DEPARTMENT OF DEVELOPMENTAL AND MOLECULAR
BIOLOGY
The department consists of the laboratories of Drs. Ruth Angeletti, Nick Baker, Teresa
Bowman, Dianne Cox, Ana Maria Cuervo, Antonio Di Cristofano, Andreas Jenny, Paraic Kenny,
Umadas Maitra, Florence Marlow, Anne Müsch, Nicholas Sibinga, Richard Stanley, Amit Verma,
Duncan Wilson, Fajun Yang, and Liang Zhu. Research interests in the Department cover four
major areas: (1) Cell determination and regulation in blood, heart, kidney, muscle, nervous and
reproductive systems development (Drs. Angeletti, Bowman, Jenny, Maitra, Marlow, Sibinga,
Stanley and Verma); (2) Signal transduction pathways regulating cellular function or
developmental interactions (Drs. Jenny, Müsch, Stanley, Verma, Yang and Zhu); (3) Cell cycle
regulation in normal and neoplastic cells (Drs. Di Cristofano, Kenny, and Zhu); and (4) Protein
synthesis, processing, targeting, intracellular vesicle trafficking and autophagy (Drs. Cuervo,
Maitra, Müsch and Wilson). Faculties frequently collaborate on projects within and among
these areas.
The strength of the Department derives from our outstanding group of PhD and
MD/PhD graduate students, who have been encouraged to, and accepted the challenge of,
working along side of our faculty in all aspects of department life. We have weekly
departmental work-in-progress (Fridays at noon with lunch) and theme-focused journal clubs
(in the morning with breakfast) that all graduate students participate along with post-doctoral
fellows and our faculty. In these meetings, we enjoy not only superb presentations but also
probing questioning-answering periods in an atmosphere of learning, appreciating, and being
critical and caring at the same time. A dearly cherished asset of the department is our spirit of
cooperation, which makes being a member of the department a fruitful and pleasant experience
that you will take with you after you finish your Degrees.
The department’s outside speakers’ seminar program contains regularly scheduled
“student-invited speaker” seminars. Students poll and vote on their most favorite speakers
every year and the students serve as hosts at the seminars and restaurant dinners.
The department retreats are another highlight of our department life. The retreats have
been held in a mountain lodge in the Shawangunk Mountains in upstate New York, Mystic
Seaport in Connecticut and Montauk on the east tip of Long Island, among many others. In
addition to formal presentations of research findings and strategies, we discuss ways of
improving the function and atmosphere of the department as well as finding time to socialize!
Besides the scientific events, we are just as proud of our recreational activities, including
BBQ and picnic at Glen Island at the beginning of the school year, and the joyful but competitive
presentations of skits by the students and the faculty at our Holidays parties, when students and
faculty try to outwit each other in making the most fun of each other!
DR. NICHOLAS E. BAKER
!
Department of Developmental and Molecular Biology
Ullmann Building – Room 805
(718) 430-2854; nicholas.baker@einstein.yu.edu
Fundamental mechanisms of growth and development
Growth and morphogenesis are regulated during the development of multicellular organisms.
Growth stops with terminal cell cycle exit and must be up-regulated for regeneration, tissue
regulation, or response to damage. Many diseases involve disorders in these events. To study
these processes in intact animals, molecular and genetic manipulation of the fruitfly Drosophila
melanogaster permits in vivo studies not yet possible in most organisms. We are also employing
mathematical models to understand cell-cell interactions fully.
Current projects include:
1) ‘Cell competition’ is a process that occurs between cells that differ in growth, for example
because of different expression levels of ribosomal protein genes or of the proto-oncogene
myc. It is thought that cell competition may exist to identify and eliminate aneuploid cells, or
progenitor cells with reduced fitness, and that cell competition suppresses cancer and
genome damage during aging. We have identified multiple genes that are required for cell
competition and are studying the molecular mechanisms of this process and and its
contributions to aging and cancer in flies and mice.
2) HLH proteins represent a well-known class of transcription factors that are important in
development. Their ubiquitously-expressed heterodimer partners are implicated in a very
wide variety of diseases. We are studying how they are controlled both to allow
differentiation and to promote or suppress progenitor cell proliferation, processes that underly
human diseases such as Pitt-Hopkins Syndrome, schizophrenia, Fuchs corneal dystrophy,
Rett syndrome, and atherosclerosis.
These basic processes that balance growth against terminal differentiation are so fundamental
to multicellular organisms that they contribute to understanding many normal and pathological
processes. The processes we study are relevant to human diseases including many cancers,
cellular senescence, diabetes, Diamond Blackfan Anemia, neuronal dendrite development,
multiple neurodegenerative diseases, polycystic kidney disease, Sjogren’s Syndrome, and
developmental changes associated with pregnancy and lactation.
For more details, and complete list
http://fruitfly4.aecom.yu.edu/index.html
of
publications,
please
see
our
website
at:
Selected Recent Publications
Baker, N.E. and Kale, A. (2015). Ribosomal protein mutations: apoptosis, cell competition and
cancer. Mol Cell Oncol in press.
Wang, L.-H. and Baker, N.E. (2015) Salvador-Warts-Hippo pathway in a developmental
checkpoint monitoring Helix-Loop-Helix proteins. Developmental Cell 32:.191-202.
Kale, A., Li, W., Lee, C.-H. and Baker, N.E (2015) Apoptotic mechanisms during competition of
ribosomal protein mutant cells: roles of the initiator caspases Dronc and Dream/Strica. Cell
Death Different 22:1300-1312.
Fullard, J.F. and Baker, N.E. (2015) Signaling by the engulfment receptor Draper: a screen in
Drosophila melanogaster implicates cytoskeletal regulators, Jun N-terminal Kinase, and Yorkie.
Genetics, 199:117-134.
Baker, N.E. and Jenny, A. (2014).
mitochondria. Cell 158: 1240-1.
Metabolism and the other fat: a protocadherin in
Ruggiero, R., Kale, A., Thomas, B. and Baker, N.E. (2012) Mitosis in neurons: Roughex and
Anaphase Promoting Complex maintain cell cycle exit to prevent cytokinetic and axonal defects
in Drosophila photoreceptor neurons. PLoS Genetics 8:e1003049.
Bhattacharya, A. and Baker, N.E. (2011). A network of broadly-expressed HLH genes regulates
tissue-specific cell fates. Cell, 147, 881-892.
Baker, N.E. (2011). Cell competition. Curr Biol 21: R11-15.
Lubensky, D.K., Pennington, M.W., Shraiman, B., and Baker, N.E. (2011). A dynamical model
of ommatidial crystal formation. Proc Natl Acad Sci, 108: 11145-11150.
DR. TERESA BOWMAN
!
Department of Developmental and Molecular Biology
Chanin Building – Room 501
(718) 430-4001; teresa.bowman@einstein.yu.edu
Bowman Laboratory
Hematopoietic stem cells (HSCs) are one of the most widely utilized stem cell populations in the
clinic today, in large part due to their robust ability to regenerate and replace the entire blood
system after myeloablative injuries. Our research focuses on uncovering the molecular
mechanisms underlying how HSCs respond to these injuries. Specifically, we are interested in
identifying the key components for directing HSC fate decisions during regeneration. Our
studies combine the advantages of zebrafish and mammalian models to explore the
development and genetic regulation of HSC stress response. Zebrafish offer powerful genetic
pliability, easily accessible in vivo imaging, numerous transplantation assays, and screening
capabilities. Through these studies, we anticipate identifying factors that are critical in the HSC
regenerative response, which can be used to inform therapeutic strategies to improve outcomes
following myeloablative treatments as well as HSC derivation from pluripotent stem cells.
RNA regulation during HSC specification and regeneration: Proper mRNA processing is
critical to HSC regeneration and development, but which components are involved is largely
unknown. Recent identification of somatic mutations in spliceosomal components in patients
with myelodysplastic syndrome (MDS) demonstrated how deregulation of splicing could lead to
a disease state. We are combining zebrafish genetics and biochemical techniques to determine
how mutations in spliceosomal factors result in aberrant hematopoiesis. Zebrafish is well-suited
for this project as mutants in a subset of splicing factors display hematopoietic stem cell
specification defects. Additionally, we are performing a synthetic lethal screen in these mutants
to determine which epigenetic co-factors work with the splicing machinery during HSC formation
and regeneration. Determining the cellular and contextual specificity of interactions between
splicing and epigenetic factors would help reveal how mutations in such "general" machinery
could elicit such specific phenotypic outcomes in vivo.
Drug discovery for regulators of HSC regeneration: Rapid hematopoietic recovery following
myeloablative treatments, such as chemotherapy or radiotherapy, is critical to minimize
complications from infection, bleeding, or anemia. Using a candidate approach, we previously
showed that transient activation of the Wnt and BMP signaling pathways can accelerate
hematopoietic regeneration. In order to find additional pathways involved in HSC regeneration,
we plan to take advantage of the screening capabilities afforded in the zebrafish. We are
developing new transgenic zebrafish models that allow hematopoietic regeneration to be
followed in real time throughout development and into adulthood. These transgenics will be
used to screen small molecule libraries for drugs that can block or accelerate hematopoietic
recovery. The mechanism of action of the drugs will be tested using genetic, cell biological, and
biochemical assays. One of the advantages to chemical screens is the ease at which chemicals
can be tested in multiple assays. Thus, once confirmed in the zebrafish, compounds will be
tested for functional conservation in murine and human models of hematopoietic regeneration.
The identified compounds could have direct therapeutic potential in treating patients following
myeloablative regimens.
Lab website: https://sites.google.com/site/thebowmanlaboratoryeinstein/
Selected Publications
Trompouki E*, Bowman TV*, Lawton LN, Fan Z, Wu D, DiBiase A, Martin CS, Cech JN, Sessa
AK, Leblanc JL, Li P, Mosimann C, Durand E, Heffner GC, Daley GQ, Paulson RF, Young RA
and Zon LI. BMP and Wnt pathway regulation of lineage-specific gene programs underlie
Hematopoietic Regeneration. Cell, 2011; 147(3): 577-89. * These authors contributed equally.
Bowman TV, McCooey AJ, Merchant AA, Ramos CA, Fonseca P, Poindexter A, Bradfute SB,
Oliveira D, Green R, Zheng Y, Jackson KA, Chambers SM, McKinney-Freeman S, Norwood
KG, Darlington G, Gunaratne P, Steffen D, and Goodell MA. Differential mRNA Processing in
Hematopoietic Stem Cells. Stem Cells, 2006; 24(3): 662-670.
Venezia TA*, Merchant AM*, Ramos CA, Whitehouse NL, Young AS, Shaw CA, and Goodell
MA. Molecular Signatures of Proliferation and Quiescence in Hematopoietic Stem Cells. PLoS
Biology, 2004; 2(10): e301. * These authors contributed equally.
Selected Reviews
Bowman TV. Zebrafish Models to Study Stem Cells. The SAGE Encyclopedia of Stem Cell
Research, Summer 2015.
Trompouki E, King KY, Will B, Lessard J, Flores-Figueroa E, Kokkaliaris K, and Bowman T.
Bloody Signals: From birth to disease and death. Experimental Hematology, 2014; 42(12): 98994.
Bowman TV. Getting to the ncor of hematopoietic stem cell emergence. Blood, 2014; 124(10):
1541-2.
Bowman TV and Zon LI. Swimming into the Future of Drug Discovery: In Vivo Chemical
Screens in Zebrafish. ACS Chemical Biology, 2010; 5(2): 159-61.
MACROPHAGE PHAGOCYTOSIS AND MOTILITY
Dr. Dianne Cox, Ph.D.
Laboratory: Gruss (MRRC) 306 Phone: 718-430-4005
E-mail: dianne.cox@einstein.yu.edu
Cryptococcus neoformans, and Legionella
pneumophila and are internalized by
macrophages, escape destruction and grow
and divide inside the macrophage. The
phagocytic processes by which these
organisms are internalized are currently
unknown. We are currently dissecting the
downstream signaling pathways that mediate
these processes during phagocytosis.
Studying the molecular mechanisms of
chemotaxis
The directed movement of cells in response to
chemoattractants involves several complex,
interrelated processes, including directional or
chemotactic sensing, polarity, and motility.
These processes are mediated by complex,
interacting signaling pathways that appear to
have many similarities but yet have distinct
characteristics
depending
on
the
chemoattractant and receptor. We are
currently dissecting the signaling pathways
required for macrophage chemotaxis towards;
Macrophage Phagocytosis and Motility
Macrophages play an important role in host
defense against invading micro-organisms
and they are also key players in initiating and
maintaining an immune response. However,
macrophages can also play negative roles,
such as in chronic inflammatory disease.
Also, tumor-associated macrophages (TAMs),
which are present in large numbers in many
tumors, appear to play an important role in
promoting the progression of solid tumors to
an
invasive,
metastatic
phenotype.
Macrophages are therefore a prime target for
therapies, but it is important to elucidate the
mechanisms by which they are recruited to
and activated in tissues.
1. CSF-1, a growth factor for macrophage
survival and differentiation produced by
many tumors and found in high
concentrations in arthritic joints.
2. Chemokines that direct monocyte
recruitment to different tissues
Studying the molecular mechanisms of
phagocytosis
Understanding the specific signaling
components required for migration towards
each of these factors will allow us to test the
importance of these signaling components in
vivo. The ability of macrophages, altered in
pathways identified in vitro, to migrate into
the tissue space will be tested in animal
models of peritoneal infection,
atherosclerosis, and breast cancer.
Phagocytosis via receptors for the Fc portion
of IgG (Fc!Rs) or for complement (CR3)
requires actin assembly, pseudopod extension,
and phagosome closure. Actin polymerization
in response to particle binding requires the
activation of members of the Rho GTPases,
either Rac or Cdc42 for Fc!R-mediated
phagocytosis or Rho in the case of CR3mediated phagocytosis. Both Rac and
The role of macrophages in the tumor
microenvironment
Cdc42 regulate the cytoskeleton in part
through the activation of the Wiskott
Aldrich Syndrome/ WASP verprolinhomologous (WASP/WAVE) family of
proteins. Several human pathogens, such as
It is now increasingly recognized that the
tissue microenvironment plays a critical role
in tumor progression, but most studies on
5
mediated matrix degradation. Eur. J. Cell Biol.
90:205-212.
tumor metastasis involve the use of endpoint
assays or in vitro studies on cell lines. It
appears that macrophages and tumor cells
participate in a paracrine interaction, with the
tumor cells secreting CSF-1 and macrophages
secreting EGF, but the precise roles of this
paracrine interaction in tumor metastasis are
unknown. We have developed a number of in
vitro assays that reconstitute paracrine
interaction
between
macrophage
and
carcinoma cells that mimic in vivo
interactions of macrophages and tumor cells
in the tumor microenvironment that have been
observed by intravital imaging (Dovas et al.,
J. Microscopy 2012). We are currently using
these assays to understand the roles of both
soluble factors and direct interaction between
macrophages and tumor cells through
tunneling nanotubes to mediate long distance
signaling to promote tumor metastasis.
Ishihara, D., Dovas A., Park, H., Isaac, B.M.,
and Cox, D. (2012) The chemotactic defect in
Wiskott-Aldrich Syndrome macrophages is due
to reduced persistence of directional
protrusions. PLoS One 7(1):e30033
Ishihara, D.*, Dovas, A.*, Hernandez, L.,
Pozzuto, M., Wyckoff, J., Segall, J., Condeelis,
J., Bresnick, B., and Cox, D. (2013) WiskottAldrich syndrome protein regulates leukocytedependent breast cancer metastasis. Cell
Reports 4:429-436 *co-first authors
Rougerie, P., Miskolci, V. and Cox, D. (2013)
Generation of membrane structures during
phagocytosis and chemotaxis of macrophages:
role and regulation of the actin cytoskeleton.
Immunol. Reviews. 256:222-239.
Selected Publications (Students in bold)
Park, H., Dovas, A., Hanna, S., Cougoule, C,
Marridoneau-Parini, I., and Cox, D. (2014)
Tyrosine phosphorylation of Wiskott-Aldrich
Syndrome protein (WASP) by Hck regulates
Macrophage Function. J. Biol. Chem.
289(11):7897-906.
Abou-Kheir, W., Gevrey, J-C., Issac, B.M.,
Yamaguchi, H., and Cox, D. (2005) A
WAVE2/Abi1 complex mediates CSF-1induced F-actin rich membrane protrusions and
migration in macrophages. J. Cell Science
118:5369-5379.
Hanna, S.*, Miskolci, V.*, Cox, D.# and
Hodgson, L.# (2014) Development of a new
single-chain genetically encoded Cdc42 FRET
biosensor. PLoS One 9(5):e96469. *co-first
authors, #co-corresponding authors
Abou-Kheir, W.G., Isaac, B., Yamaguchi H.,
and Cox, D. (2008) Membrane targeting of
WAVE2 is not sufficient for WAVE2
dependent actin polymerization: a role for
IRSp53 in mediating the interaction between
Rac and WAVE2. J. Cell Science 121:379-90.
Miskolci, V., Hodgson, L., Cox, D., and
Vancurova, I. (2014) Western Analysis of
Intracellular
Interleukin-8
in
Human
Mononuclear
Leukocytes.
Methods
in
Molecular Biology: Cytokines. 1172:285-93.
Luo, Y., Isaac, B.M., Casadevall, A., and Cox
D.
(2009)
Macrophage
phagocytosis
suppresses F-actin enriched membrane
protrusions stimulated by CX3CL1 and CSF-1.
Infect. and Immun. 77:4487-4495.
Miskolci, V., Spiering, D., Cox, D., and
Hodgson, L. (2014) A mix-and-measure assay
for determining the activation status of
endogenous Cdc42 in cell lysates. Methods in
Molecular Biology: Cytokines. 1172:173-84.
Isaac, B.M.*, Ishihara, D.*, Nusblat, L.M.,
Gevrey, J-C, Dovas A., Condeelis, J., and Cox,
D. (2010) N-WASP has the Ability to
Compensate for the Loss of WASP in
Macrophage Podosome Formation and
Function. Exp. Cell Res. 316:3406-16. *co-first
authors
Wu, B.*, Miskolci, V.*, Donnelly, S.K., Cox,
D., Singer, R.H.% and Hodgson L.% (2015)
Synonymous
modification
of
repeated
sequences in retroviral reporters. Genes Dev.
29 (8):876-86. *co-first authors
Nusblat, L.M., Dovas, A., and Cox, D. (2011)
The essential role of N-WASP in podosome6
DR. ANA MARIA CUERVO
Department of Developmental and Molecular Biology
Chanin Building – Room 504
(718) 430-2689; ana-maria.cuervo@einstein.yu.edu
Autophagy in Aging and Age-related diseases
The main focus of our laboratory is on understanding how cytosolic proteins are transported into
lysosomes for their degradation (autophagy) and how impaired autophagy contributes to aging
and age-related diseases.
A common feature of senescent cells is the accumulation of abnormal or damaged proteins in their cytosol that,
undoubtedly, impairs cellular function. Protein accumulation results, at least in part, from impaired protein
degradation with age. Among the different systems that participate in the intracellular degradation of proteins,
lysosomes are the most affected by age. We have previously identified in many tissues of aged animals a decrease
with age in the activity of a selective pathway for the degradation of cytosolic proteins in lysosomes known as
chaperone-mediated autophagy. The main goal of our research is to identify the defect(s) that lead to the
decreased activity of chaperone-mediated autophagy with age and in age related pathologies, and to analyze if the
correction of those defects and recovery of normal proteolytic activity in old cells leads to an improvement in
cellular function.
Chaperone-mediated autophagy is responsible for the
degradation of as much as 30% of cytosolic proteins, and
it is mainly activated under conditions of stress, such as
nutrient deprivation and oxidative stress. Substrate
proteins are selectively recognized by cytosolic
chaperones (hsc70 and cochaperones) that stimulate their
binding to a glycoprotein receptor in the lysosomal
membrane (LAMP-2A). The transport of the cytosolic
proteins into lysosomes for their degradation requires
also the presence of another chaperone in the lysosomal
matrix (lys-hsc70).
Our current efforts are directed to:
Autophagic pathways in mammalian cells
1. Characterization of the different components
involved in chaperone-mediated autophagy and identification of new players for this pathway.- We can isolate
intact lysosomes from several tissues (liver, kidney and spleen) of rodents. For the identification of new CMA
components we are using different immunochemical approaches and a global proteomic approach. We have also
developed a photoswitchable reporter which allows us to identify changes in CMA in intact cells. We are currently
using this reporter to perform RNAi screenings in order to discover novel unknown regulators of this pathway.
2 Understanding the consequences of the age-related defect in chaperone-mediated autophagy.- We have
generate conditional and inducible transgenic mouse models incompetent for CMA in different tissues and have
started to investigate possible tissue-dependence differences in the requirements for functional CMA. We are also
analyzed the cellular response to CMA blockage and the different compensatory mechanisms elicited. We have
found that blockage of CMA leads to important alterations in cellular quality control and cellular metabolism and
deficiencies in the response to different stressors.
3. Consequences of impaired autophagy in age related-disorders.- In collaboration with the
groups of David Sulzer (Columbia University) and Randolph Nixon (New York University), we
have been analyzing changes in autophagy in specific neurodegenerative disorders, as example
of age-related diseases. By combining
metabolic assays, cellular fractionation
procedures and our in vitro lysosomal transport
assays in different animal and cellular models
of these diseases, we have found a primary
defect in CMA in some familial forms of
Parkinson’s disease. We are currently
analyzing other neurodegenerative disorders,
Alzheimer’s and Huntington’s disease. We are
interested in identifying the primary defect, the
compensatory mechanisms elicited by the cell
and the changes that lead to this general failure Failure of chaperone-mediated autophagy
of the proteolytic systems in these and other results in lipid accumulation (green)
age-related disorders.
References:
• Kaushik, S. Cuervo AM*. Degradation of lipid droplet-associated proteins by chaperone-mediated
autophagy facilitates lipolysis. Nat. Cell. Biol. 17: 759-70, 2015
• Arias E., Koga H, Diaz A, Mocholi E, Patel B, Cuervo AM*. Lysosomal mTORC2/PHLPP1/Akt
regulate chaperone-mediated autophagy. Mol. Cell 59, 270-84, 2015
• Park C, Shu Y, Cuervo AM*.Regulated degradation of Chk1 by chaperone-mediated autophagy in
response to DNA damage. Nat. Commun. 6:6823 doi: 10.1038/ncomms7823, 2015
• Rui Y-N, Xu Z, Patel B, Chen Z, Chen D, Tito A, David G, Sun Y, Stimming ER, Bellen H, Cuervo
AM*, Zhang S*. Huntingtin functions as a scaffold for selective macroautophagy. Nat. Cell. Biol.
17: 262-75, 2015
• Schneider S, Villarroya J, Diaz A, Patel B, Urbanska AM, Thi MM, Villarroya F, Santambrogio L,
Cuervo AM*. Loss of hepatic chaperone-mediated autophagy accelerates proteostasis failure in
aging. Aging Cell, 14:249-64, 2015
• Schneider JL, Suh Y, Cuervo AM. Deficient chaperone-mediated autophagy in liver leads to metabolic
disregulation. Cell Metab. 20:417-432, 2014
• Bejarano, E, Yuste, A, Patel B, Stout, RJ, Spary, D Cuervo AM. Connexins modulate autophagosome
biogenesis. Nat. Cell. Biol. 16:401-14, 2014
• Pampliega O, Orhon I, Patel B, Sridhar S, Diaz-Carretero A, Beau I, Codogno P, Satir B, Satir P,
Cuervo AM. Functional interaction between autophagy and ciliogenesis. Nature 502:194-200, 2013
• Orenstein SJ, Kuo SH, Tasset-Cuevas I, Arias E, Koga H, Fernandez-Carasa I, Cortes, E., Honig, L.S.,
Dauer, W., Consiglio A, Raya A, Sulzer, D, Cuervo AM. Interplay of LRRK2 with chaperonemediated autophagy. Nat. Neurosci. 16:394-406, 2013
• Anguiano J, Gaerner T, Daas B, Gavathiotis E, Cuervo AM. Chemical modulation of Chaperonemediated autophagy by novel retinoic acid derivatives. Nat. Chem. Biol. 9:374-82, 2013
Reviews:
• Schneider JL, Cuervo AM. Liver autophagy: much more than taking out trash. Nat Rev Gastroenterol
Hepatol. 11:187-200, 2013
• Kauhsik S, Cuervo AM. Chaperone-mediated autophagy: a unique way to enter the lysosome world.
Trends Cell Biol 2012, 22: 407-17, 2012
DR. ANTONIO DI CRISTOFANO
Department of Developmental and Molecular Biology
Price Center – Room 302
(718) 678-1137; antonio.dicristofano@einstein.yu.edu
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DR. ANDREAS JENNY
Department of Developmental & Molecular Biology
Chanin Building- Room 503
(718) 430-4183; andreas.jenny@einstein.yu.edu
Canonical and non-canonical Wnt signaling: patterning and cell polarization
Wnt/Wingless (Wg) growth factors commonly signal through either the canonical Wnt (Wg)-Frizzled
(Fz)/!-catenin pathway or through non-canonical Wnt pathways such as the Wnt/Fx-planar cellular polarity (PCP)
pathway, resulting the polarization of cells within the pane of the epithelium. These two pathways are highly
conserved between humans, mice, fish, and flies. Canonical Wnt/!-catenin signaling is essential for many aspects
of development. For example in vertebrates, it controls the specification of the dorsal-ventral (D-V) embryonic
axis, cell proliferation in many tissues, and the maintenance of stem cells and during vascularization. In addition,
aberrant canonical Wnt signaling in humans causes cancer. Our lab studies the function of Wnk kinases, for
which we have identified a novel role in Wnt signaling in addition to their well-known role in the regulation of ion
homeostasis in the kidney, where their lack causes hypertension (Gordon syndrome).
Non-canonical Wnt signaling established polarity within the plane o an epithelium, commonly referred to
as epithelial planar cell polarity (PCP) and allows a cell to form structures that require not only positional, but also
vectorial information. Examples of PCP in vertebrates can be very obvious, as in the ordered arrangement of
scales on fish or hairs of mammalian skin. Less visible examples are the cilia of the respiratory tract and oviduct
as well as the stereocilia of the sensory epithelium of the organ of Corti in the vertebrate inner ear. Aberrant PCP
can lead to left/right asymmetry defects, open neural tubes, deafness and kidney disease. PCP signaling is,
however, best studied in Drosophila melanogaster, mainly because of the versatility of the fly as model system.
Our lab is particularly interested in how Rho kinase (Rock) is required for the migration aspect of PCP
establishment which will help to understand tumor cell migration.
A genetic model for Endosomal Microautophagy
Autophagy delivers cytosolic components to lysosomes for degradation and is thus essential for
cellular homeostasis and to cope with different stressors. As such, autophagy counteracts various human
diseases and its reduction leads to aging like phenotypes. Macroautophagy (MA) can selectively degrade
organelles or aggregated proteins, but selective degradation of single proteins has only been described for
Chaperone-mediated autophagy (CMA) and endosomal Microautophagy (eMI). These two autophagic pathways,
described to date only in mammals, are specific for proteins containing KFERQ-related targeting motifs. In
collaboration with the Cuervo lab, we have developed a fluorescent reporter to characterize an eMI or CMA-like
process in Drosophila in vivo. Our data provide evidence for a novel, starvation inducible catabolic process
resembling endosomal microautophagy in a non-mammalian species. We are thus for the first time able to
perform genetic screens for regulatory components of eMI, this only recently identified form of autophagy about
which barely anything is known.
It is our goal to use Drosophila as model system to address fundamental questions that are relevant for
development and disease in general.
Lab homepage: http://jennylab.aecom.yu.edu/
Selected References:
1. Maung, S. M. T., Jenny, A. (2011). Planar Cell Polarity in Drosophila. Edited by Carroll, T. Organogenesis
7(3) 165-179.
2. Jenny, A. (2010) Planar Cell Polarity in the Drosophila Eye. Curr. Top. Dev. Bio. Edited by Reh, T. and
Cagan, R. Curr. Top. Dev. Bio. 93, 189-227.
3. Serysheva, E., Berhane, H., Grumolato, L., Demir, K., Balmer, S., Bodak, M. , Aaronson, S., Boutros, M.,
Mlodzik*, M., Jenny, A*. (2013). Wnk kinases are positive regulators of canonical Wnt/b-catenin signaling.
EMBO Reports 14(8), 718-725.
4. Fagan, J.K., Dollar, G., Lu, Q., Barnett, A., Pechuan Jorge, J., Schlosser, A., Pfleger, C., Adler, P., Jenny,
A..* (2014) Combover/ CG10732, a novel PCP effector for Drosophila wing hair formation. PLoS One,
9(9):e107311. doi: 10.1371. PMID: 25207969.
5. Forbes, M.M., Rothhämel, S., Jenny, A., Marlow FL. (2015). Maternal dazap2 regulates germ granules via
counteracting Dynein in zebrafish primordial germ cells. Cell Reports,12 (1), 49-57.
6. Gombos, R., Migh, E., Antal, O., Mukherjee, A., Jenny, A., Mihály, J. (2015) The Formin DAAM Functions as
Molecular Effector of the Planar Cell Polarity Pathway during Axonal Development in Drosophila. J.
Neuroscience, 35 (28),10154-67.
DR. FLORENCE MARLOW
Department of Developmental and Molecular Biology
Chanin Building – Room 514
(718) 430-4208; florence.marlow@einstein.yu.edu
Polarity in the Zebrafish Ovary
Every animal starts out as a single fertilized cell, yet we do not fully understand the events that are
essential for producing that cell because they take place within the ovary of the mother. Failure to form
an egg that is capable of embryonic development can result in profound birth defects or miscarriage. In
addition, cancers of the ovary can arise from uncontrolled proliferation of the germ cells, those cells that
can become eggs, or the somatic cells, the cells that do not develop as eggs, of the ovary. In normal
ovaries, these two types of cells communicate with one another to regulate the growth and survival of
both cell populations. In most animals, the germ line stem cells undergo an asymmetric division to
generate daughter cells that will remain stem cells and others, cystoblasts that divide and eventually form
eggs. The divisions of the cystoblasts are unique because the cells do not completely separate from one
another, but instead remain attached to each other. Studies in mammals show that the connections
between cystoblasts prevent too many cells from becoming oocytes, and in humans uncontrolled and
complete separation of cystoblasts correlates with germ cell neoplasias. However, since these events
occur before or at the time of fertilization we understand little about how the genes that are
involved. Therefore, understanding how the growth and survival of these cells is regulated has important
consequences to both fertility and cancer formation.
To study relationships between interacting cells within adjacent tissues, such as germline and somatic
follicle cells, we need to analyze an animal system in which we can manipulate genes and study early
development. The zebrafish system has advantages that allow us to use embryological, biochemical, and
genetic techniques to access maternally controlled processes during vertebrate animal development. Our
studies exploit the powerful genetics and cell biological access in the zebrafish system to unravel the
mechanisms that regulate oocyte polarization and follicle cell fate in a vertebrate. Many features of
primary oocyte development are evolutionarily conserved, including humans; thus this architecture is
likely fundamental for germline development and fertility.
Our genetic and biochemical studies of oocyte polarity have led us to genes involved in mRNA
localization and polarized transport, including motor and RNA binding proteins. Like oocytes, neurons are
highly polarized cells, which rely on trafficking and post-transcriptional regulation of mRNAs to ensure
that gene products are only expressed in discrete locations. Inappropriate accumulation of proteins and
organelles due to failed trafficking and post-transcriptional regulation is associated with neuronal loss
underlying devastating neurodegenerative diseases. The rapid development, optical clarity, and ease of
generating transgenic and mutant zebrafish strains make it an ideal system for live cell tracking and
visualization of fluorescently labeled organelles, motor proteins, mRNA cargos, and the cytoskeleton in
the living animal. To better understand how trafficking is regulated in distinct polarized cell types, we are
also using the zebrafish model system to examine the cellular and molecular basis of transport in neurons.
Knowledge of how the individual transport mechanisms operating in neurons contribute to their function
has potential to uncover novel pathological mechanisms underlying neurodegenerative diseases.
Selected Publications:
Zebrafish vasa is required for germ cell differentiation and maintenance. Hartung O, Forbes
MM, Marlow FL. Molecular Reproduction and Development. In press.
Oocyte polariry requires a Bucky ball dependent feedback amplification loop. Heim A,
Rothhämel S, Hartung O, Ferriera, E Jenny A, Marlow FL. Development. 2014 Feb;141(4):842-54. doi:
10.1242/dev.090449.PMID: 24496621.
notch3 is essential for oligodendrocyte development and vascular integrity in zebrafish.
Zaucker A, Mercurio S, Sternheim N, Talbot WS, Marlow FL. Disease Models and Mechanisms. 2013
Sep;6(5):1246-59. doi: 10.1242/dmm.012005. Epub: 2013 May 29.
Analysis of zebrafish kinesin-1 kif5 heavy chain genes during zebrafish development. Campbell,
PD, Marlow FL. Gene Expression Patterns. Epub 2013 May 15. 2013 Oct;13(7):271-9. doi:
10.1016/j.gep.2013.05.002.
The adhesion GPCR Gpr125 modulates Dishevelled distribution and planar cell polarity
signaling.Li X, Roszko I, Sepich DS, Ni M, Hamm H, Marlow FL*, Solnica-Krezel L.* Development. 2013
Jul;140(14):3028-39. doi: 10.1242/dev.094839. PMID: 23821037 *cocorresponding authors.
DR. ANNE MÜSCH
Department of Developmental and Molecular Biology
Chanin Building – Room 517
(718) 430-3508; anne.muesch@einstein.yu.edu
Signaling Mechanisms Involved in the Establishment of Epithelial Cell
Polarity
Loss of epithelial cell polarity is associated with the overwhelming majority of human cancers.
This has prompted intense research on mechanisms for epithelial polarity. Over the last decades, we have
gained insight into how epithelial cell establish polarized surface domains and carry out vectorial protein
transport, how cell-cell adhesion is mediated and how simple epithelia maintain a monolayer organization.
This groundwork has set the stage for a new challenge: to identify and characterize the signaling
mechanisms that integrate these individual aspects of epithelial polarity and architecture during
morphogenesis and in pathology. My lab is interested in characterizing such novel polarity signaling
cascades for epithelial polarity by means of cell biological approaches in established model cell lines for
non-stratified epithelia of the kidney and liver.
Most of our current effort is focused on Par1, a serine/threonine kinase that was identified as
polarity determinant in the one-cell embryo of C. elegans. We have determined that dysfunction of Par1
inhibits various aspects of epithelial polarization, notably the establishment of an apical surface domain and
protein trafficking to the apical surface, the development of a columnar cell shape and the organization of
an epithelial-specific microtubule array. Moreover, we found that Par1 is targeted by Helicobacter pylori, a
bacterium that destroys the epithelial lining of the stomach, resulting in gastritis and gastric ulcers and is
implicated in gastric cancer. We are now working to identify the Par1 substrates that regulate individual
aspects of epithelial morphology and polarity. We anticipate that this will give us mechanistic insight into
how these different aspects of polarization are linked. It is known, for instance, that protein trafficking to
the apical domain is microtubule-dependent. Thus, we expect that the Par1 substrate(s) involved in
epithelial microtubule-organization also regulate apical protein targeting and the formation of the apical
cell surface.
As few Par1 substrates are known so far, we have developed an unbiased proteome-wide screen
for novel substrates of the kinase in epithelial cells. Putative Par1 targets are tested for their Par1dependent phosphorylation in vitro and in vivo. Subsequently, we are characterizing their cell biological
roles in cultured epithelial cells.
Our repertoire of approaches include the analysis of microtubule-dynamics and of apical surface
formation in intact cells by time lapse imaging of GFP and RFP-tagged proteins, immunofluorescence and
confocal laser microscopy to study cell morphology and sub-cellular protein distribution, biochemical
assays that measure the kinetics of different protein trafficking steps and biochemical assays that measure
protein-protein interactions and enzyme activities in cell lysates.
Selected Publications:
Treyer A., Müsch, A. “Hepatiocyte Polarity” (2013), Comprehensive Physiology, 3: 243-287. Ispolatov,
I., Müsch, A. " A model for the self-organization of vesicular flux and protein distributions in the Golgi
apparatus" (2013), PLoS Comput Biol. 2013 Jul;9(7):e1003125. doi: 10.1371/journal.pcbi.1003125.
Cohen D., Fernandez D., Lazaro-Dieguez, F. and Anne Müsch,. “The serine/threonine kinase Par1b regulates
epithelial lumen polarity via IRSp53-mediated cell-ECM signaling”, (2011) J Cell Biol. 192, 525-540
Zaher Zeaiter, David Cohen, Anne Müsch, Fabio Bagnoli, Antonello Covacci, and Markus Stein “Analysis
of detergent resistant membranes of Helicobacter pylori infected gastric adenocarcinoma cells reveals a
role for MARK2/Par1b in CagA-mediated disruption of cellular polarity”, (2008) J Cellular Microbiology,
10, 781-94.
Cohen D., Tian Y and Müsch, A (2007): “Par1b promotes hepatic-type lumen polarity in MDCK cells via
myosin II and E-cadherin dependent signaling”, Mol. Biol. Cell 18, 2203-15.
Elbert, M., Cohen D.and Müsch, A, (2006): “ Par1b promotes cell-cell adhesion and inhibits Dishevelledmediated transformation of MDCK cells”, Mol.Biol.Cell, 17, 3345-55.
Cohen, D., Rodriguez-Boulan, E. and Müsch, A. (2004). "Par-1 promotes a hepatic mode of apical protein
trafficking in MDCK cells". PNAS 101, 13792-97.
Cohen, D., Brennwald, P.J., Rodriguez-Boulan, E. and Müsch, A. (2004).“Mammalian PAR-1 determines
epithelial lumen polarity by organizing the microtubule cytoskeleton”. Journal Cell Biology, 164, 717-727.
DR. NICHOLAS E. S. SIBINGA
Department of Developmental & Molecular Biology
Forchheimer Building- Room G35 S
718 430-2881; nicholas.sibinga@einstein.yu.edu
Key Words: Cardiovascular disease, inflammation, growth regulation, mouse models
CONTROL OF VASCULAR CELL GROWTH, DIFFERENTIATION, AND INJURY RESPONSE
Vascular disease, the greatest single cause of morbidity and mortality in developed societies, results from
interactions between circulating inflammatory cells, the endothelium that lines the vasculature, and underlying
vascular smooth muscle cells (VSMCs) that comprise most of the arterial wall. We seek to identify new factors and
pathways that regulate disease-associated activation of these cell types.
The underlying pathogenesis is complex: factors that impinge on these cell types include reactivated
developmental pathways, innate and acquired immune responses, and changes in cell function that result from
physical stresses, perturbed blood flow, and biochemical stimuli. Our general approach is to characterize these
responses at the molecular level, in cell culture, and in mouse models that reflect specific types of vascular injury,
including atherosclerosis, restenosis after angioplasty, saphenous vein graft disease, and transplant-associated
arteriosclerosis.
One project focuses on the atypical cadherin adhesion molecule Fat1, which is strongly induced in injured arteries.
Fat1 is a member of the ancient Fat cadherin subfamily – in Drosophila, these proteins are important regulators of
growth and planar cell polarity. Our work in mammalian VSMCs shows that Fat1 interacts with and limits the
transcriptional activity of beta-catenin, the major downstream mediator of Wnt signaling, a core developmental
pathway that regulates many aspects of metazoan embryogenesis. Fat1 also interacts with Atrophin proteins to
control VSMC directional migration. Interestingly, beta-catenin-mediated signaling in smooth muscle cells is
required for vessel wall assembly during vascular development. While beta-catenin is essential in development,
Fat1 is an important regulator of VSMC growth and differentiation in injured vessels in the adult. A second project
involves the allograft inflammatory factor-1 (Aif-1), a 17kD EF-hand protein expressed primarily in activated
macrophages. We have found that Aif-1 promotes macrophage migration, phagocytosis, survival, and selected
cytokine production. We are currently evaluating how Aif-1 contributes to integrated immune responses and
metabolism, and how loss of Aif-1 function in the mouse affects the pathogenesis of multiple inflammatory
diseases, including atherosclerosis, in which this factor is strongly induced.
In collaborative work with the Stanley lab, we have characterized a role for colony stimulating factor-1 (CSF-1), the
main regulator of macrophage survival, proliferation, and differentiation, in control of transplant-associated
arteriosclerosis, the major barrier to longterm success of organ transplants. Surprisingly, this effect appears to
involve VSMC-associated CSF-1 in an autocrine/juxtacrine mechanism that is largely independent of macrophages.
Ongoing work in these areas involves defining the molecular bases for these effects. By identifying such novel
mechanisms, we hope to find new targets for therapeutic intervention to regulate VSMC activities and improve
vascular disease prevention and treatment.
Selected References:
1. Chinnasamy P, Lutz S, Casimiro I, Brosnan C, Sibinga NES. Loss of Allograft inflammatory factor-1
ameliorates experimental autoimmune encephalomyelitis by limiting encephalitogenic CD4 T cell
expansion. Molecular Medicine, 2015 Jan 6;21(1):233-41.
2. Bruder-Nascimento T, Chinnasamy P, Riascos-Bernal DF, Cau SB, Callera GE, Touyz RM, Tostes RC,
Sibinga NE. Angiotensin II induces Fat1 expression/activation and vascular smooth muscle cell migration
via Nox1-dependent reactive oxygen species generation. J Mol Cell Cardiol. 2014 Jan;66:18-26.
3. Hiroyasu S, Chinnasamy P, Hou R, Hotchkiss K, Casimiro I, Dai XM, Stanley ER, Sibinga NES. Donor
and Recipient Cell Surface Colony Stimulating Factor-1 Promote Neointimal Formation in TransplantAssociated Arteriosclerosis. Arterioscler Thromb Vasc Biol. 2013 Jan;33(1):87-95.
4. Hou R, Sibinga NES. Atrophin proteins interact with the Fat1 cadherin and regulate migration and
orientation in vascular smooth muscle cells. J Biol Chem, 2009; 284(11):6955-65.
5. Hou R, Liu L, Anees S, Hiroyasu S, Sibinga NES. The Fat1 cadherin integrates vascular smooth muscle
cell growth and migration signals. J Cell Biol, 2006; 173(3):417-29.
DR. E. RICHARD STANLEY
Department of Developmental and Molecular Biology
Chanin Building – Room 507
(718) 430-2344; richard.stanley@einstein.yu.edu
Growth Factors and Signaling in Development:
CSF-1 Biology: Colony stimulating factor-1 (CSF-1) is a growth factor which regulates the production of
macrophages, osteoclasts, Paneth cells, microglia, neuronal cells and the function of certain non-myeloid cell
types in the female reproductive tract. It is expressed as a secreted glycoprotein, secreted proteoglycan or a
membrane-spanning, cell-surface glycoprotein. Its effects are mediated via a receptor tyrosine kinase, the c-fms
protooncoprotein. CSF-1-deficient mice are osteopetrotic due to a lack of osteoclasts, have poor fertility and
several other defects related to CSF-1 regulation of macrophages that have critical scavenger and trophic roles
in the development, maintenance or function of tissues in which they reside. CSF-1R-deficient mice have a
more severe phenotype than CSF-1-deficient mice, due to the existence of a second CSF-1R ligand, interleukin34 (IL-34), which we have recently shown also acts through another receptor, protein tyrosine phosphatase-zeta.
IL-34 is highly expressed in brain where, with CSF-1, it regulates the development and maintenance of
microglia and the differentiation of neural progenitor cells. CSF-1R regulation of macrophages is also important
in innate immunity, gut development, inflammatory diseases, atherosclerosis, obesity, cognitive disorders and
within tumors, for tumor progression and metastasis. Autocrine regulation by CSF-1 has been demonstrated in
leukemia and solid tumors. Mouse genetic and other approaches are being taken to investigate the
developmental and physiological roles of CSF-1 and IL-34, as well as their roles in disease development.
CSF-1 signal transduction: Since phosphorylation of specific CSF-1R intracellular domain tyrosine residues
initiate particular signaling pathways, detailed structure-function studies of the CSF-1R are being carried out in
macrophages and macrophage progenitor cells. In the analysis of very early post-receptor events, proteins that
are rapidly phosphorylated in tyrosine in response to CSF-1 or associated with tyrosine-phosphorylated
proteins, approximately 800 in all, have been identified by mass spectrometry. A combination of genetic,
proteomic, biochemical and analytical imaging approaches are being used to elucidate the roles and interactions
of these signaling proteins in the immediate post-receptor events in CSF-1 signal transduction. Previous studies
have identified and elucidated the actions of the protein tyrosine phosphatase SHP-1, that negatively affects cell
survival in the absence of CSF-1, the cbl proto-oncogene product, that negatively regulates CSF-1 proliferation
signaling by enhancing CSF-1R endocytosis and the protein tyrosine phosphatase, PTP-phi, that mediates CSF1-induced morphological changes, adhesion and motility, via its action on a specific substrate, paxillin. Current
work is focused on Dok-1, that regulates macrophage motility and the macrophage F-actin associated and
tyrosine phosphorylated protein (MAYP/PSTPIP2). PSTPIP2 is an F-BAR protein that coordinates processes
involving the cell membrane and actin cytoskeleton. It is a negative feedback regulator of CSF-1R-mediated
osteoclast and macrophage production and loss-of-function mutations in PSTPIP2 lead to an autoinflammatory
disease involving these cell types.
Signaling by the Shark tyrosine kinase: Embryonic dorsal closure in Drosophila is a series of morphogenetic
movements involving the bilateral dorsal movement of the epidermis (cell stretching) and dorsal suturing of the
leading edge cells to enclose the viscera. The Syk family tyrosine kinase, Shark, is expressed in the epidermis
and plays a crucial role in this Jun kinase-dependent process where, with the adapter, Ddok, it acts upstream of
JNK in leading edge cells. Shark is also expressed by glial cells, where it functions downstream of the Draper
receptor and Src42A to regulate the recognition and engulfment of dying neuronal cells. Mutations in the genes
for Shark-interacting proteins, coupled with cell biological approaches, are being used to define Shark function
and the Shark signaling pathways.
Selected Publications:
Cecchini, M.G., Dominguez, M.G., Mocci, S., Wetterwald, A., Felix, R., Fleisch, H., Chisholm, O., Pollard,
J.W., Hofstetter, W. and Stanley, E.R. (1994) Role of colony stimulating factor-1 in the establishment and
regulation of tissue macrophages during post natal development of the mouse. Development 120:1357-1372.
Dai, X-M., Ryan, G.R., Hapel, A.J., Dominguez, M.G., Kapp, S., Sylvestre, V. and Stanley, E.R. (2001)
Targeted disruption of the mouse CSF-1 receptor gene results in osteopetrosis, mononuclear phagocyte
deficiency, increased splenic progenitor cell frequencies and reproductive defects. Blood 99:111-120.
Dai X-M, Zong XH, Sylvestre V and Stanley ER. (2004) Incomplete restoration of colony stimulating factor-1
(CSF-1) function in CSF-1-deficient Csf1op/Csf1op mice by transgenic expression of cell surface CSF-1. Blood
103: 1114-1123.
Nandi, S., Akhter, M.P., Seifert, M.F., Dai, X-M. and Stanley, E.R. (2006). Developmental and functional
significance of the CSF-1 proteoglycan chondroitin sulfate chain. Blood 107(2):786-795.
Huynh, D., Dai, X-M., Nandi, S., Lightowler, S., Trivett, M., Chan, C-K., Bertoncello, I., Ramsay, R.G. and
Stanley, E.R. (2009). CSF-1 dependence of Paneth cell development in the mouse small intestine.
Gastroenterology 137(1):136-144.
Yeung, Y-G. and Stanley, E.R. (2003) Proteomic approaches to analysis of the early events in CSF-1 signal
transduction. Molecular and Cellular Proteomics. 2: 1143-1155
Wei, S., Nandi, S., Chitu, V., Yeung, Y-G., Yu, W., Huang, M., Williams, L.T., Lin, H. and Stanley, E.R.
(2010). Functional overlap but differential expression of CSF-1 and IL-34 in their CSF-1 receptor-mediated
regulation of myeloid cells. J Leukoc. Biol. 88(3):495-505.
Ginhoux, F., Greter, M., Leboeuf, M., Nandi, S., See, P., Gokhan, S., Mehler, M.F., Ng, L.G., Stanley, E.R.,
Samokhvalov, I.M. and Merad, M. Fate mapping studies reveal that adult microglia derive from primitive
macrophages. Science 330(6005):841-845.
Nandi, S., Gokhan,S., Dai, X-M., Wei, S., Enikolopov, G., Lin,H., Mehler, M.F. and Stanley E.R. (2012). The
CSF-1 receptor ligands IL-34 and CSF-1 exhibit distinct developmental brain expression patterns and regulate
neural progenitor cell maintenance and maturation. Developmental Biology 367:100-13. PMID: 22542597.
Nandi, S., Cioce, M., Yeung, Y.G., Nieves, E., Tesfa, L., Lin, H., Hsu, A.W., Halenbeck R., Cheng, H.Y.,
Gokhan, S., Mehler, M.F., Stanley, E.R.(2013).Receptor-type protein tyrosine phosphatase zeta is a functional
receptor for interleukin-34. J. Biol. Chem., 288:21972-86. PMID: 23744080
Wang, Y., Yeung, Y.G., Langdon, W.Y. and Stanley, E.R. (1996) c-Cbl Is Transiently Tyrosine
phosphorylated, Ubiquitinated, and Membrane-targeted following CSF-1 Stimulation of Macrophages. J. Biol.
Chem. 271:17-20.
Lee, P.S.W., Wang, Y., Dominguez, M.G., Yeung, Y-G., Murphy, M.A. Bowtell, D.D.L. and Stanley, E.R.
(1999) The Cbl protooncoprotein stimulates CSF-1 receptor multiubiquitination and endocytosis, and
attenuates macrophage proliferation. EMBO J. 18:3616-3628.
Yeung, Y.G., Soldera, S. and Stanley, E.R. (1998) A novel macrophage actin-associated protein (MAYP) is
tyrosine phosphorylated following CSF-1 stimulation. J. Biol. Chem. 273:30638 30642.
Chitu, V., Pixley, F.J., Yeung, Y.G., Macaluso, F., Condeelis, J and Stanley, E.R. (2005). The PCH family
member MAYP/PSTPIP2 directly regulates F-actin bundling and enhances filopodia formation and motility in
macrophages. Mol. Biol. Cell. 16(6):2947-2959.
Grosse, J., Chitu, V., Marquardt, A., Hanke, P., Schmittwolf, C., Zeitlmann, L., Schropp, P., Barth, B., Yu, P.,
Paffenholz, R., Stumm, G., Nehls M., and Stanley E. R. (2006). Mutation of mouse MAYP/PSTPIP2 causes a
macrophage autoinflammatory disease. Blood 107(8):3350-3358.
Chitu, V. and Stanley E. R. (2006). Colony stimulating factor-1 in immunity and inflammation. Current
Opinion in Immunology 18(1):39-48.
Chitu, V. and Stanley, E.R. (2007). Pombe Cdc15 homology (PCH) proteins: coordinators of membranecytoskeletal interactions. Trends in Cell Biol. 17(3):145-156.
Chitu V., Nacu, V., Charles, J.F., Henne, W.M., McMahon, H.T., Nandi, S., Ketchum, H., Harris, R.,
Nakamura, M.C., Stanley, E.R. (2012) PSTPIP2 deficiency in mice causes osteopenia and increased
differentiation of multipotent myeloid precursors into osteoclasts. Blood, in press.
Pixley, F.J., Lee, P.S.W., Condeelis, J. and Stanley, E.R. (2001) Protein tyrosine phosphatase phi regulates
paxillin tyrosine phosphorylation and mediates Colony Stimulating Factor 1 induced morphological changes in
macrophages. Mol. Cell. Biol. 21:1795-1809.
Wyckoff, J., Wang, W., Lin, E.L., Wang, Y., Pixley, F.J., Stanley, E.R., Graf, T., Pollard, J.W., Segall, J. and
Condeelis, J. (2004). A paracrine loop between tumor cells and macrophages is required for tumor cell
migration in mammary tumors. Cancer Res. 64(19):7022-7029.
Patsialou, A., Wyckoff, J., Wang, Y., Goswami, S., Stanley, E.R. and Condeelis J.S. (2009). Invasion of human
breast cancer cells in vivo requires both paracrine and autocrine loops involving the colony stimulating factor-1
receptor. Cancer Research 69(24):9498-9506.
Pixley, F.J. and Stanley, E.R. (2004) CSF-1 Regulation of the Wandering Macrophage: Complexity in action.
Trends in Cell Biol. 14:628-638.
Xiong, Y., Song, D., Cai, Y., Yu, W., Yeung, Y.G., and Stanley, E.R. (2010). A CSF-1 receptor
phosphotyrosine 559 signaling pathway regulates receptor ubiquitination and tyrosine phosphorylation. J. Biol.
Chem. 286(2):952-960.
Sampaio, N., Yu, W., Cox, D., Wyckoff, J., Condeelis, J., Stanley, E.R. and Pixley, F.J. (2011).
Phosphorylation of Y721 of the CSF-1R mediates PI3K association to regulate macrophage motility and
enhancement of tumor cell invasion. J. Cell Sci. 124(Pt 12):2021-2031.
Yu, W., Chen, J., X. Ying, Pixley, F.J., Yeung, Y.G., and Stanley, E.R. (2012). Macrophage proliferation is
regulated through CSF-1 receptor tyrosines 544, 559 and 807. J. Biol.Chem. 287:13694-704. PMID: 22375015.
Fernandez, R., Takahashi, F., Liu, Z., Steward, R., Stein, D. and Stanley, E.R. (2000) The Drosophila Shark
tyrosine kinase is required for embryonic dorsal closure. Genes and Development 14:604-614.
Biswas, R., Stein, D. and Stanley, E.R (2006). Drosophila Dok is required for embryonic dorsal closure.
Development 133(2):217-227.
Zeigenfuss, J.S.*, Biswas, R.*, Avery, M.A., Sheehan, A.E., Hong, K., Yeung Y-G., Stanley, E.R.** and
Freeman, M.R.** (2008). Draper-dependent glial phagocytic activity is mediated by Src and Syk family kinase
signaling. Nature 453(7197):935-939.
DR. AMIT K. VERMA
Department of Developmental and Molecular Biology
Chanin Building – Room 302B
(718) 430-8761; amit.verma@einstein.yu.edu
Key Words: cytokines, hematopoiesis, epigenetics, mds, leukemia, esophageal cancer,
pancreatic cancer
1.
Targeting signal transduction in hematologic malignancies: Cytokines play important roles in the
regulation of normal hematopoiesis and a balance between the actions of hematopoietic growth factors
and myelosuppressive factors is required for optimal production of different hematopoietic cell lineages.
We study the role of MAP kinases in the regulation of hematopoiesis and have shown that the p38 MAPK
signaling pathway is the dominant cytokine regulated inhibitory pathway in human hematopoiesis and is
overactivated in MDS. The myelodysplastic syndromes (MDS) are collections of heterogeneous
hematologic diseases characterized by refractory cytopenias due to ineffective hematopoiesis. These
preleukemic disorders are common causes of anemia in the elderly and are rapidly increasing in incidence.
We have also demonstrated that the TGF-b pathways is overactivated in MDS. We are trying to study the
molecular mechanisms that lead to the activation of these pathways in MDS and are using small molecule
inhibitors in mouse models to target these pathways.
2.
Epigenomic analysis of tumors: We are using high throughput assays to analyze DNA methylation
across the genome and have optimized these assays to work with low amounts of DNA. We have
successfully used these assays in conducting an integrative analysis of epigenetic and genetic alterations
in MDS and esophageal cancer. We are now exploring alterations in DNA methylation in pancreatic
cancer, myeloproliferative neoplasms and renal cell cancer.
3.
Clinical studies in Myelodysplastic syndromes: We have a “center of excellence” clinic for
patients with Myelodysplatic syndromes. We offer a variety of clinical trials for these patients
(www.mdstreatment.com).
Selected References:
1.
Zhou L, Opalinska J, Sohal D, Yu Y, Mo Y, Bhagat T, Abdel-Wahab O, Fazzari M, Figueroa M, Alencar C,
Zhang J, Kanbhampati S, Parmar S, Nischal S, Heuck C, Suzuki M, Friedman E, Pellagatti A, Boultwood J, Steidl
U, Sauthararajah Y, Yajnik V, Gore SD, Platanias LC, Levine R, Melnick A, Greally JM, Verma A. Aberrant
epigenetic and genetic marks are seen in myelodysplastic leucocytes and reveal DOCK4 as a candidate
pathogenic gene on chr7q. J Biol Chem. 2011 Apr 30. PMID: 21532034
2.
Alvarez H, Opalinska J, Zhou L, Sohal D, Fazzari M, Yu Y, Montagna C, Montgomery E, Canto M, Dunbar K,
Wang J, Roa J, Mo Y, Bhagat T, Ramesh K, Cannizzaro L, Mollenhauer J, Thompson R, Suzuki M, Meltzer S,
Melnick A, Greally JM, Maitra A, Verma A Widespread hypomethylation occurs early and synergizes with
gene amplification during esophageal carcinogenesis. PLOS Genetics 2011 Mar;7(3) PMID: 21483804
4.
L Zhou, C McMahon, T Bhagat, C Alencar, Y Yu, M Fazzari, D Sohal, C Heuck, K Gundabolu, C Ng, Y Mo, W
Shen, A Wickrema, G Kong, E Friedman, L Sokol, G Mantzaris, A Pellagatti , J Boultwood, LC. Platanias. U
Steidl, L Yan, JM Yingling, MM Lahn, A List, M Bitzer and A Verma Reduced SMAD7 leads to overactivation
of TGF-beta signaling in MDS that can be reversed by a specific inhibitor of TGF-beta receptor I kinase.
Cancer Research 2011 Feb 1;71(3):955-63.PMID: 21189329
5.
Sohal D, Yeatts A, Ye K, Pellagatti A, Zhou L, Pahanish P, Mo Y, Bhagat T, Mariadason J, Boultwood J, Melnick
A, Greally JM, Verma A. Meta-analysis of microarray studies reveals a novel hematopoietic progenitor cell
signature and demonstrates feasibility of inter-platform data integration. PLOS One 2008 Aug 13;3(8):e2965
PMID: 18698424
6.
Zhou L, Nguyen AN, Sohal D, Ma JY, Pahanish P, Gundabolu K, Hayman J, Chubak A, Mo Y, Bhagat T, Das B,
Haghnazari E, Navas T, Parmar S, Kambhampati S, Pellagati A, Braunchweig I, Zhang YE, Wickrema A,
Boultwood J, Platanias LC, Higgins LS, List A, Bitzer M, Verma A. Inhibition of Transforming Growth Factor
beta receptor I kinase can stimulate hematopoiesis in MDS Blood 2008 Oct 15;112(8):3434-43 PMID:
18474728
7.
Navas T, Mohindru M, Estes M, Ma JY, Sokol L, Pahanish P, Parmar S, Haghnazari E, Zhou L, Collins RC, Kerr
I, Nguyen A, Xu Y, Platanias LC, List AA, Higgins LS, Verma A. Inhibition of overactivated p38 MAPK can
restore hematopoiesis in myelodysplastic syndrome progenitors Blood 2006, 15;108(13):4170-7 PMID:
16940419
DR. MARGARITA VIGODNER
!
Department of Developmental and Molecular Biology
Chanin Building – Room 501
(718) 430-4001; teresa.bowman@einstein.yu.edu
Understanding the role of sumoylation during spermatogenesis
Post-translational modification by Small Ubiquitin-like Modifiers or SUMO proteins has been
identified as an important regulatory event that is implicated in several cellular processes,
seemingly based on the cell type. Recent findings from our laboratory suggest diverse and
potentially multiple roles of SUMO in testicular function and spermatogenesis however, SUMO
targets remained uncharacterized in the testis due to the complex multicellular nature of
testicular tissue, the inability to maintain and manipulate spermatogenesis in vitro, and the
technical challenges involved in identifying low-abundance endogenous SUMO targets. My
laboratory performed cell-specific identification of sumoylated proteins using concentrated cell
lysates prepared with de-sumoylation inhibitors from germ cells and sperm. Hundred and twenty
proteins were uniquely identified in specific germ cell fractions. The identified proteins are
involved in the regulation of transcription, stress response, microRNA biogenesis, regulation of
major enzymatic pathways, nuclear-cytoplasmic transport, cell cycle control, acrosome
biogenesis, and other processes. Several proteins with important roles during spermatogenesis
were chosen for further characterization by co-immunoprecipitation, co-localization and in-vitro
sumoylation studies. Many of these proteins have a unique role in spermatogenesis, particularly
during meiotic progression. Interestingly, several kinases have been identified as sumoylated
targets. This research opens a novel avenue for numerous further studies of SUMO at the level
of individual targets. Studies are currently underway to understand how sumoylation affects
activity of several targets, in particular the activity of kinases and phosphatases important for
spermatogenesis.
Characterization of new regulators of WNT signaling in lung diseases
Wnt signaling is required for airway epithelial differentiation and healing. A decrease in Wnt
target genes and an increase in Wnt inhibitors was reported in bronchial epithelial cells of
smokers and COPD (Chronic obstructive pulmonary disease) patients, and in experimental
mouse models of emphysema. Notably, preventive and therapeutic WNT/!-catenin activation
led to a significant reduction of experimental emphysema. Therefore, targeting the Wnt pathway
is a promising approach to the development of therapies for lung diseases; however, the
question of where and how to target Wnt pathway remains open. CDK14 was identified by our
group as a gene significantly down-regulated by tobacco exposure in vivo in testes ands lungs.
It has recently been discovered that Wnt signaling is activated by Cdk14/ cyclin Y complex
through phosphorylation of LRP6 co-receptor, a key regulator of the WNT pathway. We have
recently shown that CDK14 is expressed in bronchial and alveolar epithelium and that treatment
of lung cell lines including primary normal human bronchial cells (NHBE) with CS decreased the
level of CDK14; COPD-derived human bronchial cell (DHBE) showed reduced CDK14 levels
versus NHBE. Furthermore, preliminary observations suggest a lower number of CDK14
positive cells in emphysema versus normal lungs. The research in our laboratory currently
focuses on 1) performing a screening and quantitative analysis of COPD samples as well as
smokers versus non-smokers for the levels of CDK14/ Cyc Y and Wnt pathway activity;
2).Testing whether down-regulation of CDK14 and/or CycY gene expression inhibits Wnt
signaling in NHBE and small airway epithelial cells (SAEC), and whether restoration of the
normal level of CDK14/CycY in COPD-derived cells would activate Wnt signaling.3)
comparative analysis of quantitative changes in phosphoproteome of NHBE before and after
the down-regulation of CDK14 and/or CycY in order to identify CDK14 /CycY downstream
targets and molecular pathways. The results from this research will lead to a better
understanding of the molecular pathology of COPD in order to use the WNT pathway as a
diagnostic and therapeutic target for lung diseases.
Lab homepage: http://einstein.yu.edu/labs/margarita-vigodner/research-projects/
Selected Publications
•
•
•
Vigodner, M., Shrivastava V, Gutstein LE, *Schneider J, Nieves E, Goldstein M,
Feliciano M, Callaway M., Localization and identification of sumoylated proteins in
human sperm: excessive sumoylation is a marker of defective spermatozoa. Hum
Reprod, 2013. 28(1): p. 210-23. PMID:23077236.
Xiao Y, Pollack D, Nieves E, Winchell A, Callaway M, Vigodner M. Can your protein be
sumoylated? A quick summary and important tips to study SUMO-modified proteins.
Anal Biochem. 2014 Nov 29. pii: S0003-2697(14)00527-2. PMID:25454506.
Pollack D, XiaoY, Shrivasatava V, Levy A, Andrusier M, Jeanine D'Armiento
J, Holz MK, and Vigodner M.; CDK14 expression is down-regulated by cigarette smoke
in vivo and in vitro, Toxicology Letters, 2015; 234 :120-30. PMID: 25680692
DR. DUNCAN W. WILSON
Department of Developmental and Molecular Biology
Chanin Building – Room 513
(718) 430-2305; duncan.wilson@einstein.yu.edu
!
Viruses, cancer and the nervous system
Humans are infected by at least eight different species of herpes viruses. These pathogens cause several
forms of cancer, severe birth defects or miscarriage, and are a leading cause of organ transplant rejection.
Our interests are focused on the Kaposi’s sarcoma herpes virus (KSHV) and Herpes Simplex Virus (HSV).
KSHV: a cancer-causing virus of humans
Kaposi’s sarcoma-associated herpesvirus (KSHV) is the etiological agent of AIDS-associated and endemic
Kaposi’s sarcoma (KS), primary effusion lymphoma (PEL) and multicentric Castleman’s disease. In all these
cancers the KSHV-encoded protein K15p plays a pivotal role. In KSHV-infected B cells, the principal reservoir
of the virus in infected individuals, K15p drives cell proliferation and B cell-transformation to primary effusion
lymphomas. K15p then keeps these lymphoma cells alive by “pretending” to be an activated B-cell receptor
(BCR), ensuring continued cell-proliferation and suppressing apoptosis. In Kaposi’s sarcoma (the archetypal
malignancy of AIDS patients) K15p adopts a different disguise; this time impersonating an activated vascular
endothelial growth factor receptor (VEGF-R) in KSHV-infected endothelial cells. This drives angiogenesis and
results in the extensive vascularization that is characteristic of KS tumors. We are studying the unusual and
dramatic mechanism by which K15p is processed to its active receptor-mimicking form (Fig. 1).
Fig. 1. K15p: the “Great Pretender” of Kaposi’s sarcoma virus.
K15p is initially synthesized as a ~45kDa protein with twelve
membrane-spanning domains and a C-terminal cytoplasmic tail that
recruits cellular signaling molecules (orange ovals). Lipid bilayer is
in pale green. An unknown processing mechanism, the focus of our
interests, cleaves the carboxy-terminal region of the molecule (in
blue) from the rest of the molecule (in green) to generate an active
mimic of the B-cell and VEGF-Receptors (see text).
HSV: a pathogen of the nervous system
Herpes Simplex Virus (HSV) is a leading cause of blindness, fetal mortality and severe neurodevelopmental
and other birth defects. These diseases are a direct result of the ability of the virus to invade, manipulate and
traffic within the human nervous system. Our laboratory is dissecting the molecular machinery that HSV uses
to achieve assembly and transport within neurons (Fig. 2) (1, 2).
Fig. 2. HSV makes a break for it. Left hand image: HSV initially assembles clusters of capsids in the
neuronal cell nucleus. These then fill with viral DNA, visible as a dense core (arrow at right). They then traffic
to, and bud into, the nuclear membranes (left hand arrow) to enter the cytoplasm and neuronal axons. Right
hand image: Similar to left hand but at higher magnification, showing a DNA-containing HSV capsid punching
through the nuclear membranes.
Once in the cell cytoplasm HSV capsids dock with, and become enveloped by, cytoplasmic organelles
to assemble the mature infectious virus. These then traffic along neuronal microtubules to travel within the
nervous system. The events of virus assembly, and the detailed molecular structure of assembly and
trafficking intermediates are very poorly understood. Most recently, in collaboration with the analytical imaging
facility (AIF) here at the Albert Einstein College of Medicine we have pioneered the application of Correlative
Light and Electron Microscopy to the study of HSV assembly in the neuronal cell body (Fig. 3) (3).
Fig. 3. What to do when more light doesn’t help you see better. Correlative light and electron microscopy
makes it possible to observe HSV assembly simultaneously by fluorescence microscopy and electron
microscopy. Rows A, B and C represent three different examples of HSV capsids assembling onto cellular
organelles in the cytoplasm of infected neurons. In each row a light-microscopic fluorescence image is shown
in the left hand panel, an electron-microscopic image of the same field is in the right hand panel and an
alignment of the two is in the middle panel. Green light is being emitted by HSV capsids engineered to contain
molecules of Green Fluorescent Protein (GFP). Red light is coming from the membranes of cellular organelles
that have been stained by a fluorescent lipophilic dye. Yellow shows where Green and Red light overlap. Until
the application of this technology our understanding of HSV assembly was limited to the level of resolution
shown in the leftmost panels. Scale bars: 500nM.
Selected References
1.
2.
3.
Kharkwal H., Smith C.G., Wilson D.W. (2014). Blocking ESCRT-mediated envelopment inhibits
microtubule-dependent trafficking of alphaherpesviruses in vitro. Journal of Virology 88:14467-14478.
Shanda S.K., Wilson D.W. (2008). UL36p is required for efficient transport of membrane-associated
herpes simplex virus type 1 along microtubules. Journal of Virology 82:7388-7394.
Kharkwal H., Shanda S.K., Wilson D.W. (2015). HSV capsid/organelle association in the absence of
the tegument protein UL36p. Journal of Virology. Manuscript Submitted.
DR. FAJUN YANG
Department of Developmental & Molecular Biology
Price Center - Room 377
(718) 678-1142; fajun.yang@einstein.yu.edu
Key Words: gene expression, lipid metabolism, metabolic syndrome, obesity
Dysregulation of lipid homeostasis is common in major human diseases, including type 2 diabetes, obesity, fatty liver
diseases, atherosclerosis and some types of cancer. Our laboratory is interested in the transcriptional control of lipid
metabolism. In addition to DNA-binding transcription factors, gene expression in eukaryotic cells often requires a series
of transcriptional cofactors. We use biochemical and genetic approaches to study the physiological and pathophysiological
functions and regulation of the network of transcription factors and their cofactors in the liver (hepatocytes) and adipose
tissues (white, brown and beige adipocytes). A focus of our work is the multi-subunit Mediator complex, which is a highly
conserved transcriptional cofactor. The mammalian Mediator is one of the cofactors for the sterol regulatory element binding proteins (SREBP), the master regulators of both fatty acid/triglyceride and cholesterol biosynthesis. SREBP
proteins are synthesized as inactive precursors that are tethered to the endoplasmic reticulum membrane. When lipid
biosynthesis is demanded, SREBP precursors are proteolytically processed to mature forms of transcription factors that
migrate into the nucleus, interact with cofactors including the Mediator complex, and activate transcription of target
genes, which encode the rate-limiting enzymes in synthesizing fatty acids/triglycerides and cholesterol. Through the
protein-protein interactions of different subunits with SREBP as well as other transcription factors, the Mediator complex
critically regulates lipid metabolism. Moreover, dynamic remodeling of the Mediator complex conveys the metabolic
signals to transcriptional outputs, providing another layer of regulation of gene expression. With the ultimate goal of
identifying novel targets for preventing or treating metabolic syndrome (including type 2 diabetes, obesity, fatty liver
disease, hyperlipidemia and hypertension), our research will advance our understanding of how lipid homeostasis is
regulated at the molecular level.
Selected Publications:
Abdulla A, Zhang Y, Hsu FN, Xiaoli AM, Zhao X, Yang ES, Ji JY, Yang F. Regulation of lipogenic gene expression by
lysine-specific histone demethylase-1 (LSD1). J Biol Chem. 2014, 289(43): 29937-47.
Zhao X, Feng D, Wang Q, Abdulla A, Xie XJ, Zhou J, Sun Y, Yang ES, Liu LP, Vaitheesvaran B, Bridges L, Kurland IJ, Strich
R, Ni JQ, Wang C, Ericsson J, Pessin JE, Ji JY, Yang F. Regulation of lipogenesis by cyclin-dependent kinase 8-mediated
control of SREBP-1. J Clin Invest. 2012, 122(7): 2417-27.
Mulligan P, Yang F, Di Stefano L, Ji JY, Ouyang J, Nishikawa JL, Toiber D, Kulkarni M, Wang Q, Najafi-Shoushtari SH,
Mostoslavsky R, Gygi SP, Gill G, Dyson NJ, Näär AM. A SIRT1-LSD1 Corepressor Complex Regulates Notch Target
Gene Expression and Development. Mol Cell. 2011, 42(5): 689-699.
Walker AK, Yang F, Jiang K, Ji JY, Watts JL, Purushotham A, Boss O, Hirsch ML, Ribich S, Smith JJ, Israelian K, Westphal
CH, Rodgers JT, Shioda T, Elson SL, Mulligan P, Najafi-Shoushtari H, Black JC, Thakur JK, Kadyk LC, Whetstine JR,
Mostoslavsky R, Puigserver P, Li X, Dyson NJ, Hart AC, Näär AM. Conserved role of SIRT1 orthologs in fastingdependent inhibition of the lipid/cholesterol regulator SREBP. Genes & Development, 2010, 24(13): 1403-1417.
Morris EJ!, Ji JY!, Yang F, Di Stefano L, Herr A, Moon N, Kwon E, Haigis KM, Näär AM, Dyson NJ. E2F1 represses bcatenin transcription and is antagonized by both pRB and CDK8. Nature, 2008, 455(7212): 552-556. (!Equal
contribution)
Thakur JK!, Arthanari H!, Yang F!, Pan SJ, Fan X, Breger J, Frueh DP, Gulshan K, Li DK, Mylonalis E, Struhl K, MoyeRowley WS, Cormack BP, Wagner G, Näär AM. A nuclear receptor-like pathway regulating multidrug resistance in fungi.
Nature (Article), 2008, 452(7187): 604-609. (!Equal contribution)
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DR. LIANG ZHU
Department of Developmental and Molecular Biology
Ullmann Building – Room 521
(718) 430-3320; liang.zhu@einstein.yu.edu
Key Words: tumor suppressor, cell cycle, cell death, cancer, inhibiting cancer, metabolism
and obesity, mouse models
CONTROL OF TUMORIGENESIS, OBESITY, AND REGENERATION BY
TUMOR SUPPRESSORS AND ONCOPROTEINS
A common feature shared by cancer, obesity, and tissue regeneration is deregulation of cellular homeostasis,
including cell proliferation, cell growth, cell metabolism, and cell death. Tumor suppressors such as pRb and p53,
oncoproteins such as Ras and Myc, and organ size regulators such as YAP, are the major regulators of these
aspects of cellular homeostasis. We are studying how these regulators regulate these aspects.
In the cancer field, we are identifying treatment strategies for tumors that have permanently lost the tumor
suppressor pRb or both pRb and p53 using mouse tumor models. pRb and p53 are the two major tumor
suppressors. Their functions are activated by oncogenic events and they implement most and best cells’ antitumor
mechanisms to safeguard against tumorigenesis. When tumorigenesis succeeds, pRb, p53, or both is frequently
inactivated. In reverse, reactivation of pRb and p53 are rationale for most antitumor therapeutics. However,
when pRb and p53 are genetically inactivated, cells irreparably lose the antitumor mechanisms afforded by them,
which may explain why advanced cancers are difficult to treat. We generated mouse tumor models in which the
genes for pRb or both pRb and p53 are knocked out to identify mechanisms that can still inhibit these tumors. We
showed that combining deletion of Skp2, which is a target of repression by pRb, completely blocked tumorigenesis
in the absence of pRb or both pRb and p53 (refs 2, 5, 7, 8). Ongoing studies aim to target functions of Skp2 in
regulating p27 degradation, regulating cancer cell metabolism, and regulating epithelial-to-mesenchymal transition
to inhibit pRb and p53 doubly deficient tumors (ref 9).
In the obesity field, we are studying the function of pRb in hypothalamus POMC neurons. These neurons negatively
regulate feeding, and it has been suggested that high fat feeding could damage these neurons, which results in more
desire to eat. This feed-forward circle may explain why obesity is difficult to treat. We determined that high fat
feeding induced or activated a kinase in POMC neurons to phosphorylate and, therefore, functionally inactivate
pRb. Interestingly, in this context, inactivation of pRb did not induce tumorigenesis. On the contrary, it induced
degenerative changes in POMC neurons. When we deleted pRb in POMC neurons, the mice increased their food
intake and become obese, demonstrating that pRb functions to maintain POMC neuron homeostasis to suppress
obesity. To our surprise, in AGRP neurons, which reside together with POMC neurons in hypothalamus and
functionally antagonize POMC neurons to reduce the desire to feed, pRb function is not important. Deletion of
pRb in AGRP neurons did not harm AGRP neuron homeostasis. Based on these findings (ref 6, in collaboration of
with Dr. Chua of the Einstein Diabetes Center), we are identifying the kinases that phosphorylate pRb in POMC
neurons after high fat feeding, and establishing techniques to prevent pRb phosphorylation following high fat
feeding. Through these studies, we aim to block the feed-forward circle to treat obesity.
The liver has remarkable ability to regenerate when more than 70% of it is resected. This regenerative capability
has important implication to tumorigenesis and regenerative medicine. Since the supply of donor liver is far
smaller than the demand, hepatocyte transplantation is the best approach to treating liver failure and liver defects.
We showed that by deleting the cyclin-dependent kinase inhibitor p27, hepatocytes were stimulated to proliferate
more in the host liver to save mice from liver failure (ref 1). Unfortunately, deleting p27 also increased liver
cancer burden during liver carcinogenesis by chronic HBV or chemical DEN (ref 3, 4). In collaboration with Dr.
Shafritz of the Einstein Liver Center, we are now studying the liver size regulator YAP to determine its ability to
increase hepatocyte proliferation following transplantation and its associated liver cancer risk.
Selected References:
1.
Karnezis, A.N., Dorokhov, M., Grompe, M. and Zhu, L. (2001) Loss of p27Kip1 enhances the
transplantation efficiency of hepatocytes transferred into diseased livers. J Clin Invest. 108:383-390.
2.
Ji, P., Jiang, H., Rekhtman, K., Bloom, J., Ichetovkin, M., Pagano, M., and Zhu, L. (2004) An Rb-Skp2-p27
pathway mediates acute cell cycle inhibition by Rb and is retained in a partial penetrance Rb mutant. Mol
Cell. 16:47-58.
3.
Sun, D., Ren, H., Oertel, M., Sellers, R.S., and Zhu, L. (2007) Loss of p27Kip1 enhances tumor
progression in chronic hepatocyte injury-induced liver tumorigenesis with widely ranging effects on Cdk2
or Cdc2 activation. Carcinogenesis. 28:1859-1866.
4.
Sun, D., Ren, H., Oertel, M., Sellers, R.S., Shafritz, D.A., and Zhu, L. (2008) Inactivation of p27Kip1
promotes chemical mouse liver tumorigenesis in the resistant strain C57BL/6J. Mol Carcinog. 47:47-55.
5.
Wang, H., Bauzon, F., Ji, P., Xu, X., Sun, D., Locker, J., Sellers, R.S., Nakayama, K., Nakayama, K.I.,
Cobrinik, D., and Zhu, L. (2010) Skp2 is required for survival of aberrantly proliferating Rb1-deficient
cells and for tumorigenesis in Rb1
+/-
mice. Nat Genet. 42:83-887.
6.
Lu, Z., Marcelin, G., Bauzon, F., Wang, H., Fu, H., Dun, S.L., Zhao, H., Li, X., Jo, Y.H., Wardlaw, S., Dun,
N., Chua, S. Jr., and Zhu, L. (2013) pRb is an obesity suppressor in hypothalamus and high-fat diet
inhibits pRb in this location. EMBO J. 32:844-857.
7.
Zhao, H., Bauzon, F., Fu, H., Lu, Z., Cui, J., Nakayama, K., Nakayama, K.I., Locker, J., and Zhu, L.
(2013) Skp2 deletion unmasks a p27 safeguard that blocks tumorigenesis in the absence of pRb and
p53 tumor suppressors. Cancer Cell. 24:645-659.
8.
Lu, Z., Bauzon, F., Fu, H., Cui, J., Zhao, H., and Zhu, L. (2014) Skp2 suppresses apoptosis in Rb1
deficient tumors by limiting E2F1 activity. Nat Commun. 5:3463.
9.
Zhu, L., Lu, Z., and Zhao, H. (2014) Antitumor mechanisms when pRb and p53 are genetically
inactivated. Oncogene. in press