L.J., Suk, J.Y., Huff, M.E., Lim, H.J., Venable, J., Yates, J., Kelly, J.W.,
Balch, W.E. Metalloendoprotease cleavage triggers gelsolin amyloidogenesis. Embo
J. 24:4124, 2005.
Stagg, S.M., Gurkan, C., Fowler, D.M., LaPointe, P., Foss, T.R., Potter, C.S.,
Carragher, B., Balch, W.E. Structure of the Sec13/31 COPII coat cage. Nature
439:234, 2006.
Suk, J.Y., Zhang, F., Balch, W.E., Linhardt, R.J., Kelly, J.W. Heparin accelerates
gelsolin amyloidogenesis. Biochemistry 45:2234, 2006.
Wang, X., Venable, J., LaPointe, P., Hutt, D.M., Koulov, A.V., Coppinger, J., Gurkan,
C., Kellner, W., Matteson, J., Plutner, H., Riordan, J.R., Kellly, J.W., Yates, J.R. III,
Balch, W.E. Hsp90 cochaperone rescue of misfolding disease. Cell, in press.
Wiseman, R.L., Balch, W.E. A new pharmacology: drugging stressed folding pathways. Trends Mol. Med. 11:347, 2005.
Molecular Mechanisms of
Olfactory Perception and Neural
Circuit Formation
K.K. Baldwin, S. Tate, B. Fields, S. Ghosh
n mammals, the sense of smell is critical for survival. Scents trigger suckling at birth, distinguish
food from poison, provide warning of predators, and
identify attractive mates. A primary goal of neurobiology is to discover how neural circuits link these types
of sensory inputs to appropriate behavioral outputs.
Surprisingly little is known about how neural circuits
specific to one set of inputs are organized or built.
We take advantage of the unique architecture and
genetic tractability of the mouse olfactory system to
study specific olfactory circuits at the first 2 levels of pro-
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THE SCRIPPS RESEARCH INSTITUTE
27
cessing. Our goal is to genetically label the neurons that
respond to specific odors in the nose and the olfactory
bulb, to trace the projections of the neurons into the cortical regions where inputs converge, and to identify the
molecular mechanisms that govern the formation of specific neural circuits. We anticipate that our findings will
reveal mechanisms common to neural circuit formation
throughout the brain and provide insight into genetic
bases of human cognitive and behavioral disorders.
CLONING MICE FROM NEURONS
In contrast to gene activation in other sensory systems, odorant receptor genes are activated by stochastic
mechanisms. Stochastic gene activation in the immune
system is due to irreversible DNA rearrangements. We
searched for chromosomal rearrangements by cloning
mice from the nuclei of olfactory sensory neurons. We
found that odorant receptor choice is reversible, as is
neuronal differentiation. We will now clone mice from
other types of neurons to determine whether irreversible chromosomal alterations accompany neuronal diversification in the brain.
VISUALIZING OLFACTORY INPUTS TO THE BRAIN
A major challenge is to understand how olfactory
information is integrated in the olfactory cortex. An
important first step is to describe the anatomy of the
second-order circuit. This endeavor has been hindered
by the lack of specific promoters for the output neurons
(mitral cells) of the olfactory bulb. We identified a gene
that is expressed specifically in mitral cells and have
produced mice in which subsets of these cells express
fluorescent proteins. We use confocal and 2-photon
microscopy to map the projections of individual mitral
cells into the brain. By visualizing the second-order
olfactory circuit, we can begin to understand how the
brain recognizes odors.
N E U R A L D I V E R S I T Y A N D C I R C U I T F O R M AT I O N
Although genes that regulate axon guidance and
large-scale brain patterning have been identified, the
genes that endow neurons with the precise patterns of
neuronal synaptic connectivity remain enigmatic. We
reasoned that genes expressed in subsets of mitral cells
would be good candidates to direct the formation of
specific neuronal circuits. Using bioinformatics and
single-cell gene profiling, we have identified about 70
genes that can be used to subdivide mitral cells into
different classes. We will use gene targeting to test the
role of these genes in neural circuit formation.
One class of genes that diversifies mitral cells is the
large family of approximately 60 clustered genes for pro-
28 CELL BIOLOGY
2006
tocadherins. Protocadherins are expressed throughout
the nervous system. Intriguingly, each neuron seems to
express a distinct combination of several protocadherin
proteins. We have produced mice that do not express one
subfamily of protocadherins. These mice have behavioral
abnormalities consistent with defects in neuronal function. We are investigating the cellular and physiologic
consequences of loss of protocadherin diversity.
Automated Molecular Imaging
B. Carragher, C.S. Potter, A. Cheng, D. Fellmann, G. Lander,
S. Mallick, P. Mercurio, J. Pulokas, J. Quispe , S. Stagg,
C. Yoshioka
uring the past decade, electron cryomicroscopy
has emerged as a powerful method for determining the structure of large macromolecular
complexes. Elucidating the structure and mechanism
of action of these “molecular machines” is an emerging
frontier in understanding how the information in the
genome is transformed into cellular activities. Examples
of the machines include ribosomes, transcription complexes, track-motor complexes, and membrane-embedded pumps and channels.
In electron cryomicroscopy, the macromolecular
specimen is preserved in a thin layer of vitreous (glassy)
ice and imaged in an electron microscope by using low
doses of electrons. The low signal-to-noise ratio of the
resulting images means that averaging is required to
recover the signal and reconstruct a 3-dimensional
map of the structure.
In 2002, we established the National Resource for
Automated Molecular Microscopy (NRAMM) to develop,
test, and apply technology for automating the processes
involved in using electron cryomicroscopy to solve
macromolecular structures. The goal of automation is
not only to facilitate the process of molecular microscopy, although this facilitation is a welcome benefit,
but also to expand the scope of accessible problems
and push experimental frontiers by making possible
investigations deemed too difficult or high risk because
of the considerable effort involved in using manual
methods. An additional goal of automation is to enable
much higher throughput of data and thus improve resolution for single-particle reconstructions by increasing
the numbers of particles that contribute to the average
3-dimensional map. Another mission of NRAMM is to
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THE SCRIPPS RESEARCH INSTITUTE
use the infrastructure developed to open up the sometimes esoteric practices of electron cryomicroscopy to
a much wider group of researchers, including investigators in cell biology, x-ray crystallography, and materials science.
During the past 3 years, the new techniques and
technologies that we developed included a new grid
substrate designed to improve quality and throughput
for vitreous ice specimens; a prototype of a robotic
grid-handling system used for screening; Leginon, an
automated system for microscope control and image
acquisition; a relational database that tracks and manages data acquired by Leginon and tools for viewing
and delivering the data via Web browsers; and ACE, a
program for the automated measurement and correction
of contrast transfer function. These technologies all
contributed in demonstrating the potential for automated
high-throughput data acquisition and analysis in an
experiment in which images of more than 280,000
particles of GroEL, a molecule involved in protein folding, were acquired in a single 25-hour session at the
microscope and subsequently subjected to completely
automated procedures to reconstruct a 3-dimensional
map to a resolution better than 8 Å (Fig. 1).
F i g . 1 . More than 280,000 particles of GroEL were acquired
from a single grid by using Leginon in a period of 25 hours. These
particles were sorted by ice thickness and used to reconstruct a
3-dimensional density map to a resolution of approximately 8 Å.
Visualization by Scott Stagg and Mike Pique.
These technological developments have been
designed for and used in a number of collaborative
research projects, including reconstruction of a minimal coatomer complex II cage and reconstruction of
an intact infectious P22 virion. The infrastructure has
also, in accordance with our mission, made electron
cryomicroscopy accessible to a much wider commu-
CELL BIOLOGY
2006
nity, leading to publications from research groups in
chemistry, x-ray crystallography, materials science,
and industry. NRAMM currently provides support for
more than 35 collaborative and service projects, and
the Leginon software, including the database, has been
distributed to about 30 laboratories outside Scripps
Research. We are also distributing ACE, a variety of
other software packages, and the novel grid substrates.
These efforts are complemented by training activities
that include small-group training, a biennial large
training course in electron cryomicroscopy, and small
workshops focused on various aspects of automation.
An additional project, sponsored by the National
Science Foundation, is the development of automated
data collection techniques for imaging serial sections 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 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
Cheng, A., Fellmann, D., Pulokas, J., Potter, C.S., Carragher, B. Does contamination
buildup limit throughput for automated cryoEM? J. Struct. Biol. 154:303, 2006.
Fellmann, D., Banez, R., Carragher, B., Potter, C.S. Temperature monitoring of an
EM environment. Microsc. Today 14:24, January 2006.
Stagg, S.M., Gurkan, C., Fowler, D.M., LaPointe, P., Foss, T.R., Potter, C.S., Carragher, B., Balch, W.E. Structure of the Sec13/31 COPII coat cage. Nature
439:234, 2006.
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29
Stagg, S.M., Lander, G.C., Pulokas, J., Fellmann, D., Cheng, A., Quispe, J.D.,
Mallick, S.P., Avila, R.M., Carragher, B., Potter, C.S. Automated cryoEM data
acquisition and analysis of 284,742 particles of GroEL. J. Struct. Biol., in press.
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. 151:41, 2005.
Regulation of
Cytomechanochemical Systems
G. Danuser, A. Bacconi, J. Dorn, K. Jaqaman, L. Ji,
J. Kunken, D. Loerke, M. Machacek, A. Matov, M. Sabouri,
K. Thompson, G. Yang
e study how force-generating molecular
machines are spatially and temporally regulated to mediate complex cell functions,
including migration, division, and intracellular transport
of organelles and vesicles. Specifically, we investigate
the relationships between assembly and contraction of
the actin cytoskeleton and the dynamic coupling of actin
filaments with other components of the cytoskeleton
during cell migration. We also study how assembly
and disassembly of microtubules and motor-driven sliding of microtubule bundles are orchestrated to symmetrically segregate replicated DNA from the dividing
mother cell into 2 daughter cells.
In the past year, we expanded our research program with 2 new, collaborative projects. In the first
project, we aim to establish the requirements for local
regulation of cortical actin mechanics during endocytosis. In the second, we are analyzing the modes of
interaction between microtubule plus end– and minus
end–directed motor families in vesicle transport along
neuronal axons.
To examine molecular systems, we develop computational models to predict the relationship between
the dynamics of molecular-level component processes
and cellular-level outputs. Subsequently, we validate
the models and estimate unknown parameters by fitting the parameters to measurements of cell dynamics. The challenges in such data-driven, multiscale
modeling are 2-fold: the precise and complete characterization of cell dynamics in space and time and the
implementation of numerical tools for fitting cellularlevel data to models with molecular resolution.
In our studies of cell migration, we made 2 major
advancements. First, in collaboration with C. WatermanStorer, Department of Cell Biology, we extended fluores-
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30 CELL BIOLOGY
2006
cent speckle microscopy to accomplish an integrated,
correlative multiparameter analysis of cytoskeleton
dynamics. We can now determine accurately how cell
movements depend on molecular processes such as
the assembly, disassembly, and transport of cytoskeleton components or adhesions, and we can use biosensor probes to visualize activation of signals. We recently
used our analysis framework in studies in which we dissected the roles of several signaling cascades in the
regulation of cell motility and the involvement of adhesion molecules in the transient, integrin-mediated coupling of the actin cytoskeleton to the extracellular matrix.
A second breakthrough was achieved in our effort
to reconstruct intracellular force distributions from light
microscopic measurements of cytoskeleton deformation.
We established a unique method to probe the relationship between spatially distributed force generation and
the resulting cell morphologic outputs, for example,
during cell migration. We will use this tool to dissect
the mechanism of force regulation by signals and identify feedback interactions between force transduction
and signal activation, which are a central element in
molecular systems control, not only in cell motility but
also in a broad set of other cell functions.
To study chromosome segregation, we use fluorescent speckle microscopy to analyze the dynamics of
microtubule scaffolds associated with the spindle apparatus in animal cells and 3-dimensional, high-resolution
light microscopy to analyze the dynamics of single chromosomes in yeast. The first approach should reveal how
microtubule assembly and disassembly across the spindle are coregulated with motor-mediated generation of
force. The second approach should allow us to identify
the functions of proteins in the kinetochore, a molecular complex that regulates the attachment of chromosomes to spindle microtubules.
We have developed fully automated, image-based
approaches of unprecedented sensitivity for investigations of phenotype microtubule and chromosome dynamics. In collaboration with E.D. Salmon, University of
North Carolina, T. Kapoor, Rockefeller University, and
P. Sorger, Massachusetts Institute of Technology, we are
using the data obtained to systematically characterize
the involvement of spindle- and kinetochore-associated
proteins in the regulation of chromosome motion
throughout the cell cycle.
PUBLICATIONS
Cameron, L.A., Yang, G., Cimini, D., Canman, J.C., Kisurina-Evgenieva, O.,
Khodjakov, A., Danuser, G., Salmon, E.D.S. Kinesin 5-independent poleward flux
of kinetochore microtubules in Ptk1 cells. J. Cell Biol. 173:173, 2006.
THE SCRIPPS RESEARCH INSTITUTE
Danuser, G. Coupling the dynamics of two actin networks: new views on the
mechanics of cell protrusion. Biochem. Soc. Trans. 33:1250, 2005.
Danuser, G., Waterman-Storer, C.M. Quantitative fluorescent speckle microscopy
of cytoskeleton dynamics. Ann. Rev. Biophys. Biomol. Struct. 35:361, 2006.
deRooij, J., Kerstens, A., Danuser, G., Schwartz, M.A., Waterman-Storer, C.M.
Integrin-dependent actomyosin contraction regulates epithelial cell scattering. J.
Cell Biol. 171:153, 2005.
Dorn, J.F., Jaqaman, K., Rines, D.R., Jelson, G.S., Sorger, P.K., Danuser, G. Yeast
kinetochore microtubule dynamics analyzed by high-resolution three-dimensional
microscopy. Biophys. J. 89:2834, 2005.
Ji, L., Danuser, G. Tracking quasi-stationary flow of weak fluorescent features by
adaptive multi-frame correlation. J. Microsc. 220:150, 2005.
Lussi, J.W., Tang, C., Kuenzi, P.A., Staufer, U., Csucs, G., Voros, J., Danuser, G.,
Hubbell, J.A., Textor, M. Selective molecular assembly patterning at the nanoscale:
a novel platform for producing protein patterns by electron-beam lithography on
SiO2/indium tin oxide-coated glass substrates. Nanotechnology 16:1781, 2005.
Machacek, M., Danuser, G. Morphodynamic profiling of protrusion phenotypes.
Biophys. J. 90:1439, 2006.
Meijering, E., Smal, I., Danuser, G. Tracking in molecular bioimaging. IEEE Signal
Process. Mag. 23:46, May 2006.
Ponti, A., Matov, A., Adams, M., Gupton, S., Waterman-Storer, C.M., Danuser, G.
Periodic patterns of actin turnover in lamellipodia and lamellae of migrating epithelial cells analyzed by quantitative fluorescent speckle microscopy. Biophys. J.
89:3456, 2005.
Shah, S., Yang, G., Danuser, G., Goldstein, L.S.B. Axonal transport: imaging and
modeling of a neuronal process. Springer Lecture Notes in Physics. In: The Nobel
Symposium. Springer, New York, in press.
Yang, G., Matov, A., Danuser, G. Reliable tracking of large scale dense antiparallel
particle motion for fluorescence live cell imaging. In: Proceedings of the 2005 IEEE
Computer Society Conference on Computer Vision and Pattern Recognition (CVPR’05)
Workshops. IEEE Computer Society, Washington, DC, 2005, Vol. 3, p. 138.
Synthetic Protein Chemistry
P.E. Dawson, A. Dirksen, F. Brunel, M. Churchill, F. Hansen,
E. Lempens, N. Metanis, T. Shekhter, T. Tiefenbrunn
e use chemical synthesis to design and engineer proteins with novel structures and functions. We continue to develop methods to
link fully unprotected peptides and carbohydrates via
native and nonnative linkages. During the past year,
we focused on synthesizing mimics of the HIV envelope
protein gp41, the oxidoreductase enzyme glutaredoxin,
a glycosylated form of monocyte chemoattractant protein-3, and carbohydrate-binding proteins. We are also
using these chemoselective ligation reactions to label
proteins such as thrombin and nanoparticle quantum
dots. Overall, our goal is to use synthetic chemistry to
understand the molecular basis of protein structure
and function.
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S E L E N O G L U TA R E D O X I N
Selenoenzymes have a central role in maintaining
cellular redox potential. These enzymes have selenylsul-
CELL BIOLOGY
2006
fide bonds in their active sites that catalyze the reduction
of peroxides, sulfoxides, and disulfides. The preparation
of enzymes containing selenocysteine is experimentally
challenging. As a result, little is known about the kinetic
role of selenols in enzyme active sites, and the redox
potential of a selenylsulfide or diselenide bond in a protein has not been experimentally determined.
To fully evaluate the effects of selenocysteine on
oxidoreductase redox potential and kinetics, we synthesized glutaredoxin 3 (Grx3; Fig. 1) and all 3 seleno-
F i g . 1 . Chemical synthesis of Grx3 by using folding to accelerate
the ligation reaction. Selective oxidation of the active-site disulfide
allowed alkylation of the cysteine residue at the ligation site.
cysteine variants of the enzyme’s conserved 11CXX14C
active site. Grx3, Grx3(C11U), and Grx3(C14U) had
redox potentials of –193, –259 and –273 mV, respectively. The position of redox equilibrium between
Grx3(C11U-C14U) (–308 mV) and thioredoxin (–270
mV) suggests a possible role for diselenide bonds in
biological systems. Kinetic analysis indicated that the
lower redox potentials of the selenocysteine variants is
due primarily to the greater nucleophilicity of the activesite selenium rather than to the role of the selenium
as either a leaving group or a “central atom” in the
exchange reaction. The 100- to 10,000-fold increase
THE SCRIPPS RESEARCH INSTITUTE
31
in the rate of thioredoxin reduction by the seleno-Grx3
analogs indicates that compared with their sulfide counterparts, oxidoreductases containing either selenylsulfide
or diselenide bonds can have physiologically compatible
redox potentials and enhanced reduction kinetics.
H I V VA C C I N E D E S I G N
The transmembrane protein gp41 is an attractive
target for the development of an HIV vaccine. We are
collaborating with M.B. Zwick and D.R. Burton, Department of Immunology, and I.A. Wilson, Department of
Molecular Biology, to design peptides that mimic the
gp41 epitopes of known neutralizing antibodies. The
membrane-proximal external region of gp41 contains
several neutralizing epitopes, including 4E10 and Z13e1.
On the basis of our previous work on 4E10, we designed
and synthesized peptides to map the Z13 epitope and
performed an alanine scan to identify key elements
within the sequence. Structural constraints are being
introduced into the peptides to obtain an antigen capable of eliciting both 4E10- and Z13e1-like antibodies.
To better mimic the molecular environment of
native gp41, we plan to introduce steric constraints
such as polyethylene glycol and carbohydrates. To
mimic the viral membrane, we have appended a
transmembrane helix and have incorporated the peptide into soluble lipid bilayers. Recently, neutralizing
antibodies to the N-heptad repeat of gp41 have been
discovered. We designed and synthesized 3-helix bundles to mimic this region of gp41, and we are using
them to identify these epitopes and map the key binding interactions.
PUBLICATIONS
Brunel, F.M., Zwick, M.B., Cardoso, R.M., Nelson, J.D., Wilson, I.A., Burton, D.R.,
Dawson, P.E. Structure-function analysis of the epitope for 4E10, a broadly neutralizing human immunodeficiency virus type 1 antibody. J. Virol. 80:1680, 2006.
Cremeens, M.E., Fujisaki, H., Zhang, Y., Zimmermann, J., Sagle, L.B., Matsuda,
S., Dawson, P.E., Straub, J.E., Romesberg, F.E. Efforts toward developing direct
probes of protein dynamics. J. Am. Chem. Soc. 128:6028, 2006.
Delehanty, J.B., Medintz, I.L., Pons, T., Brunel, F.M., Dawson, P.E., Mattoussi, H.
Self-assembled quantum dot-peptide bioconjugates for selective intracellular delivery. Bioconjug. Chem. 17:920, 2006.
Medintz, I.L., Clapp, A.R., Brunel, F.M., Tiefenbrunn, T., Uyeda, H.T., Chang,
E.L., Deschamps, J.R., Dawson, P.E., Mattoussi, H. Proteolytic activity monitored
by fluorescence resonance energy transfer through quantum-dot-peptide conjugates. Nat. Mater. 5:581, 2006.
Sagle, L.B., Zimmermann, J., Matsuda, S., Dawson, P.E., Romesberg, F.E. Redoxcoupled dynamics and folding in cytochrome c. J. Am. Chem. Soc. 128:7909. 2006.
Yamamoto, N., Takayanagi, A., Sakakibara, T., Dawson, P.E., Kajihara, Y. Highly
efficient synthesis of sialylglycopeptides overcoming unexpected aspartimide formation during activation of Fmoc-Asn(undecadisialyloligosaccharide)-OH. Tetrahedron
Lett. 47:1341, 2006.
32 CELL BIOLOGY
2006
Regulation of Actin Dynamics in
Morphogenesis and Development
V.M. Fowler, T. Fath, R.S. Fischer, C. McKeown, J. Moyer,
R. Nowak, J. Palomique, K. Weber
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 architecture and actin-based morphogenetic processes in development. We focus on the
tropomodulin family of proteins that cap the pointed
ends of actin filaments. Tropomodulins are a conserved
family of proteins of about 40 kD that bind to tropomyosin and actin. The tropomodulins are expressed in
a tissue-specific and developmentally regulated fashion in vertebrates, flies, and worms.
In vertebrates, the tropomodulin 1 isoform is associated with stable architectural arrays of actin filaments
such as thin filaments in striated muscle myofibrils and
actin filaments in the membrane skeleton of red blood
cells (RBCs) and on the lateral membranes of the fiber
cells of the eye lens. Previous research indicated that
tropomodulin 1 regulates the dynamics of actin pointed
ends and thus the length and stability of thin filaments
in myofibrils of cultured cardiac muscle cells. Tropomodulin 3, the isoform in the cytoplasm, is associated
with dynamic actin filaments in the lamellipodia of
crawling endothelial cells, where it is a negative regulator of cell migration.
Our goal is to tie the molecular and cellular regulation of the dynamics of actin pointed ends by tropomodulins to the in vivo functions of the proteins in
actin-based morphogenetic processes in development.
We use mouse genetic models to study the function of
tropomodulins in myofibril assembly and cardiac development, the biogenesis and stability of the RBC membrane
skeleton, and the morphogenesis and transparency of
fiber cells in the eye lens.
The structure and function of tropomodulins is best
understood for tropomodulin 1, which consists of 2
domains: an unstructured, flexible N-terminal domain
and a compact, folded C-terminal domain composed
of 5 leucine-rich repeats. The N-terminal domain binds
tropomyosin and is regulated by tropomyosin to cap
tropomyosin-actin pointed ends with nanomolar affinity.
The C-terminal domain caps actin pointed ends with
submicromolar affinity and is unaffected by tropomyosin.
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THE SCRIPPS RESEARCH INSTITUTE
Despite the high level of sequence conservation
(~70%) among vertebrate tropomodulins, comparisons of
their actin-binding activities reveals that tropomodulin 3,
but not tropomodulin 1, binds actin monomers and
nucleates actin filament assembly in addition to capping
pointed ends. Tropomodulin 3 can be chemically crosslinked to actin in a 1:1 complex, providing a tool to
identify the amino acids at the tropomodulin 3–actin
binding interface. Initial results from tryptic digestion
and mass spectrometry indicate that tropomodulin 3
interacts with actin monomers via a unique interface on
the actin and on the tropomodulin 3. Site-directed mutagenesis plus structural and functional interaction studies
are in progress to further define the tropomodulin
3–actin binding interface and to develop tropomodulin
mutants for studies of cellular functions in vivo.
To investigate the in vivo function of tropomodulin 1
in myofibril assembly and cardiac development, we are
using 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 investigate the primary defect in myofibril
assembly, we examined nascent myofibrils on myocyte
membranes in embryos at 4–5 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.
More recently, we found that cardiac development fails
specifically at the stage of looping morphogenesis, at
an earlier stage than that observed in all other mice
that lack genes for contractile proteins. We are testing
the hypotheses that defective myofibril assembly in
absence of tropomodulin 1 may lead directly or indirectly to aberrant cell-cell contacts or polarity, and/or
to defective cell proliferation, leading to failure of looping morphogenesis.
To investigate the consequences in RBCs of deleting the gene for tropomodulin 1, we prevented death
in the embryos of the mutant mice by expressing a
tropomodulin 1 transgene solely in the heart. The result
CELL BIOLOGY
2006
was viable mice with no tropomodulin 1 in their RBCs.
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 fragile
than normal RBCs. Western blotting indicated increased
levels of tropomodulin 3 in the tropomodulin 1–deficient RBCs.
Using these tropomodulin 1–deficient RBCs, we
can test the effects of tropomodulin 3 on the length
and dynamics of actin filaments and the consequences
for stability of the membrane skeleton, RBC survival,
and function in vivo. Mice with the transgene also provide an opportunity to examine the function of tropomodulin 1 in vivo in other tropomodulin 1–expressing
cells and tissues such as the eye lens, neurons, and
kidney. We are also producing mice that lack the gene
for tropomodulin 3 to obtain mice with RBCs deficient
in both tropomodulin 1 and tropomodulin 3 to assess
the consequences of complete lack of tropomodulin on
RBC structure and function.
PUBLICATIONS
Fowler, V.M., McKeown, C.R., Fischer, R.S. Nebulin: does it measure up as a
ruler? Curr. Biol. 16:R18, 2006.
Angiogenesis-Dependent
Disease and Membrane
Protein Topogenesis
M. Friedlander, E. Aguilar, E. Banin, F. Barnett, R. Bautchek,
M. Dorrell, M. El-Kalay, S.F. Friedlander, S. Hanekamp,
R. Jacobson, A. Johnson, V. Machetti, M. Ritter, L. Scheppke,
J. Trombley, H. Uusitalo-Jarvinen, V. Marchetti, W. Ruf
ANGIOGENESIS-DEPENDENT DISEASE
ost diseases that cause catastrophic loss of
vision do so as a result of abnormal growth
of blood vessels. Similarly, tumors depend on
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.
M
THE SCRIPPS RESEARCH INSTITUTE
33
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 this process.
In collaboration with P.R. Schimmel, Department of
Molecular Biology, we found that fragments of tryptophan
tRNA synthetase are potent angiostatics that significantly reduce retinal neovascularization. The synthetase
fragments are also angiostatic in vivo when delivered
by a cell-based method.
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 therapies for diseases of retinal ischemia, we explored the potential usefulness of
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 both lineage-negative hematopoietic progenitors and CD44 hi -expressing myeloid
progenitors specifically target activated retinal astrocytes, incorporate into and around new vessels, and,
in a mouse model of retinal degeneration, rescue and
stabilize a degenerating retinal vasculature.
We also showed that both types of stem cells have
a profound neurotrophic effect when injected into 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 rescue retinal vasculature subject to hypoxic
stress and may be useful in the treatment of ischemic
retinal abnormalities such as diabetic retinopathy and
retinopathy of prematurity. The mechanism of rescue
is not clear, but it is related to high levels of heat-shock
proteins found in these populations of cells. We also
defined a role for CD44 hi-derived microglia in facilitating vascular recovery in models of retinal ischemia.
34 CELL BIOLOGY
2006
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 a 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 are 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 eukaryotic 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 mutated exchanger is expressed
and mice that lack the gene for the exchanger.
THE SCRIPPS RESEARCH INSTITUTE
PUBLICATIONS
Banin, E., Dorrell, M.I., Aguilar, E., Ritter, M.R., Aderman, C.M., Smith, A.C.H.,
Friedlander, J., Friedlander, M. T2-TrpRS inhibits preretinal neovascularization and
enhances physiological vascular regrowth in OIR as assessed by a new method of
quantification. Invest. Ophthalmol. Vis. Sci. 47:2125, 2006.
Dorrell, M., Uusitalo-Jarvinen, H., Aguilar, E., Friedlander, M. Ocular angiogenesis; basic mechanisms and therapeutic advances. Surv. Ophthalmol., in press.
Dorrell, M.I., Friedlander, M. Mechanisms of endothelial cell guidance and vascular
patterning in the developing mouse retina. Prog. Retin. Eye Res. 25:277, 2006.
Friedlander, M. Stem cells and retinal disease. In: Retina, 4th ed. Ryan, S.J.
(Editor-in-Chief). St. Louis, Mosby, 2006, Vol. 1, p 23.*
Friedlander, S.F., Ritter, M.R., Friedlander, M. Recent progress in our understanding of
the pathogenesis of infantile hemangiomas. Lymphat. Res. Biol. 3:219, 2005.
Jin, H., Aiyer, A., Su, J., Borgstrom, P., Stupack, D., Friedlander, M., Varner, J. A
homing mechanism for bone marrow-derived progenitor cell recruitment to the neovasculature. J. Clin. Invest. 116:652, 2006.
Ritter, M., Aguilar, E., Banin, E., Scheppke, L., Uusitalo-Jarvinen, H., Friedlander, M.
Three-dimensional in vivo imaging of the mouse ocular vasculature during development and disease. Invest. Ophthalmol. Vis. Sci. 46:3021, 2005.
Ritter, M., Banin, E., Aguilar, E.A., Dorrell, M.I., Moreno, S.K., Friedlander, M.
Myeloid progenitors differentiate into microglia and promote vascular repair in a
model of ischemic retinopathy. J. Clin. Invest., in press.
Ritter, M., Friedlander, M. Integrins in ocular angiogenesis. In: Ocular Angiogenesis:
Diseases, Mechanisms, and Therapeutics. Tobran-Tink, J., Barnstable, C. (Eds.).
Humana Press. Totowa, NJ, 2006, p. 279.
Ritter, M., Reinisch, J., Friedlander, S.F., Friedlander, M. Myeloid cells in infantile
hemangioma. Am. J. Pathol. 168:621, 2006.
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, E. Choi, K. Datta, T. Guan,
M. Huber, 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
CELL BIOLOGY
2006
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 complexes are translocated through the NPC by receptor
binding 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 small-molecule inhibitors.
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 collaboration with a research team at Scripps Research to iden-
THE SCRIPPS RESEARCH INSTITUTE
35
tify small-molecule inhibitors of Rev transport and function; the goal is to find compounds for developing new
drugs to inhibit HIV replication in humans.
NUCLEAR LAMINA AND HIGHER LEVEL NUCLEAR
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 functions, as indicated by the recent findings that
more than 15 inherited diseases in humans, including
several 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, cell signaling,
and gene expression. Until recently, only about 12
transmembrane proteins specific to the nuclear envelope had been identified.
To determine the full complement of proteins in the
nuclear envelope, we carried out a proteomics analysis
of the nuclear envelope of rodent liver cells in collaboration with J.R. Yates, Department of Cell Biology. We
identified 67 novel putative nuclear envelope transmembrane proteins. Almost all members of this group
that we have examined are authentic components of
the nuclear envelope.
Currently, we are analyzing nuclear envelope transmembrane proteins in muscle, because this is the tissue most sensitive to disruption of lamina function by
disease-causing mutations. Using transcriptional profiling of cultured myoblasts, we found that the genes
for 6 of the nuclear envelope transmembrane proteins
are strongly upregulated in myoblast differentiation. The
genes also are highly expressed in muscle in adults,
consistent with a role of the genes in muscle differentiation and/or maintenance. We have confirmed that
these nuclear envelope transmembrane proteins are
authentic nuclear envelope proteins; we are using gene
silencing approaches to analyze their requirement in
muscle cell function. Our goals are to identify novel
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 directs nuclear structure and functions.
PUBLICATIONS
Ospina, J.K., Gonsalvez, G.B., Bednenko, J., Darzynkiewicz, E., Gerace, L., Matera,
A.G. Cross-talk between snurportin1 subdomains. Mol. Biol. Cell 16:4660, 2005.
Schirmer, E.C., Gerace, L. The nuclear membrane proteome: extending the envelope. Trends Biochem. Sci. 30:551, 2005.
36 CELL BIOLOGY
2006
Wodrich, H., Cassany, A., D’Angelo, M.A., Guan, T., Nemerow, G., Gerace, L.
Adenovirus core protein pVII is translocated into the nucleus by multiple import
receptor pathways. J. Virol. 80:9608, 2006.
Organization and Function of
the Neuronal Cytoskeleton
S. Halpain, J. Braga, B. Calabrese, L. Dehmelt, E. Hwang,
J. Koehler, 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. 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
used to uncover structure-function relationships for cytoskeletal proteins and the consequences of the dysfunction of the proteins. The results of these projects have
contributed to our understanding of molecular events
in normal brain development and in regeneration of
neuronal structure after injury and disease.
D
M I C R O T U B U L E - A S S O C I AT E D P R O T E I N S
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 fluorescence-based
time-lapse imaging and high-resolution confocal microscopy, we tracked the behaviors of microtubules 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 microtubules toward the cell periphery. This dynein-dependent
activity provides a key force that pushes the cell membrane outward during neurite initiation. Currently, we
are using proteomic approaches and high-content, microscopy-based screening technology to identify other cytoskeletal proteins and signal transduction pathways
crucial to the initiation of neurites.
DENDRITIC SPINES
A second project concerns the regulation of dendritic spines, specialized cellular protrusions that form
THE SCRIPPS RESEARCH INSTITUTE
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 also discovered that the membrane-associated
protein myristoylated alanine-rich C kinase substrate
(MARCKS), a major substrate of the signaling enzyme
protein kinase C, is a key molecule in the regulation
of spine shape and stability. Alterations in MARCKS
are implicated in Alzheimer’s disease and in major
depression. We have extensively characterized the
effects of either depleting MARCKS or overexpressing
various mutant forms of MARCKS in hippocampal neurons. The results revealed several key insights into
MARCKS function and novel forms of synaptic plasticity in young neurons.
PUBLICATIONS
Calabrese, B., Wilson, M.S., Halpain, S. Development and regulation of dendritic
spine synapses. Physiology (Bethesda) 21:38 2006.
Halpain, S., Dehmelt, L. MAP1 family proteins. Genome Biol., in press.
Function of Nuclear Receptors
in Stress and Mitochondrial
Homeostasis
A. Kralli, J. Cardenas, B. Hazen, M.B. Hock, F. Jaramillo,
C. Tiraby-Nguyen, J. Villena
W
e are interested in the molecular mechanisms
that relay metabolic stress signals to a network of transcriptional regulators and the
CELL BIOLOGY
2006
ensuing transcriptional outputs that mediate adaptive
metabolic responses to the stress signals. In particular,
we focus on the coactivators peroxisome proliferator–
activated receptor γ coactivator-1α (PGC-1α) and
PGC-1β and the orphan nuclear receptors of the estrogen-related receptor (ERR) subfamily, which control
mitochondrial biogenesis and energy homeostasis. Our
goals are to elucidate the biology of this specific transcriptional network, understand how deregulation of the
network leads to disease, and ultimately identify the
components of the network that are most suitable for
drug intervention to counteract metabolic disease.
R E G U L AT I O N O F T H E P G C - 1 / E R R N E T W O R K
Levels of PGC-1α and PGC-1β change in response
to signals that relay metabolic needs. The coactivators
then relay such signals, via interactions with ERRs and
other factors, to regulate the expression of specific target genes. We are interested in the mechanisms that
regulate PGC-1s at the postranslational level via covalent modifications of or interaction with other proteins,
and thereby control the properties of the PGC-1/ERR
network. This past year, in collaboration with M. Stallcup, University of Southern California, we showed that
PGC-1α is methylated by the protein arginine methyltransferase 1 and that this methylation increases the
activity of PGC-1α. As a result, protein arginine methyltransferase 1 and PGC-1α act cooperatively to activate
ERRα and to induce ERRα target genes with roles in
mitochondrial biogenesis. Currently, we are investigating the molecular mechanisms by which methylation
regulates PGC-1α activity.
ROLE OF ERRα IN MITOCHONDRIAL FUNCTION
Our previous studies suggested that the effects of
PGC-1α and PGC-1β on mitochondrial biogenesis are
mediated primarily by ERRα. To determine the physiologic relevance of ERRα for mitochondrial function, we
are studying brown adipose tissue. This tissue is rich
in mitochondria, it expresses high levels of ERRα, and its
function is readily assayed in the context of the whole
organism. Compared with wild-type mice, mice lacking ERRα have a decrease in the expression of genes
associated with oxidative phosphorylation, lipid oxidation, and the tricarboxylic acid cycle. Morphologic analysis by electron microscopy revealed that brown adipose
tissue from mice lacking ERRα has reduced mitochondrial density and increased lipid accumulation. When
exposed to cold, mice lacking ERRα had impaired adaptive thermogenesis, despite normal induction of the
uncoupling protein UCP-1. Adipocytes isolated from
THE SCRIPPS RESEARCH INSTITUTE
37
the mice had reduced oxidative capacity, suggesting that
the effect of ERRα on mitochondrial function is cell
autonomous. This research establishes for the first time
that ERRα is an important component of the regulatory
network that supports high levels of mitochondrial biogenesis and oxidative metabolism in vivo.
Interestingly, whereas ERRα is an important downstream effector of PGC-1α in the stimulation of mitochondrial biogenesis and oxidative capacity, it counteracts
the ability of PGC-1α to induce gluconeogenic genes
in hepatocytes. Notably, it is required for proper suppression of gluconeogenic enzymes in the liver of fed
mice. Mitochondrial dysfunction has been implicated
as an underlying cause of insulin resistance and type
2 diabetes. Moreover, derepression of hepatic gluconeogenesis contributes to high glucose levels in diabetes.
The opposing effects of ERRα on genes important for
mitochondrial oxidative capacity vs genes important in
gluconeogenesis suggest that enhancing ERRα activity
could have beneficial effects on glucose metabolism
in patients with diabetes via 2 distinct mechanisms:
increasing mitochondrial oxidative capacity in peripheral
tissues and suppressing inappropriate glucose production in the liver.
PUBLICATIONS
Cartoni, R., Léger, B., Hock, M.B., Praz, M., Crettenand, A., Pich, S., Ziltener, J.,
Luthi, F., Dériaz, O., Zorzano, A., Gobelet, C., Kralli, A., Russell A.P. Mitofusins
1/2 and ERRα expression are increased in human skeletal muscle after physical
exercise. J. Physiol. 567(Pt. 1):349, 2005.
Herzog, B., Cardenas, J., Hall, R.K., Villena, J.A., Budge, P.J., Giguère, V.,
Granner, D.K., Kralli, A. Estrogen-related receptor α is a repressor of phosphoenolpyruvate carboxykinase gene transcription. J. Biol. Chem. 281:99, 2006.
Teyssier, C., Ma, H., Emter, R., Kralli, A., Stallcup, M.R. Activation of nuclear
receptor coactivator PGC-1α by arginine methylation. Genes Dev. 19:1466, 2005.
Structural and Functional
Proteomics
P. Kuhn, J. Nieva,* E. Abola, A. Brooun, R. Bruce, C. Chen,
J. Chrencik, P. Clark, S. Coon, J. Dupuy, S. Foster, J. Joseph,
L. Kim, A. Kolatkar, M. Kraus, N. Lazarus, M. Leach,
D. Marrinucci, E. Rayon, K. Saikatendu, V. Subramanian,
A. Tang, M. Yadav
* Department of Molecular and Experimental Medicine, Scripps Research
uring the past 3 years, we have focused on 4
major research programs: detection of cancer
cells in circulation, structural proteomics of
cancer drug targets, structural and functional proteomics
D
38 CELL BIOLOGY
2006
THE SCRIPPS RESEARCH INSTITUTE
of the coronavirus that causes severe acute respiratory syndrome (SARS-CoV), and development of novel
approaches in miniaturization, integration, and automation to lower the overall cost of moving from the synthesis of genes to examination of the structures encoded
by the genes. These programs involve collaborations
with R.C. Stevens, Department of Molecular Biology;
other researchers at Scripps Research; and scientists
at the Palo Alto Research Center and Lyncean Technologies in Palo Alto, California, the Novartis Institutes for
Biomedical Research, Cambridge, Massachusetts, and
deCODE biostructures, Bainbridge Island, Washington.
D E T E C T I N G R A R E C E L L S I N T H E C I R C U L AT I O N
Many clinically important cells in blood occur at frequencies of less than 1 cell per 1 million cells. Detecting
and characterizing these rare cells requires the development of new technology that operates with exceptional
specificity. Scientists at the Scripps-PARC Institute for
Advanced Biomedical Sciences have developed an
instrument, based on fiber-optic array scanning technology (FAST), that provides rapid and accurate identification of rare cells in the circulation in humans. Potential
clinical applications include finding circulating tumor
cells, circulating endothelial cells, and circulating fetal
cells in the maternal circulation.
Malignant cells from solid tumors begin to circulate
at the earliest stages in cancer formation. Because the
circulating cells are quite rare, technology to detect and
characterize them can be valuable in screening for cancer and in guiding individualized cancer therapy. During
the past year, we used FAST to accurately enumerate
circulating cancer cells in blood samples obtained from
patients with metastatic breast or lung cancer (Fig. 1).
In collaboration with J. Kroener, Scripps Clinic, we found
that accurate detection of circulating tumor cells by
FAST can be used to predict prognosis in patients with
metastatic breast cancer. We are developing molecular
tools to characterize these rare tumor cells after they
have been detected. Our goal is to determine their tissue of origin, their potential anatomic destination, and
their metastatic potential.
STRUCTURAL PROTEOMICS AND DRUG DISCOVERY
We are collaborating with the Novartis Institutes for
Biomedical Research, Cambridge, Massachusetts, in
a project to understand and modulate therapeutically
relevant protein-protein interactions. Analysis of an
initial subset of 5 therapeutically relevant protein-protein interaction pairs will provide the guiding principles
for the selection of a feasible set of drug targets to be
examined. We used a high-throughput approach to
F i g . 1 . An image of a single circulating tumor cell from a
patient with breast cancer identified from a background of 50 million peripheral blood mononuclear cells by using a FAST cytometer.
rapidly identify ideal constructs for expression, crystallization, and biophysical studies. The results are providing general insights into the range of different physical
interactions for a given protein-protein interaction.
The Eph-ephrin interaction is particularly interesting, because it has been implicated in cancer progression and in pathologic forms of angiogenesis. We have
solved the crystal structure of the ligand-binding domain
of EphB4 in complex with an antagonistic peptide that
inhibits ephrin binding and has antitumorigenic properties in vivo. We have also solved the cocrystal structure of an EphB4–ephrin-B2 complex. Structural and
biophysical analysis are providing the first insights into
how we can modulate pathways involved in tumorigenesis and angiogenesis that rely on EphB4–ephrin-B2
signaling.These results will deepen our understanding
of the basic biology behind protein-protein interactions
and aid in the development of novel therapeutic
approaches for modulating protein-protein interactions.
STRUCTURAL AND FUNCTIONAL PROTEOMICS
A N A LY S I S O F S A R S - C o V
We are generating a structure-function-interaction
map of the SARS-CoV proteome and its interactions
with the host cell to provide a comprehensive set of
targets for rational, structure-based drug and vaccine
design. We use bioinformatics, structural biology, genetic
methods, and functional assays.
CELL BIOLOGY
2006
So far, we have determined the structures of 7
SARS-CoV proteins. We used crystallography for 3
nonstructural proteins, nuclear magnetic resonance for
3 other nonstructural proteins, and electron cryomicroscopy for a large surface glycoprotein spike. These
studies are providing important information on the formation of the replicase complex, transcription, and
RNA-processing events unique to the SARS-CoV life
cycle. A total of 7 other proteins have been successfully expressed in soluble, folded forms, and their
structures are being determined. Together, the results
from these studies should enable identification of
compounds that may be effective agents for treatment
of infections caused by SARS-CoV.
A C C E L E R AT E D T E C H N O L O G I E S C E N T E R F O R G E N E
TO 3D STRUCTURE
Scientists at the Accelerated Technologies Center
for Gene to 3D Structure are simultaneously developing, operating, and deploying 3 key technologies to
improve the costs of using x-ray crystallography to
determine the structure of experimental proteins. The
first technology, computer-aided design of expressionoptimized synthetic genes and protein constructs for
crystallography, improves the success rate for gene
isolation and allows researchers to engineer the gene
sequence of interest to be optimized for protein production in a desired heterologous expression system. The
second technology, microfluidic plug-based nanovolume
protein crystallization in microcapillaries for in situ xray screening and data collection, is economical and
greatly broadens the range of useful quantities of proteins required for crystal growth. This technology also
allows for fine control over chemical gradients in crystal
growth, thereby expanding the coverage of crystallization space without consuming large quantities of protein.
The third technology, the compact light source, is
a tunable laboratory x-ray source with peak intensity
at x-ray wavelengths that span selenium anomalous
absorbance. Having a tunable laboratory x-ray source
in the same facility where a crystal inventory is held
will greatly enhance the ability to efficiently solve new
protein crystal structures.
The future integration of such technologies in a single facility at Scripps Research will enable efficient gene
design for improved protein production, small-volume
crystallization with in situ x-ray diffraction screening,
and tunable x-ray data collection in a single laboratory.
Our target focus is the structural elucidation of human
proteins of biomedical relevance (or eukaryotic homo-
THE SCRIPPS RESEARCH INSTITUTE
39
logs thereof) that belong to superfamilies of integral
membrane proteins and transcription factors.
PUBLICATIONS
Chrencik, J.E., Brooun, A., Kraus, M.L., Recht, M.I., Kolatkar, A.R., Han, G.W.,
Seifert, J.M., Widmer, H., Auer, M., Kuhn, P. Structural and biophysical characterization of the EphB4–ephrinB2 protein-protein interaction and receptor specificity.
J. Biol. Chem. 281:28185, 2006.
Chrencik, J.E., Brooun, A., Recht, M.I., Kraus, M.L., Koolpe, M., Kolatkar, A.R.,
Bruce, R.H., Martiny-Baron, G., Widmer, H., Pasquale, E.B., Kuhn, P. Structure
and thermodynamic characterization of the EphB4/ephrin-B2 antagonist peptide
complex reveals the determinants for receptor specificity. Structure 14:321, 2006.
Hsieh, H.B., Marrinucci, D., Bethel, K., Curry, D.N., Humphrey, M., Krivacic,
R.T., Kroener, J., Kroener, L., Ladanyi, A., Lazarus, N., Kuhn, P., Bruce, R.H.,
Nieva, J. High speed detection of circulating tumor cells. Biosens. Bioelectron.
21:1893, 2006.
Joseph, J.S., Saikatendu, K.S., Subramanian, V., Neuman, B.W., Brooun, A.,
Griffith, M., Moy, K., Yadav, M.K., Velasquez, J., Buchmeier, M.J., Stevens, R.C.,
Kuhn, P. Crystal structure of nonstructural protein 10 from the severe acute respiratory syndrome coronavirus reveals a novel fold with two zinc-binding motifs. J.
Virol. 80:7894, 2006.
Marrinucci, D.C., Bethel, K., Bruce, R.H., Curry, D.N., Hsieh, B., Humphrey, M.,
Krivacic, R.T., Kroener, J., Kroener, L., Ladanyi, A., Lazarus, N.H., Nieva, J.,
Kuhn, P. Case study of the morphologic variation of circulating tumor cells. Hum.
Pathol., in press.
Neuman, B.W., Stein, D.A., Kroeker, A.D., Chruchill, M.J., Kim, A.M., Kuhn, P.,
Dawson, P., Moulton, H.M., Bestwick, R.K., Iverson, P.L., Buchmeier, M.J. Inhibition, escape, and attenuated growth of severe acute respiratory syndrome coronavirus treated with antisense morpholino oligomers. J. Virol. 79:9665, 2005.
Peti, W., Johnson, M.A., Herrmann, T., Neuman, B.W., Buchmeier, M.J., Nelson,
M., Joseph, J., Page, R., Stevens, R.C., Kuhn, P., Wüthrich, K. Structural genomics of the severe acute respiratory syndrome coronavirus: nuclear magnetic resonance structure of the protein nsP7. J. Virol. 79:12905, 2005.
Ratia, K., Saikatendu, K.S., Santarsiero, B.D., Barretto, N., Baker, S.C., Stevens,
R.C., Mesecar, A.D. Severe acute respiratory syndrome coronavirus papain-like
protease: structure of a viral deubiquitinating enzyme. Proc. Natl. Acad. Sci. U. S.
A. 103:5717, 2006.
Saikatendu, K.S., Joseph, J.S., Subramanian, V., Clayton, T., Griffith, M., Moy, K.,
Velasquez, J., Neuman, B.W., Buchmeier, M.J., Stevens, R.C., Kuhn, P. Structural
basis of severe acute respiratory syndrome coronavirus ADP-ribose-1′′-phosphate
dephosphorylation by a conserved domain of nsP3. Structure 13:1665, 2005.
Yadav, M.K., Gerdts, C.J., Sanishvili, R., Smith, W.W., Roach L.S., Roach, R.F.,
Ismagilov, F., Kuhn, P., Stevens, R.C. In situ data collection and structure refinement
from microcapillary protein crystallization. J. Appl. Crystallogr. 38:900, 2005.
Vascular Imaging and Tumor
Targeting With Virus-Based
Nanoparticles
M. Manchester, G. Destito, M. Estrada, M.J. Gonzalez,
K. Koudelka, E. Powell, C. Rae, P. Singh, D. Thomas
urrent treatment of cancer typically involves
chemotherapies that have severe adverse effects.
The requirement that patients must withstand the
toxic effects of treatment often limits the effectiveness
of the therapy. Further, many promising anticancer
C
40 CELL BIOLOGY
2006
compounds that are highly effective in vitro are too
toxic to be used in vivo.
The ability to specifically target therapies to the site
of a developing tumor while avoiding healthy tissue is
an important goal for cancer research. Similarly, a
tremendous need exists to identify, image, and monitor
tumors, particularly at early stages and during treatment.
Recently, “smart” nanoparticles, which combine these
multiple targeting, imaging, and drug delivery functions,
have been developed. Therapies based on nanoparticles
have tremendous potential to increase the sensitivity
and specificity of diagnostic imaging and treatment.
Many different classes of nanoparticles are currently in
development, including dendrimers, liposomes, paramagnetic nanoparticles, and quantum dots.
We focus on virus-based nanoparticles as platforms
for the development of tissue-specific targeting and
imaging agents in vivo. Two of the viruses we study are
cowpea mosaic virus (CPMV) and canine parvovirus.
CPMV AS A NOVEL BIOMOLECULAR SENSOR FOR
VA S C U L A R I M A G I N G
CPMV is an icosahedral, 31-nm particle that is
produced easily and inexpensively in black-eyed pea
plants. In contrast to the structure of most other nanomaterials, the structure of the CPMV capsid 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. CPMV is bioavailable and nontoxic, and the capsids are highly stable
to temperature, pH, and the conditions required for
chemical reactions.
By conjugation to surface lysine residues, CPMV
can be labeled with fluorophores at high densities,
resulting in an extremely bright, nontoxic material that
is an outstanding tool for imaging vasculature in live
animals. Working with H. Stuhlmann, Department of
Cell Biology, we showed that CPMV can be used to
effectively image the complete vasculature in the
embryos of several species and that it is superior to
other imaging particles such as lectins, fluorescent
dextrans, or polystyrene microspheres.
CPMV particles have also been highly useful in highlighting angiogenesis in developing tumors. Uptake of
particles into endothelial cells occurs, yielding a bright
imaging signal that can be used to differentiate between
arterial and venous vessels. Such endothelial uptake is
mediated by a cellular membrane protein, and uptake
can be observed in the endothelium of a variety of
THE SCRIPPS RESEARCH INSTITUTE
healthy tissues as well as in disease states such as
atherosclerosis.
T U M O R TA R G E T I N G W I T H V I R U S - B A S E D
NANOPAR TICLES
We have designed virus-based nanoparticles that
can specifically target tumors in vivo. In collaboration
with M.G. Finn, Department of Chemistry, we bioconjugated CPMV to tumor ligands such as transferrin and
folic acid, whose receptors are upregulated on metabolically active tumor cells. The targeted particles had
a high degree of specificity for the tumor ligand and for
uptake by tumor cells. In a separate study, we showed
that canine parvovirus, which has a natural affinity for
the transferrin receptor and thus for tumor cells, could
specifically deliver small molecules to tumor cells.
These studies will allow the further design of antitumor agents that can provide localized, highly specific
imaging and therapy in vivo. Use of virus-based nanoparticles may help us visualize and eliminate small tumors
before the tumors have a chance to metastasize. In
addition, the ability of the particles to focus toxic effects
to the site of the malignant cells, thereby expanding
the range of effective therapies that can be used in
vivo, holds great promise for reducing cancer-related
morbidity and mortality.
PUBLICATIONS
Hsu, C., Singh, P., Ochoa, W., Manayani, D.J., Manchester, M., Schneemann, A.,
Reddy, V.S. Characterization of polymorphism displayed by the coat protein
mutants of tomato bushy stunt virus. Virology 349:222, 2006.
Lewis, J.D., Destito, G., Zijlstra, A., Gonzalez, M.J., Quigley, J.P., Manchester,
M., Stuhlmann, H. Viral nanoparticles as tools for intravital vascular imaging. Nat.
Med. 12:354, 2006.
Manchester, M., Singh, P. Virus-based nanoparticles (VNPs): platform technologies
for diagnostic imaging. Adv. Drug Dev. Rev., in press.
Rae, C.S., Khor, I.W., Wang, Q., Destito, G., Gonzalez, M.J., Singh, P., Thomas,
D.M., Estrada, M.N., Powell, E., Finn, M.G., Manchester, M. Systemic trafficking
of plant virus nanoparticles in mice via the oral route. Virology 343:224, 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. 192:1047, 2005.
Sen Gupta, S., Kuzelka, J., Singh, P., Lewis, W.G., Manchester, M., Finn, M.G.
Accelerated bioorthogonal conjugation: a practical method for the ligation of diverse
functional molecules to a polyvalent virus scaffold. Bioconjug. Chem. 16:1572,
2005.
Singh, P., Destito, G., Schneemann, A., Manchester, M. Canine parvovirus-like
particles, a novel nanomaterial for tumor targeting. J. Nanobiotechnol. 4:2, 2006.
Singh, P., Gonzalez, M.J., Manchester, M. Viruses and their uses in nanotechnology. Drug Dev. Res., in press.
CELL BIOLOGY
2006
Translational Regulation in
Chloroplasts and Expression of
Human Monoclonal Antibodies
in Eukaryotic Algae
THE SCRIPPS RESEARCH INSTITUTE
41
Compared with bacterial 70S ribosomes, the chloroplast ribosome has unique structural domains located
primarily on the small ribosomal subunit (Fig. 1). This
S.P. Mayfield, D. Barnes, A. Manuell, M. Beligni,
J. Marin-Navarro, M. Muto, M. Tran, D. Siefker, R. Henry
ene expression in chloroplasts is primarily controlled during the translation of plastid mRNAs
into proteins, and understanding how this process is regulated is key to understanding plant development and function. Controlling chloroplast translation
is also an essential component in optimizing the production of human therapeutic proteins in algae.
Using proteomics and bioinformatics analyses, we
identified the set of proteins that function in chloroplast
translation. These studies indicated that the core translational apparatus of chloroplasts is highly related to
that of bacteria but that chloroplasts have incorporated
additional protein components that allow more complex regulatory mechanisms. Some of these additional
components are ribosomal proteins; others are protein
translation factors. Chloroplast mRNAs also contain a
number of RNA regulatory elements that are not found
in bacteria, as well as conserved RNA elements, such
as ribosome-binding sequences, but even these conserved elements appear to function in chloroplast translation differently than in bacterial translation. The unique
components of chloroplast translation provide the opportunity for regulation of chloroplast translation, for example in response to exposure to light, that cannot be
achieved in simpler bacterial systems.
To better understand translation in plants, we are
examining the structure of both the chloroplast and
cytoplasmic ribosomes from Chlamydomonas reinhardtii,
a unicellular photosynthetic eukaryote. Using electron
cryomicroscopy and single-particle reconstruction, we
determined the structure of the C reinhardtii cytoplasmic 80S ribosome and found that it is nearly identical
to ribosomes from animals, including the human 80S
ribosome. Parallel proteomics analysis supported this
finding. We also determined the structure of the chloroplast ribosome to 20 Å and found that although it is
conserved with bacterial 70S ribosomes, it has large
unique structural domains, as predicted by our proteomics analysis.
G
F i g . 1 . Structure of the chloroplast ribosome from C reinhardtii
calculated to 20-Å resolution. Structures identified as chloroplast
unique through comparison with bacterial ribosomes are labeled.
The chloroplast-unique structures are found on the path of mRNA
through the ribosome, which is labeled as mRNA entrance and exit
tunnels. These structures are positioned for interaction with mRNAs
before and during translation; such interactions most likely are
used to select and position mRNA for initiation of translation and
to increase the rate and fidelity of mRNA translation.
finding is supported by our proteomics results that
indicated that the mass of the small (30S) subunit of
the chloroplast ribosome is 25% larger than the bacterial 30S subunit. Chloroplast-unique structures are
found on the solvent side of the small subunit; the
large subunit interacting face is similar to that in bacterial ribosomes.
The largest of the chloroplast-unique domains occurs
in the vicinity of the mRNA exit tunnel but also extends
up alongside the head and down across the platform.
This structure has multiple lobes and possibly spans the
entire solvent-exposed face of the chloroplast small ribosomal subunit, connecting with a small region of chloroplast-unique density below the shoulder. Another
distinct region of chloroplast-unique density is located
in the beak region of the ribosome, near the mRNA
entrance tunnel. These chloroplast-unique ribosomal
structures are poised to interact with chloroplast
mRNAs early in message recognition, a key point for
translational regulation. These studies have revealed
the structural basis from which we can pursue identification of the molecular and biochemical interactions
of mRNAs, translation factors, and the chloroplast
ribosome that result in regulated translation of chloroplast mRNAs.
42 CELL BIOLOGY
2006
In addition to these basic studies on translation, we
have developed a system for the expression of recombinant proteins, including human therapeutic proteins,
in C reinhardtii chloroplasts. We have expressed a number of mammalian proteins, including human monoclonal antibodies. We recently expressed 83K7C, a
human monoclonal antibody that binds to protective
antigen 83 of anthrax toxin. We showed that 83K7C
assembles in the cell to form functional antibodies that
bind the antigen and thus potentially could block the
toxic effects of Bacillus anthracis during infection. We
also showed that this algal-based system can be used
to produce high levels of 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 at large scale. Our continued genetic, biochemical, and structural studies should lead to a greater
understanding of the mechanism of chloroplast translation
and enable us to design appropriate transgenes to achieve
higher levels of expression of therapeutic proteins.
PUBLICATIONS
Barnes, D., Franklin, S., Schultz, J., Henry, R., Brown, E., Coragliotti, A., Mayfield, S.P. Contribution of 5′- and 3′-untranslated regions of plastid mRNAs to the
expression of Chlamydomonas reinhardtii chloroplast genes. Mol. Genet. Genomics
274:625, 2005.
Fletcher, S.P., Muto, M., Mayfield, S.P. Optimization of recombinant protein
expression in the chloroplast of green algae. In: Transgenic Microalgae as Green
Cell Factories. León, R., Gaván, A., Fernández, E. (Eds.). Landes Bioscience,
Austin, TX, in press.
Manuell, A.L., Mayfield, S.P. A bright future for Chlamydomonas. Genome Biol.
7:327, 2006.
THE SCRIPPS RESEARCH INSTITUTE
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. We use genetic manipulation in mice to investigate the molecular events
involved in learning and memory.
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.
We found not only that the formation of new memories is 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. We also examined where calcium/calmodulin-dependent protein kinase functions within cells. We
found that the synthesis of this kinase from RNA located
specifically at synapses is necessary for the stabilization
of memories that last several months.
GENETIC MODELS OF DISEASE
Manuell, A.L., Yamaguchi, K., Haynes, P.A., Milligan, R.A., Mayfield, S.P. Composition and structure of the 80S ribosome from the green alga Chlamydomonas reinhardtii: 80S ribosomes are conserved in plants and animals. J. Mol. Biol.
351:266, 2005.
Molecular Basis of Cognitive
Function and Dysfunction
M. Mayford, E. Korzus, 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
T
The determination of the complete sequences of
the mouse and human genomes indicates that humans
are highly similar to mice at the genetic level. One
approach for investigating a genetic disease 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
CELL BIOLOGY
2006
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 possible
treatment for Rubenstein-Taybi syndrome 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 the 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 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 have used this approach to study extinction, a
process used in the treatment of phobias by which memories are weakened by repeated exposure to a relevant
stimulus. We found that the neurons originally activated
by a fearful stimulus were no longer activated after
extinction. This finding suggests that extinction training actually erases or interferes with some component
of the original memory trace.
PUBLICATIONS
Colvis, C.M., Pollock, J.D., Goodman, R.H., Impey, S., Dunn, J., Mandel, G.,
Champagne, F.A., Mayford, M., Korzus, E., Kumar, A., Renthal, W., Theobald,
D.E., Nestler, E.J. Epigenetic mechanisms and gene networks in the nervous system. J. Neurosci. 25:10379, 2005.
Reijmers, L.G., Coats, J.K., Pletcher, M.T., Wiltshire, T., Tarantino, L.M., Mayford,
M. A mutant mouse with a highly specific contextual fear-conditioning deficit found in
an N-ethyl-N-nitrosourea (ENU) mutagenesis screen. Learn. Mem. 13:143, 2006.
Yasuda, M., Mayford, M.R. CaMKII activation in the entorhinal cortex disrupts previously encoded spatial memory. Neuron 50:309, 2006.
Regulation of the Plasminogen
Activation System
L.A. Miles, A. Baik, J.W. Mitchell, H. Bai, F.J. Castellino,*
R.J. Parmer**
* University of Notre Dame, Notre Dame, Indiana
** University of California, San Diego, California
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 upreg-
A
THE SCRIPPS RESEARCH INSTITUTE
43
ulated when monocytoid cells undergo apoptosis. Therefore, we are investigating the ability of plasminogen to
modulate monocyte apoptosis.
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 normal 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.
Because monocytoid cells synthesize plasminogen
activators, we examined the role of plasmin proteolytic
activity in the antiapoptotic effects of plasminogen. A
plasminogen active-site mutant did not recapitulate the
cytoprotective effect of wild-type plasminogen. In addition, the antiapoptotic activity of plasminogen was
blocked by increasing concentrations of α2-antiplasmin,
with full reversal at a 2-µM concentration of α2-antiplasmin, suggesting that the cytoprotective effect of plasminogen requires activation of plasminogen to plasmin.
Furthermore, antibodies against protease-activated receptor 1 blocked the antiapoptotic effects of plasminogen.
Our results suggest that plasminogen protects monocytic cells from apoptosis via a mechanism that requires
the proteolytic activity of plasmin and that the protection
is mediated by protease-activated receptor 1. Because
monocyte apoptosis regulates inflammation and atherosclerosis, these results provide insight into a novel role
for plasminogen in these processes.
An emerging area of research has indicated a novel
role for the plasminogen activation system in regulating
the release of neurotransmitters. Prohormones, secreted
by cells within the sympathoadrenal system, are processed by plasmin to bioactive peptides that mediate
feedback inhibition of secretagogue-stimulated release
of neurotransmitters. Catecholaminergic cells of the
sympathoadrenal system are prototypic prohormonesecreting cells. Processing of prohormones by plasmin
is enhanced in the presence of catecholaminergic cells,
and the enhancement requires binding of plasmin(ogen)
to cellular receptors. Consequently, modulation of the
local cellular fibrinolytic system of catecholaminergic
cells results in substantial changes in catecholamine
release. However, mechanisms for enhancing prohor-
44 CELL BIOLOGY
2006
mone processing and cell-surface molecules that mediate the enhancement on catecholaminergic cells have
not been investigated.
We found that plasminogen activation was enhanced
more than 6.5-fold on catecholaminergic cells. Treatment with carboxypeptidase B decreased cell-dependent
plasminogen activation by almost 90%, suggesting that
the binding of plasminogen to proteins exposing C-terminal lysines on the cell surface is required to promote
plasminogen activation. Using a novel strategy of targeted
specific proteolysis with carboxypeptidase B combined
with a proteomics approach involving 2-dimensional gel
electrophoresis, radioligand blotting, and tandem mass
spectrometry, we identified catecholaminergic plasminogen receptors required for enhancing plasminogen activation. Two major plasminogen-binding proteins that
exposed C-terminal lysines on the cell surface contained
amino acid sequences corresponding to β- and γ-actin.
A monoclonal antibody to actin inhibited cell-dependent
plasminogen activation and also enhanced nicotinedependent catecholamine release. Our results suggest
that forms of actin expressed on the cell surface bind
plasminogen, thereby promoting plasminogen activation and increased prohormone processing leading to
inhibition of neurotransmitter release.
PUBLICATIONS
Mitchell, J.W., Baik, N., Castellino, F.J., Miles, L.A. Plasminogen inhibits TNFα
apoptosis in monocytes. Blood 107:4383, 2006.
Structure and Action of
Molecular Machines
R.A. Milligan, J. Chappie, P. Chowdhury, R. Coleman, T. Dang,
S. Falke,* E. Gogol,** M.B. Lee, S. Mulligan, M. Reedy,***
M.K. Reedy,*** A.B. Ward, E.M. Wilson-Kubalek, C. Yoshioka
* William Jewel College, Liberty, Missouri
** University of Missouri, Kansas City, Missouri
*** 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 pro-
M
THE SCRIPPS RESEARCH INSTITUTE
vide 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 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, such as
KinI, 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 analoginduced depolymerization products provided a snapshot
of the disassembly machine at the microtubule ends.
CELL BIOLOGY
2006
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. Our current research focuses on understanding
the role of the second head in these dimeric molecules.
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
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.
We are now investigating the binding of proteins that
track with the tips of dynamic microtubules.
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 2dimensional 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 highresolution 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
THE SCRIPPS RESEARCH INSTITUTE
45
that we are using to study the virulence factor perfringolysin O from Clostridium perfringens. Perfringolysin O is a cytolysin, an important class of proteins
that oligomerize and embed within membranes as part
of the proteins’ lytic functions. We obtained helical
crystals of wild-type and several mutant forms of the
cytolysin 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 pore formation by perfringolysin O 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., Milligan, R.A., Tweten, R.K., Wilson-Kubalek, E.M. Helical crystallization on nickel-lipid nanotubes: perfringolysin O as a model protein. J. Struct. Biol.
152:129, 2005.
Endres, N.F., Yoshioka, C., Milligan, R.A., Vale, R.D. A lever-arm rotation drives
motility of the minus-end-directed kinesin Ncd. Nature 439:875, 2006.
Manuell, A.L., Yamaguchi, K., Haynes, P.A., Milligan, R.A., Mayfield, S.P. Composition and structure of the 80S ribosome from the green alga Chlamydomonas reinhardtii: 80S ribosomes are conserved in plants and animals. J. Mol. Biol.
351:266, 2005.
CNS Development and
Mechanosensory Perception
U. Müller, C. Barros, R. Belvindrah, F. Conti, S. Franco,
N. Grillet, S. Hankel, P. Kazmierczak, R. Radakovits,
C. Ramos, A. Reynolds, A. Sczaniecka, M. Schwander,
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.
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46 CELL BIOLOGY
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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
THE SCRIPPS RESEARCH INSTITUTE
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: integrin functions in glial cells. In: Integrins and Development. Danen, E.H.J. (Ed.). Landes Bioscience, Austin, TX, 2006, p. 185.
Belvindrah, R., Müller, U. Integrin signaling and central nervous system development. In: Extracellular Matrix in Development and Disease. Miner, J.H. (Ed.). Elsevier, St. Louis, 2005, p. 153. Advances in Developmental Biology and
Biochemistry; Vol. 15.
Belvindrah, R., Nalbant, P., Chuanyue, W., Bokoch, G.M., Müller, U. Integrinlinked kinase regulates Bergmann glial differentiation during cerebellar development. Mol. Cell. Neurosci., in press.
Escher, P., Lacazette, E., Courtet, M., Blindenbacher, A., Landmann, L., Bezakova, G., Lloyd, K., Müller, U., Brenner H.R. Synapses form in skeletal muscles
lacking neuregulin receptors. Science 308:1920, 2005.
PERCEPTION, AND DEAFNESS
Mechanosensation, the transduction of mechanical
force into an electrochemical signal, allows living organisms to detect touch, hear, register movement and
gravity, 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 molecules secreted into the extracellular
matrix and membrane-anchored cell adhesion molecules.
Mutations in the genes for the cell adhesion molecule
cadherin 23 in mice and humans cause deafness. Our
findings provide strong evidence that cadherin 23 is a
Naylor, M.J., Li, N., Cheung, J., Lowe, E.T., Lambert, E., Marlow, R., Wang, P.,
Schatzmann, F., Wintermantel, T., Schuetz, G., Clarke, A.R., Müller, U., Hynes,
N.E., Streuli, C.H. Ablation of β1 integrin in mammary epithelium reveals a key
role for integrin in glandular morphogenesis and differentiation. J. Cell Biol.
171:717, 2005.
Senften, M., Schwander, M., Kazmierczak, P., Lillo, C., Shin, J.B., Hasson, T.,
Geleoc, G.S.G., Gillespie, P.G., Williams, D., Holt, J.R., Müller, U. Physical and
functional interaction between protocadherin 15 and myosin VIIa in mechanosensory hair cells. J. Neurosci. 26:2060, 2006.
Molecular Mechanisms of
Thermosensation
A. Patapoutian, M. Bandell, A. Dhaka, A. Dubin, T. Earley,
J. Grandl, H. Hu, L. Macpherson, T. Miyamoto, V. Uzzell
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 tempera-
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2006
ture. 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 1
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,
allicin (garlic), 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. In mice, high levels of TRPV3 occur solely
in skin keratinocytes, suggesting that skin cells might be
able to “sense” temperature and then communicate this
information to neurons in dorsal root ganglia. 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, data
from mice lacking the gene for TRPV3 suggest that
TRPV3 is indeed required 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 showed that the Drosophila ortholog of TRPA1
is an ion channel activated by warm temperatures, suggesting an evolutionarily 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
THE SCRIPPS RESEARCH INSTITUTE
47
analysis of the thermoTRPs, will provide 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 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
Bandell, M., Dubin, A.E., Petrus, M.J., Orth, A., Mathur, J., Hwang, S.W., Patapoutian, A. High-throughput random mutagenesis screen reveals TRPM8 residues
specifically required for activation by menthol. Nat. Neurosci. 9:493, 2006.
Dhaka, A., Viswanath, V., Patapoutian, A. TRP ion channels and temperature sensation. Annu. Rev. Neurosci. 29:135, 2006.
Macpherson, L.J., Geierstanger, B.H., Viswanath, V., Bandell, M., Eid, S.R.,
Hwang, S.W., Patapoutian, A. The pungency of garlic: activation of TRPA1 and
TRPV1 in response to allicin. Curr. Biol. 15:929, 2005.
Functional Proteins in Tumor
Metastasis and Angiogenesis
J.P. Quigley, E.I. Deryugina, A. Zijlstra, J.P. Partridge,
T. Kupriyanova, M. Madsen, V. Ardi, M.C. Subauste
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 small interfering RNA molecules
directed against specific expressed genes and applied
directly into the models 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 function-blocking monoclonal antibodies, in combination with
immunoproteomics enables us to identify specific antigenic molecules that are functionally active in metas-
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48 CELL BIOLOGY
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tasis and angiogenesis. Finally, use of activity-based
protein profiling, in collaboration with B.F. Cravatt,
Department of Cell Biology, enables us to detect, isolate, and identify active proteolytic enzymes that are
distinctively and differentially activated during metastasis and angiogenesis.
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
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
tumor cells in organs of chick embryos. The screening
procedures include direct inoculation of primary tumor
cells that have been transfected with various small interfering RNA constructs to silence specific genes that might
contribute to metastatic dissemination. Inoculating monoclonal antibodies directly into the tumor-bearing embryo
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.
THE SCRIPPS RESEARCH INSTITUTE
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 enables us to detect and measure the
newly sprouting blood vessels responding to an angiogenic stimulus such as a specific growth factor or a
growing tumor. 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 specific proteolytic event
and found that specific collagen-cleaving metalloproteinases are implicated directly in the outgrowth of
new vessels.
We also found that another metalloproteinase,
MMP-9 (gelatinase B), most likely is involved in angiogenic tissue remodeling. The proteolytic activity of this
enzyme 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 MMP13 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 specific
molecules and cells that link the inflammatory response
to the 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 tumor cells.
I N T R AVA S AT I O N
We are also investigating intravasation, the entry
of primary tumor cells into the host vasculature, often
the vasculature that is newly formed during tumor angiogenesis. Intravasation appears to be the least-studied
process in the metastatic cascade but most likely is a
rate-limiting step in tumor dissemination. We recently
isolated 2 isogenic variants of a human fibrosarcoma
cell line that differ 100-fold in their ability to enter the
vasculature in vivo and in their ability to metastasize.
We are using array technology, proteomics approaches,
and intravital microscopy in the cellular and molecular
analysis of these 2 variants. The results should indi-
CELL BIOLOGY
2006
cate specific molecules that are functionally important
in interactions between tumor cells and the vasculature and contribute to intravasation. Using activitybased protein profiling, we found that the proteolytic
enzyme urokinase is differentially activated during
intravasation and catalytically contributes to the
enhanced entry of tumor cells into the vasculature.
PUBLICATIONS
Deryugina, E.I., Zijlstra, A., Partridge, J.J., Kupriyanova, T.A., Madsen, M.A.,
Papagiannakopoulos, T., Quigley, J.P. Unexpected effect of matrix metalloproteinase
down-regulation on vascular intravasation and metastasis of human fibrosarcoma
cells selected in vivo for high rates of dissemination. Cancer Res. 65:10959, 2005.
Lewis, J.D., Destito, G., Zijlstra, A., Gonzalez, M.J., Quigley, J.P., Manchester,
M., Stuhlmann, H. Viral nanoparticles as tools for intravital vascular imaging. Nat.
Med. 12:354, 2006.
Madsen, M.A., Deryugina, E.I., Niessen, S., Cravatt, B.F., Quigley, J.P. Activitybased protein profiling implicates urokinase activation as a key step in human
fibrosarcoma intravasation. J. Biol. Chem. 281:15997, 2006.
Zijlstra, A., Seandel, M., Kupriyanova, T.A., Partridge, J.J., Madsen, M.A., HahnDantona, E.A., Quigley, J.P., Deryugina, E.I. Proangiogenic role of neutrophil-like
inflammatory heterophils during neovascularization induced by growth factors and
human tumor cells. Blood 107:317, 2006.
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 receptorligand complexes, deform the membrane, invaginate,
and eventually pinch off, forming clathrin-coated vesicles
(CCVs). The major components involved in formation of
CCVs are clathrin, adaptor proteins, and dynamin, an
atypical GTPase.
Clathrin self-assembles into a polygonal lattice
and serves as a scaffold for the formation of coated
pits. Adaptor protein-2 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. We view dynamin as the
master regulator of endocytosis.
Previously, we developed a cell-free assay in which
CCVs are reconstituted from sheets of purified plasma
membranes from rat liver. Using this in vitro reconstitu-
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THE SCRIPPS RESEARCH INSTITUTE
49
tion system and a new biochemical complementation
assay, we explored the limiting cytosolic requirements
for endocytosis of the low-density lipoprotein receptor–related protein (LRP) from isolated plasma membranes. LRP, also known as a scavenger receptor,
binds multiple, distinct ligands and participates in
constitutive endocytosis and signal transduction.
We found that clathrin, adaptor protein-2, and
dynamin do not support efficient LRP uptake; additional
factors present in a 30% ammonium sulfate supernatant
fraction of bovine brain cytosol are required. Fractionation of the supernatant revealed that multiple and
redundant factors are required to support LRP endocytosis. Our data suggest that LRP, which has several
distinct endocytic motifs in its cytoplasmic domain,
may use multiple pathways for endocytosis in vitro.
The factors we identified, 70-kD heat-shock cognate
protein, synaptojanin, and collapsin response mediator
protein-2, are all implicated in clathrin-mediated endocytosis in vivo, thus validating the assay. However, we
found that these factors were sufficient but not necessary for formation of CCVs. Thus, functional redundancy and complexity make this assay biochemically
intractable, and we have chosen, at least for the
moment, to abandon it.
We continue to analyze the structure and function
of dynamin. Dynamin is a multidomain protein consisting of an N-terminal GTPase domain, a middle
domain of previously unknown function, a PH domain
that binds phosphatidylinositol 4,5-biphosphate, a
GTPase effector domain (GED) required for dynamin
self-assembly that functions as an assembly-dependent
GTPase-activating protein for dynamin, and a C-terminal
proline-arginine–rich domain. Dynamin self-assembles in
vitro into spirals on liposomes containing phosphatidylinositol 4,5-biphosphate to tubulate the liposomes,
resulting in a greater than 100-fold stimulation of
GTPase activity. Self-assembly and assembly-stimulated GTPase activity at the necks of deeply invaginated coated pits are thought to drive conformational
changes that mediate membrane fission.
Our collaborators recently solved the crystal structure of the isolated, unoccupied GTPase domain of rat
dynamin-1. Unlike the situation in other GTPases, the
normally unstructured switch 1 and switch 2 regions
that surround the unoccupied GTP binding pocket were
ordered. On the basis of the structure, 2 arginines near
the active site were predicted to be required for catalysis, but our enzymatic analysis of lysine and alanine
50 CELL BIOLOGY
2006
substitutions of these residues suggested otherwise.
Thus, the mechanism of GTP hydrolysis remains obscure.
However, the structure revealed the existence of a hydrophobic groove created by the N- and C-terminal α-helices
of the GTPase domain, which was suggested to be a
docking site for the GED.
In a recent study, we identified mutations in the
GED that resulted in reduced GTPase activity without
affecting self-assembly. Because these mutations mapped
to a predicted amphipathic helix at the extreme C terminus of GED, we speculated that this GED helix formed a
3-helical bundle with the GTPase domain helices. To
test this hypothesis, we have generated a construct consisting of the GTPase of dynamin together with a C-terminal extension composed of a short sequence of
turn-preferring residues followed by the 20 amino acid
C-terminal GED helix. This construct, unlike GTPase
domain or GED constructs is largely soluble when
expressed in Escherichia coli and has basal GTPase
activity equivalent to that of full-length dynamin.
These exciting results suggest that we have fully
reconstituted the GED-GTPase interactions. We are
currently expressing the GTPase-GED peptide construct
for high-resolution x-ray crystallography studies, and
mutagenesis is under way to probe the mechanisms of
GED-stimulated GTPase activity.
We have also identified a new class of mutants in
the middle domain that alter the quaternary structure
of dynamin. Native dynamin exists as a tetramer, but
analytical ultracentrifugation and gel filtration chromatography coupled to multiangle light scattering have confirmed that the middle domain mutants are dimeric.
Kinetic studies established that the basal GTPase activity
of dynamin requires a highly cooperative, GTP-dependent conformational change in dynamin tetramers. The
dimeric dynamin mutants are defective in both activation in the basal state and self-assembly into higher
order structures.
Finally, in following up our earlier discovery that
actin dynamics are required for multiple stages of
clathrin-mediated endocytosis, we found that the protein sorting nexin 9 is a molecular link between dynamin
and actin assembly. Sorting nexin 9 binds dynamin
through the nexin’s SH3 domain and activates neural
Wiskott-Aldrich syndrome protein to trigger actin-related
protein 2/3–dependent actin assembly into branched
filaments. Thus, sorting nexin 9 may trigger the burst
of actin assembly that accompanies the scission of
coated vesicles.
THE SCRIPPS RESEARCH INSTITUTE
PUBLICATIONS
Leonard, M., Song, B.D., Ramachandran, R., Schmid, S.L. Robust colorimetric
assays for dynamin’s basal and stimulated GTPase activities. Methods Enzymol.
404:490, 2005.
Miwako, I., Schmid, S.L. A cell-free biochemical complementation assay reveals
complex and redundant cytosolic requirements for LRP endocytosis. Exp. Cell Res.
312:1335, 2006.
Miwako, I., Schmid, S.L. Clathrin-coated vesicle formation from isolated plasma
membranes. Methods Enzymol. 404:503, 2005.
Reubold, T.F., Eschenburg, S., Becker, A., Leonard, M., Schmid, S.L., Vallee,
R.B., Kull, F.J., Manstein, D.J. Crystal structure of the GTPase domain of rat
dynamin 1. Proc. Natl. Acad. Sci. U. S. A. 102:13093, 2005.
Molecular Biology of Olfaction
L. Stowers, I.S. Bharati, P. Chamero, J. Cruz, K. Flanagan,
J. Lin, D. Logan, T. Marton, C. Ramos
very breath samples the environment for olfactory chemical information, determining the quality of food, warning of danger, and confirming
safety. The neurons that mediate olfaction are of 2 types:
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 (TRP2)
channels. We are using a molecular genetic approach
to characterize the function of these pheromone-responsive neurons.
Through electrophysiologic recordings, we have
shown that mutant mice lacking C-type TRP2 channels
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 pheromone-responsive neurons, and through genetic
ablation, biochemistry, and electrophysiology, we are
assigning 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 C-type TRP2 signaling components. Analysis of these neurons by transcriptional
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2006
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 their complex
natural sources. To obtain these important molecules,
we are fractionating natural sources of pheromones and
then using biobehavioral assays to identify the molecules that initiate activity. The results will enable us to
both 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.
THE SCRIPPS RESEARCH INSTITUTE
51
in vitro differentiation of embryonic stem cells into
embryoid bodies. Our results indicate that Vezf1 affects
vascular differentiation by regulating cell proliferation,
differentiation, and deposition of extracellular matrix.
We are examining the molecular pathways of Vezf1
function. In collaborative studies with L. Benjamin, Beth
Israel Deaconess Medical Center, Boston, we found that
VEZF1, the protein encoded by Vezf1, interacts with
Rho GTPases to modulate the function of Rho in the
endothelium. Microarray cDNA analysis with RNA from
wild-type embryos and embryos lacking Vezf1 suggested that genes for fibrinogen and claudin and several genes involved in metabolite transport are target
genes for Vezf1.
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
THEIR PROGENITORS
Molecular Regulation of
Vascular System Development
in Mammals
H. Stuhlmann, M.J. Fitch, Z. Zou, S. Chitnis, J.D. Lewis,
A. Durrans, W. LeVine
stablishment of a functional circulatory system
during development is crucial for the delivery of
nutrients and oxygen to embryos. Defects in the
development of blood vessels result in death before birth
or in congenital cardiovascular abnormalities. We examine the molecular and genetic pathways that regulate
the 3 principal processes of vascular development: determination of vascular lineage, 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. Using an expression-based “gene
trap” screen in mouse embryonic stem cells and
embryos, we identified 2 novel genes involved in these
processes: Vezf1 and Egfl7.
E
Expression of a second endothelial gene identified
in our screen, Egfl7, is restricted to the vascular endothelium and endothelial progenitors in the yolk sac
mesoderm. Egfl7 is also expressed in multipotent stem
cells in embryos, in primordial germ cells, and during
spermatogenesis. In the quiescent vasculature in adults,
overall Egfl7 expression is downregulated. During physiologic angiogenesis in the uterus during pregnancy and
in the regenerating endothelium after vascular injury,
expression of Egfl7 is upregulated. EGFL7, the protein
encoded by Egfl7, is partially secreted and acts as a
chemoattractant on both endothelial cells and embryonic fibroblasts in in vitro migration assays. EGFL7 is
a compact 278 amino acid protein with an amino-terminal signal peptide; an EMI domain; and 2 central
epidermal growth factor–like domains, one of which
contains a putative Notch interaction domain. Importantly, recent collaborative studies with J. Kitajewski,
Columbia University, New York City, provide support
for our hypothesis that EGFL7 binds to Notch receptors and acts as an agonist for Notch signaling during
vascular development.
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
A ZINC FINGER GENE ESSENTIAL FOR NORMAL
TA R G E T I N G A N D I M A G I N G
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
In collaboration with M. Manchester, Department
of Cell Biology, we have developed viral nanoparticles
based on the cowpea mosaic virus (CPMV) for noninvasive imaging and targeting of the mammalian cardiovascular system. CPMV can be fluorescently labeled to
high densities with no quenching, resulting in bright
particles that allow high-resolution intravital imaging
of the vascular endothelium and blood flow deep inside
Vezf1 is the gene for an early zinc finger transcription factor that controls the development of blood vessels and the lymphatic system in mice. Using functional
genetic studies, we previously showed that Vezf1 plays
an essential and dosage-dependent role in the proliferation, remodeling, and integrity of the developing vasculature. We recently extended these studies by using
52 CELL BIOLOGY
2006
mouse or chick embryos for up to 72 hours. Using a
human fibrosarcoma model for tumor angiogenesis, we
found that fluorescent CPMV can be used to distinguish
between arterial and venous vessels and to monitor the
neovascularization of the tumor microenvironment. We
are extending these studies by conjugating peptides to
the CPMV capsid to target the peptides to the vasculature, both during development and in disease models.
PUBLICATIONS
Lewis, J.D., Destito, G., Gonzalez, M.J., Zijlstra, A., Quigley, J.P., Manchester,
M., Stuhlmann, H. Viral nanoparticles as tools for intravital vascular imaging. Nat.
Med. 12:354, 2006.
Kuhnert, F., Stuhlmann, H. Role of Vezf1 during vascular and lymphatic development. In: Endothelial Biomedicine. Aird, W.C. (Ed.), Cambridge University Press,
New York, in press.
Zijlstra, A., Lewis, J.D., DeGryse, B., Stuhlmann, H., Quigley, J.P. Inhibition of
tumor cell intravasation and subsequent metastasis through the regulation of
CD151-mediated in vivo tumor cell motility. Cancer Cell, in press.
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 the structures of the proteins trapped
in different physiologic states. Current studies center
on the nicotinic acetylcholine receptor at the nervemuscle 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 postsynaptic membranes isolated from
the (muscle-derived) electric organ of the 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 receptors for serotonin 5-HT3, γ-aminobutyric-acids A and C, and glycine. 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
I
THE SCRIPPS RESEARCH INSTITUTE
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
structural 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 the orientations of the
sheets 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 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 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 the related γ-aminobutyric acid,
serotonin, glycine, and neuronal acetylcholine receptors.
PUBLICATIONS
O’Brien, J., Unwin, N. Organization of spines on the dendrites of Purkinje cells.
Proc. Natl. Acad. Sci. U. S. A. 103:1575, 2006.
CELL BIOLOGY
2006
Microscopes and Motility:
Systems Integration in
Cell Migration
C.M. Waterman-Storer, O. Rodriguez, S.L. Gupton, K. Kita,
R. Littlefield, K. Hu, A. Wheeler, W. Shin, M.L. Gardel,
I.C. Schnieder, A. Parapera, J. Lim
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 microtubule and actin cytoskeletal polymers, the endomembrane trafficking system,
and focal adhesions to the extracellular environment.
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.
Recently, we extended the technology to multispectral
total internal fluorescence reflection fluorescence microscopy, allowing analysis of the integration of proteins
within focal adhesions with the actin cytoskeleton during cell migration. In collaboration with G. Danuser,
Department of Cell Biology, we developed correlational
fluorescent speckle microscopy to measure the coupling
of focal adhesion proteins to the actin cytoskeleton.
We found that different classes of focal adhesion
structural and regulatory molecules have different degrees
of correlated motions with actin filaments, indicating differential transmission of actomyosin motion through focal
adhesions. Our results suggest that transient interactions
between focal adhesion proteins and actin filaments constitute a friction clutch between the cytoskeleton and the
extracellular environment that is regulated during the morphodynamic transitions of cell migration.
On the basis of a mathematical model, researchers
predicted 15 years ago that migration speed would
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THE SCRIPPS RESEARCH INSTITUTE
53
have a biphasic response to increasing strength of cell
adhesion, with slow migration occurring at low and high
strengths and fast migration at intermediate strength.
The assumption of the model was that migrating cells
have an asymmetry in adhesion strength from the front
part of the mass of cells to the rear part, with the cells
connected by symmetric contractile elements, but
dynamic organizational states of F-actin and focal
adhesions were not considered. This biphasic dependence of migration velocity on increasing adhesion
strength has since been supported experimentally, and
a front-to-rear gradient in cell adhesion strength has
also been shown.
We sought to determine if distinct organizational
states of F-actin, myosin II, and focal adhesions accompany adhesion-dependent changes in velocity. We
characterized a unique phenotype for optimal migration at intermediate adhesion strength, entailing rapid
flow convergence and local depolymerization of F-actin,
local activation of myosin II, rapid renewal of the components of focal adhesions, and intermediate lifetime
and turnover rates of focal adhesions. We recapitulated
this phenotype and fast migration at a nonoptimal adhesion strength by manipulating the activity of myosin II.
In contrast to the results with simple models, we found
that a complex spatiotemporal integration and feedback between F-actin, myosin II, and focal adhesions
mediates the classically observed biphasic migration
velocity response to increasing adhesion strength, so
that a specific balance between adhesion and contraction induces maximal migration velocity.
The microtubule and actin cytoskeletons interact
in cells to promote a coordinated effort to drive protrusion of the leading edge of cells during cell migration.
The tumor suppressor protein adenomatous polyposis
coli and its binding partner EB1 accumulate on the
ends of microtubules. The tumor suppressor protein
specifically collects on microtubules in cell protrusions,
suggesting that it may promote cell protrusion. We
simultaneously visualized dynamics of the suppressor
protein and microtubules in living cells. We found that
the association of the protein with the ends of microtubules correlates with the increased growth stability
of the microtubules and that this stabilization can occur
independent of the association of the protein with EB1.
We also found that the protein and EB1 associate with
the ends of microtubules by distinct mechanisms. Thus,
cancer-causing mutations in this tumor suppressor
protein may arise from defects in microtubule stability
and cell migration.
54 CELL BIOLOGY
2006
PUBLICATIONS
Danuser, G., Waterman-Storer, C.M. Quantitative fluorescent speckle microscopy
of cytoskeleton dynamics. Annu. Rev. Biophys. Biomol. Struct. 35:361, 2006.
deRooij, J., Kerstens, A., Danuser, G., Schwartz, M.A., Waterman-Storer, C.M.
Integrin-dependent actomyosin contraction regulates epithelial cell scattering. J.
Cell Biol. 171:153, 2005.
Gupton, S.L., Waterman-Storer, C.L. 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., et al. (Eds.). Academic
Press, San Diego, 2005, Vol. 3, p. 137.
Gupton, S.L., Waterman-Storer, C.L. Spatiotemporal feedback between actomyosin
and focal-adhesion systems optimizes rapid cell migration. Cell 125:1361, 2006.
Kita, K., Wittmann, T., Nathke, I.S., Waterman-Storer, C.L. Adenomatous polyposis coli on microtubule plus ends in cell extensions can promote microtubule net
growth with or without EB1. Mol. Biol. Cell 17:2331, 2006.
Ponti, A., Matov, A., Adams, M., Gupton, S., Waterman-Storer, C.M., Danuser, G.
Periodic patterns of actin turnover in lamellipodia and lamellae of migrating epithelial cells analyzed by quantitative fluorescent speckle microscopy. Biophys. J.
89:3456, 2005.
Systems Biology and Malaria
E.A. Winzeler, C. Kidgell, V. Ramachandran, N. Kato,
K. Henson, J. Young, J. Johnson
espite the widespread impact of malaria on the
world’s health and economies, relatively little
is known about the function of the majority of
the 5300 genes in the genome of Plasmodium falciparum, the causative agent of the most severe form of
malaria in humans. This lack of knowledge retards the
development of drugs and vaccines against the parasite.
We use systematic discovery-based approaches to predict the function of uncharacterized Plasmodium genes;
our goal is to facilitate the discovery of new treatments.
We are using mRNA and protein expression to reveal
genetic regulatory networks and to suggest protein-protein interactions. We are also developing new methods
that can be used in systems biology research.
In addition to our past work on blood-stage parasites,
we have characterized the expression program of the
sexual stages of malarial parasites. These stages, which
are essential for the mosquito transmission of the disease, are the focus of the development of drugs and
vaccines that block transmission. To better understand
genes important to sexual development, we used a fullgenome high-density oligonucleotide microarray to profile
the transcriptomes of P falciparum gametocytes. To
interpret this transcriptional data, we developed and
used a novel knowledge-based data-mining algorithm
termed ontology-based pattern identification. With this
algorithm, published or custom gene classifications
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are used to optimize normalization methods and cluster
boundaries so that the largest number of any given gene
type is found in the smallest cluster size.
This analysis resulted in the identification of a sexual
development cluster containing 246 genes, of which
approximately 75% were unclassified and which contained most known sexual stage genes. These genes had
highly correlated, gametocyte-specific expression patterns. Statistical analysis of the upstream promoter
regions of these 246 genes revealed putative cis regulatory elements. In addition, we extended the ontologybased pattern identification by using current annotations
provided by the Gene Ontology Consortium to identify
380 statistically significant clusters containing genes with
expression patterns characteristic of various biological
processes, cellular components, and molecular functions.
We are also studying genetic diversity by using
hybridization-based approaches to further characterize
parasite genes. By performing a full genome scan of
allelic variability of 14 field and laboratory strains of
P falciparum, we showed that 10% of the genome
has higher than neutral rates of diversity at tens of
thousands of loci. We found that whereas many genes
are exceptionally well conserved across parasite isolates, paralog genes (i.e., genes related by duplication
within a genome that have different functions), genes
near the ends of chromosomes, genes that encode proteins that are trafficked to the surface of the infected
red cell, and genes that encode known and potential
drug targets are exceptionally diverse. These data suggest that rates of mitotic recombination are elevated
among genes with paralogs and that selection pressure on those without paralogs is strong.
We also revealed gene amplification events, including one associated with pfmdr1, the gene for multidrug
resistance in P falciparum, and a previously uncharacterized amplification centered on the gene for GTP
cyclohydrolase, the first enzyme in the folate biosynthesis pathway. Although GTP cyclohydrolase is not the
known target of any current drugs, downstream members of the pathway are targeted by several widely used
antimalarial agents. We propose that amplification of
the GTP cyclohydrolase enzyme in the folate biosynthesis pathway may facilitate increased flux through this
pathway and increase resistance to antifolate drugs.
These data and recent publications indicating that
90% of a small eukaryote’s genetic variation can be
captured in a single microarray hybridization, suggest
that population genomics will be a fruitful approach
CELL BIOLOGY
2006
THE SCRIPPS RESEARCH INSTITUTE
55
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 measure
global changes in protein abundance and how to characterize complex posttranslational modifications.
hypothesized that a subset of insulin-signaling targets,
specifically, daf-2 (the gene for an insulin receptor)
and daf-16 (the gene for a transcription factor negatively regulated by daf-2) mutants, might be differentially expressed in wild-type C elegans and thus could
be identified by using our quantitative proteomics
approach. We identified 104 proteins that were present in higher or lower levels in daf-2 mutants than in
wild-type and daf-16 mutant organisms, including the
known targets superoxide dismutase 3 and catalases.
Gene ontology analysis revealed that the upregulated
proteins in daf-2 mutants were overrepresented in the
metabolism of reactive oxygen species, metabolism of
carbohydrates, and biosynthesis of amino acids; the
downregulated proteins were enriched in proteins
involved in translation and lipid transport.
We confirmed by genetics analysis that some of the
possible insulin-signaling components identified in the
study play a role in regulating life span and/or formation
of dauer larvae, both of which are regulated by C elegans insulins. Among the confirmed targets is a protein
phosphatase. Using a green fluorescent protein as a
label, we found that the phosphatase was upregulated in
worms in which daf-2 was inactivated via RNA interference, consistent with the results of mass spectrometry.
Further genetic analysis indicated that this phosphatase
acted upstream of and/or in parallel to the protein DAF16 in regulation of aging and dauer formation.
Taken together, our data suggest that this protein
phosphatase is both a downstream target and a regulator of insulin signaling. Therefore it may be part of a
feedback loop. This study illustrates the effectiveness
of combining quantitative mass spectrometry and C
elegans genetics. Such an approach can be extended
to other studies beyond insulin signaling.
Q U A N T I TAT I V E P R O T E O M I C A N A LY S I S O F I N S U L I N
P R O T E I N S U M O Y L AT I O N
SIGNALING
The characterization of posttranslational modifications is also an emerging application of mass spectrometry–based proteomics. We are developing proteomic
tools to study the family of small ubiquitin-like modifiers (SUMOs). Recently, we focused on Smt3p, the
budding yeast homolog of a human SUMO. Although
genetic studies have indicated that Smt3p is required
for proper regulation of a variety of different cellular
processes, including transcription, intracellular transport, progression of the cell cycle, and the maintenance of genome integrity, the mechanisms by which
it does so remain largely unknown.
Using proteomic approaches, we addressed 2 major
areas in protein sumoylation: the large-scale identifi-
for discovering new determinants of drug resistance in
a variety of infectious agents.
PUBLICATIONS
Carret, C.K., Horrocks, P., Konfortov, B., Winzeler, E., Qureshi, M., Newbold, C.,
Ivens, A. Microarray-based comparative genomic analyses of the human malaria
parasite Plasmodium falciparum using Affymetrix arrays. Mol. Biochem. Parasitol.
144:177, 2005.
Kidgell, C., Winzeler, E.A. Using the genome to dissect the molecular basis of drug
resistance. Future Microbiol. 1:185, 2006.
Winzeler, E.A. Applied systems biology and malaria. Nat. Rev. Microbiol. 4:145,
2006.
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.
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, B.W. Lu, I. McLeod, D. McClatchy,
R. Park, E. Romijn, H. Prieto, C.I. Ruse, J. Venable,
J. Wohlschlegel, C. Wong, T. Xu
M
Quantitative mass spectrometry–based proteomics
relies on internal isotopic standards. Metabolic labeling with nitrogen 15 is a preferred method of introducing internal standards. It produces a standard for
every peptide or protein to be characterized. In addition, it is stable, nonradioactive, less error-prone than
chemical labeling, and relatively inexpensive. We have
used metabolic labeling with nitrogen 15 and quantitative mass spectrometry to study insulin signaling in
the worm Caenorhabditis elegans.
Insulin regulates a wide range of processes, including metabolism, development, and aging, but only a
handful of its downstream targets are known. We
56 CELL BIOLOGY
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cation of sumoylation targets and the development of
strategies for mapping SUMO modification sites. By
combining different affinity chromatography strategies
with our multidimensional protein identification platform, we identified 271 new SUMO targets. These
substrates play roles in a diverse set of biological processes and greatly expand the known scope of SUMO
regulation in 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. We are also characterizing the mechanism that
underlies this observation.
Characterization of many of the new substrates identified in our study was limited by difficulties in identifying
SUMO attachment sites in the target of interest. To solve
this problem, we recently developed a method for the
rapid and efficient identification of SUMO attachment
sites in cellular proteins. In this method, different
SUMO mutants are used in combination with various
protease digestion strategies, and then mass spectrometry is used to directly and specifically map the
locations of the modified lysine residues. We showed the
usefulness of this method by identifying SUMO modification sites in an assortment of model SUMO substrates and in complex mixtures. The development of
a method for identifying SUMO attachment sites will
be a powerful tool for the characterization of the new
substrates identified in our initial global analysis.
PUBLICATIONS
Cantin, G.T., Venable, J.D., Cociorva, D., Yates, J.R. III. Quantitative phosphoproteomic analysis of the tumor necrosis factor pathway. J. Proteome Res. 5:127, 2006.
Chen, E.I., Hewel, J., Felding-Habermann, B., Yates, J.R. III. Large scale protein
profiling by combination of protein fractionation and multidimensional protein identification technology (MudPIT). Mol. Cell. Proteomics 5:53, 2006.
Sadygov, R., Wohlschlegel, J., Park, S.K., Xu, T., Yates, J.R. III. Central limit theorem
as an approximation for intensity-based scoring function. Anal. Chem. 78:89, 2006..
Venable, J.D., Xu, T., Cociorva, D., Yates, J.R. III. Cross-correlation algorithm for
calculation of peptide molecular weight from tandem mass spectra. Anal. Chem.
78:1921, 2006.
Wohlschlegel, J.A., Johnson, E.S., Reed, S.I., Yates, J.R. III. Improved identification of SUMO attachment sites using C-terminal SUMO mutants and tailored protease digestion strategies. J. Proteome Res. 5:761, 2006.
THE SCRIPPS RESEARCH INSTITUTE
Macromolecular Assemblies
Visualized by Electron
Cryomicroscopy and Image
Analysis: Membrane Proteins
and Viruses
M. Yeager, R. Abagyan,**** B.D. Adair, K. Baker, K. Altieri,
A. Cheng, M.J. Daniels, K.A. Dryden, B. Ganser, J. Harless,
Y. Hua, M. Matho, 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. Schneemann,****
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 structural 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
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CELL BIOLOGY
2006
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. Threedimensional density maps are obtained by digital image
processing of the high-resolution electron micrographs.
The rich detail in the density maps exemplifies 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.
Ongoing research projects 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
[HBV], rotavirus, astrovirus); and (3) viruses used as
model systems to understand mechanisms of pathogenesis (arenaviruses, reoviruses, nodaviruses, tetraviruses and sobemoviruses). The following sections
highlight selected projects that exemplify the themes
of our research program.
THE SCRIPPS RESEARCH INSTITUTE
57
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 α-helices 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 using electron cryocrystallography.
Although individually, none of these approaches provided high-reso-
GAP JUNCTION MEMBRANE CHANNELS
lution information, their sum yielded an atomic model that predicts
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.
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 coordi-
how connexin mutations (spheres), which result in diseases such
as nonsyndromic deafness and Charcot-Marie-Tooth disease, may
interfere with formation of functional channels by disrupting helixhelix packing.
nates 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
biological processes such as development, cell adhesion, angiogenesis, wound healing, and neoplastic
transformation. The ectodomain of the integrin αvβ3
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 frag-
58 CELL BIOLOGY
2006
ment of fibronectin containing type III domains 7–10
and the EDB domain. Electron microscopy and singleparticle image analysis were used to determine the
3-dimensional structure of this complex (Fig. 2).
THE SCRIPPS RESEARCH INSTITUTE
core protein has been studied intensively. However, little is known about the structure and assembly of native
capsids present in infected cells, and even less is known
about the structure of mature virions. We used electron
cryomicroscopy and image analysis to examine HBV
virions (also called Dane particles) isolated from the
serum of a patient with hepatitis B and capsids positive and negative for HBV DNA isolated from the livers
of transgenic mice (Fig. 3).
F i g . 2 . The 3-dimensional density map (grayscale 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 proteins 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 fibronectin-bound
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.
This study suggests that the ectodomain of αvβ3 has
a bent conformation that can stably bind a physiologic
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.
F i g . 3 . A model of the HBV virion (diameter ~450 Å) based on
electron cryomicroscopy and image analysis. A, The double-stranded
DNA genome is encapsidated by an icosahedral capsid shell composed of 120 spikes. The surface is studded with glycoproteins
spaced about 60 Å apart that bind to membrane receptors on liver
cells. B, In the close-up view, the x-ray crystal structure of a
recombinant capsid has been docked into the electron cryomicroscopy density map of the virion capsid. The core spikes are in
close apposition but do not penetrate the envelope.
H E PAT I T I S B V I R U S
HBV currently infects more than 350 million people, of which 1 million will die every year. The infectious virion is an enveloped capsid containing the viral
polymerase and the double-stranded DNA genome. The
structure of the capsid assembled in vitro from expressed
Both types of capsids assembled as icosahedral
particles indistinguishable from previous image reconstructions of capsids. Likewise, the virions contained
capsids with either T = 3 or T = 4 icosahedral symmetry. Projections extending from the lipid envelope
CELL BIOLOGY
2006
were attributed to surface glycoproteins. The packing
of the projections was unexpectedly nonicosahedral but
conformed to an ordered lattice. These structural features distinguish HBV from other enveloped viruses.
PUBLICATIONS
Daniels, M.J., Wood, M.R., Yeager, M. In vivo functional assay of a recombinant
aquaporin in Pichia pastoris. Appl. Environ. Microbiol. 72:1507, 2006.
Daniels, M.J., Yeager, M. Phosphorylation of aquaporin PvTIP3;1 defined by mass
spectrometry and molecular modeling. Biochemistry 44:14443, 2005.
Dryden, K.A., Wieland, S.F., Whitten-Bauer, C., Chisari, F.V., Yeager, M. Native
hepatitis B virions and capsids visualized by electron cryomicroscopy. Mol. Cell
23:843, 2006.
Greig, S.L., Berriman, J.A., O’Brien, J.A., Taylor, J.A., Bellamy, A.R., Yeager, M.,
Mitra, A.K. Structural determinants of rotavirus subgroup specificity mapped by
cryo-electron microscopy. J. Mol. Biol. 356:209, 2006.
Wiedenheft, B., Mosolf, J., Willits, D., Yeager, M., Dryden, K.A., Young, M., Douglas,
T. An archaeal antioxidant: characterization of a Dps-like protein from Sulfolobus
solfataricus. Proc. Natl. Acad. Sci. U. S. A. 102:10551, 2005.
THE SCRIPPS RESEARCH INSTITUTE
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