Joint Molecular Biosciences Graduate Student

Association

6th Annual Graduate

Student Symposium

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“Whenever I found out anything remarkable, I

have thought it my duty to put down my discovery

on paper, so that all ingenious people might be

informed thereof.”

-Antonie Van Leeuwenhoek

Cover photo: FOXP2 (forkhead box protein P2) transcription factor acting on a strand of DNA. This

protein is necessary for proper brain and lung development; mutations in the FOXP2 locus have been

linked to severe speech and language disorders. Photo Credit: Ramon Andrade 3DCIENCIA / Science

Photo Library

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Letter from the Organizers

Welcome to the 6th Annual Graduate Student Symposium hosted by the Joint Molecular

Biosciences Graduate Student Association (JMBGSA) of UMDNJ/Rutgers University. We are

delighted to have you join us today to support the outstanding work that graduate students here at

Rutgers-UMDNJ produce. Through this symposium, we hope to not only showcase the graduate

student research, but also to provide a platform for professional interaction between students,

faculty and administration.

As a student organization, the goal of JMBGSA is to facilitate the professional development of

graduate students, and provide them with opportunities for social interaction with their peers. By

allowing them to present their work to a critical audience in a conducive environment, graduate

students are able to hone their presentation skills, and receive input on their work from faculty

from various departments. With these goals in mind, we organize the annual symposium, and each

year we look to the university community to make it a success.

We have received tremendous help and support from our peers and faculty alike in putting this

symposium together. We thank our advisor Dr. Janet Alder for her help and encouragement during

the planning process. We also thank Dr. Nancy Walworth, Dr. Richard Padgett and Dr. Terri Kinzy,

whose constant support has made the JMBGSA symposia an integral part of the molecular

biosciences program.

In order to make this symposium a scientific forum, several faculty members have taken time out

from their busy schedules to participate as judges and give students feedback on their work. We

gratefully acknowledge Drs. Nancy Walworth, Paul Copeland, Donald Winkelmann, Debabrata

Banerjee, Smita Thakkar-Varia, and Miguel Zaratiegui. Since this event would not be possible

without the participation of our fellow-graduate students, we applaud their efforts and thank them

for their poster presentations and talks. We’d like to offer a very special thank you to all our

generous sponsors as well for supporting graduate student research at Rutgers-UMDNJ.

Thank you for joining us today. We sincerely hope that you will enjoy the 6th Annual Graduate

Student Symposium and we hope to see you next year.

JMBGSA organizers.

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The JMBGSA welcomes you to the

6th Annual Graduate Student Symposium

Friday, April 20th, 2012 9:00 am – 5:00 pm Life Sciences Building Atrium

PROGRAM:

9:00 – 9:30

9:30 – 10:00 10:00 – 11:00 11:00 – 12:00

Breakfast and Registration Opening Remarks Oral Presentation – I Keynote Address

Dr. Thomas Papathomas Professor of Biomedical Engineering

Associate Director of the Laboratory of Vision Research Editor-in-chief of Early Vision and Beyond

12:00 – 1:00 1:00 – 2:00 2:00 – 3:00 3:00 – 4:00 4:00 – 4:30

Lunch Poster Session, Vendor Show - I Oral Presentations - II Poster Session, Vendor Show – II Awards Presentation

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Platform Presentations

Session I

10:00 –10:12: Pinpointing the Function of the VapC Family of Toxins from Mycobacterium

tuberculosis

Cruz, J.W. , Sharp, J.D., Husson, R.N., and Woychik, N.A

10:12 – 10:24: Role of BECN1 in Mammary Differentiation

Michelle Turek, Rumela Chakarbarti, Fred Lozy, Yibin Kang, and Vassiliki Karantza

10:24 – 10:36: Rescuing Murine Leukemia Virus p12 Mutants with DNA Tethering Domains

Jonathon Brzezinski, William M Schneider, Mercedes Gyuricza and Monica J Roth

10:36 – 10:48: Dab2ip Regulates Radial Neuronal Migration in the Developing Neocortex

Gum Hwa Lee, Sun Hong Kim, Ramin Homayouni, and Gabriella D’Arcangelo

10:48 – 11:00: Structural Basis of RNA Recognition and Activation by Innate Immune Receptor

RIG-I Anand Ramanathan, Fuguo Jiang, Matthew T. Miller, Guo-Qing Tang, Michael Gale Jr, Smita S. Patel, Joseph Marcotrigiano.

Session II

2:00 –2:12: Functional Characterization of the Selenocysteine-specific Domain in the

Translation Elongation Factor eEFSec Jonathan N. Gonzalez-Flores, Nirupama Gupta, Louise DeMong, Paul R. Copeland.

2:12 – 2:24: Cdx1 Regulates Neural Fold Fusion Through Retinoic Acid (RA) And Wnt Signaling

Bo Li, Paul G. Matteson, Alejandro Q. Nato, Jr., Tara C. Matise, Vikas Nanda and James H. Millonig

2:24 – 2:36: A Role of TRPM7 in Trace Metal Homeostasis

Omayra González-Pagán and Loren W. Runnels

2:36 – 2:48: A Novel Strategy for the Expression of Hepatitis C Virus Envelope Protein Using

Mammaliam Cell Culture Jillian Whidby, Abdul Khan, John Shires, and Joseph Marcotrigiano

2:48 – 3:00: Disruption of the mTORC2 Complex Results in Increased Insulin-like Growth

Factor-1 Receptor Expression and Decreased Insulin Receptor Expression Nicole M. Vega-Cotto, Won Jun Oh, Sung Jin Kim, Michael DeStefan and, Estela Jacinto

Keynote Speaker

Dr. Thomas Papathomas

Department of Biomedical Engineering, Rutgers University

Dr. Papathomas joined Rutgers in 1989 as an Associate Professor (promoted to full

Professor in 1997) in the Department of Biomedical Engineering and is the Associate

Director of the Laboratory of Vision Research. His research interests are in human and

machine vision, neural mechanisms in vision, image processing, imaging systems, and

scientific visualization techniques. He is investigating the mechanisms that underlie the

perception of visual motion, stereopsis, texture, and the deployment of attention in vision.

In particular, he studies the role of visual attributes (color, luminance, shape, spatial

frequency, etc.) in perception, with the ultimate goal of developing quantitative models of biological mechanisms in these modalities.

He is the editor-in-chief of Early Vision and Beyond, a volume of interdisciplinary

research in psychophysics, neurophysiology, and computational vision, published by MIT

Press in 1995. He has designed several exhibits in science museums, and his work was

exhibited at the New York Arts Biennial in 1997. He is a member of the Editorial Board of the International Journal of Imaging Systems and Technology, 1993 - present.

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Platform Presentations

T1. Pinpointing the Function of the VapC Family of Toxins from Mycobacterium tuberculosis Cruz, J.W. 1, Sharp, J.D. 1,2, Husson, R.N. 2, and Woychik, N.A. 1

1Department of Molecular Genetics, Microbiology and Immunology, UMDNJ-RWJMS; 2Division of Infectious Diseases, Children’s Hospital Boston, Harvard Medical School

Mycobacterium tuberculosis is a devastating pathogen that causes the disease tuberculosis. In 2007, there were over nine million new cases of tuberculosis and nearly two million deaths. M. tuberculosis has the unique ability to persist for long periods of time in its host as a latent infection. It is estimated that approximately two billion people, about one third of the world’s population, have latent tuberculosis. While the molecular switches that trigger M. tuberculosis latency are poorly understood, increasing evidence suggests a role for toxin-antitoxin (TA) systems. Bacterial TA systems are small operons encoding an intracellular stable toxin and a labile antitoxin. The TA toxin inhibits an essential intracellular process, such as DNA replication or translation, in the cell where it was produced. The antitoxin binds to the toxin and inhibits its action. Detailed characterization of TA systems in Escherichia coli has revealed that TA toxins impart a quasi-dormant state with striking similarities to the slow growth and dormancy exhibited by M. tuberculosis in latent tuberculosis. Curiously, the M. tuberculosis genome harbors an expanded number of TA systems relative to E. coli, with approximately two-thirds belonging to the VapBC family (VapB antitoxin and VapC toxin). The mechanism of action of VapC remains unclear. We are studying a small subset of these VapC toxins to determine the mechanism of action of the family as a whole. We are currently testing our hypothesis that VapC toxins inhibit translation through cleavage of tRNA. To build on our initial findings, we are now using in vitro and in vivo approaches to investigate the extent and position of VapC cleavage on predicted tRNA targets.

T2. Role of BECN1 in Mammary Differentiation Michelle Turek1,3,4, Rumela Chakarbarti2, Fred Lozy1,3,4, Yibin Kang2, and Vassiliki Karantza1,3 1 CINJ; 2Princeton University; 3 UMDNJ; 4Rutgers University Autophagy is a catabolic process used by cells for recycling of intracellular organelles and protein building blocks. Mice with one allele of an essential autophagy regulator, Becn1, develop mammary hyperplasias. BECN1 is reportedly allelically lost in 50% of human breast cancers, and low BECN1 mRNA levels correlate with the basal-like breast cancer subtype. Recent evidence has identified the mammary progenitor population as the transforming populations for basal-like breast cancer.Keratin 6 (K6), a well-known bipotent mammary progenitor cell marker, is preferentially expressed in immortalized mouse mammary epithelial cells, mammary tumors, mammary glands, and in transplants derived from mammary stem cell (MaSC)-enriched populations that are Becn1+/- vs. Becn1+/+. Both mammary glands from 6.5 week-old Becn1+/- mice and transplants generated by MaSC-enriched Becn1+/- mammary populations exhibit increased mammary fat pad filling and ductal tree branching, indicating that Beclin1 likely plays a critical role in mammary gland morphogenesis and development. The mechanism by which allelic Becn1 loss leads to a mammary differentiation defect and whether this defect is autophagy-related or Beclin1-specific are currently under investigation. This research is supported by Daymon Runyon Cancer Research Foundation and NIH-NCI.

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T3. Rescuing Murine Leukemia Virus p12 Mutants with DNA Tethering Domains Jonathon Brzezinski, William M Schneider, Mercedes Gyuricza and Monica J Roth

Department of Biochemistry, UMDNJ-RWJMS

The murine leukemia virus (MLV) p12, is a mostly unstructured gag protein. Viral infection with

mutant p12 (PM14) suggests p12 plays a role between reverse transcription and integration, and

does not produce virus. The association of p12 with the pre-integration complex (PIC) supports this

role. Within related retroviruses, viral and host proteins have been shown to tether the PIC to the

host DNA. This includes the host LEDGF protein for HIV. We have examined the ability of alternative

viral DNA tethering domains to complement replication-defective MLV with the p12 PM14

mutation. These domains from a herpesvirus, a prototype foamy virus, and a human papillomavirus

were inserted into p12 and functional complementation was examined in cell culture. Insertion of

any of these domains was able to rescue the early block in infection and produce virus, while

nuclear localization domains could not. Furthermore, mutating the residues required for DNA

tethering in these inserted domains resulted in the loss of the complementation. Also, p12-GFP

fusion constructs show the inserted domains tethering GFP to mitotic DNA. These results suggest

p12 is involved in tethering the viral DNA and PIC to the host DNA and might be manipulated to

alter the insertion site selection of MLV, possibly making MLV a safer gene therapy vector. This

research is supported by the NIH.

T4.

Dab2ip Regulates Radial Neuronal Migration in the Developing Neocortex

Gum Hwa Lee1, Sun Hong Kim2, Ramin Homayouni,2,3 and Gabriella D’Arcangelo1 1Department of Cell Biology and Neuroscience, Rutgers University; 2 Department of Anatomy and

Neurobiology, University of Tennessee Health Science Center, Memphis, and 3 Department of

Biology, University of Memphis, Memphis.

Dab2ip (DOC-2/DAB2 interacting protein) is a member of the Ras GTPase-activating protein (GAP)

family that interacts with the Dab1 and Dab2 adaptor proteins. Dab2ip has been previously

reported to function as a tumor suppressor in several forms of cancer. However, high expression in

brain and several genetic studies suggested a role in central nervous system. To discover the

function of Dab2ip in brain development, we knock down Dab2ip expression in neuronal precursors

in the embryonic mouse neocortex using the in utero electroporation technique. We found that the

downregulation of Dab2ip disrupts neuronal migration and cellular layer formation. Dab2ip appears

to play a role in the transition phase from multipolar to bipolar morphology during radial neuronal

migration, and this deficit may be caused by the transient decrease in expression of major neuronal

microtubule-associated proteins (MAPs), such as Map2, Map1b, and Tau. Our study for the first

time identifies a role for Dab2ip in controlling neuronal migration in the developing mammalian

brain. This research is supported by a research grant from the New Jersey Governor’s Council for

Medical Research and Treatment of Autism (G.D.).

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T5. Structural Basis of RNA Recognition and Activation by Innate Immune Receptor RIG-I Anand Ramanathan1, Fuguo Jiang2, Matthew T. Miller2, Guo-Qing Tang1, Michael Gale Jr3, Smita S. Patel1, Joseph Marcotrigiano2. 1UMDNJ; 2Rutgers University; 3University of Washington School of Medicine, Seattle, WA RIG-I-like receptors (RLRs) of the innate immune system are the cell’s principal detector of RNA

viruses. These proteins distinguish between cellular and viral RNAs by recognition of Pathogen

Associated Molecular Pattern (PAMP) motifs that are associated with viral RNAs. RIG-I (Retinoic

acid Inducible Gene - I) is a cytosolic pathogen recognition receptor that recognizes viral RNA

motifs and triggers an immune signaling cascade resulting in type-I interferon induction. RIG-I

consists of three domains; the N-terminal CAspase Recruitment Domains (CARD), the central

helicase domain and the C-terminal repressor domain (RD). The helicase and RD of RIG-I recognize

double-stranded (ds) RNA and 5'-triphosphate RNA as foreign and activate the RIG-I CARD for

signaling. However, the nature of RIG-I:RNA interaction remains unclear. To understand how the

RIG-I helicase binds RNA and leads to activation, we have determined the structure of the human

RIG-I helicase-RD domain bound to dsRNA and ADP•BeF3. The structure of the ternary complex

reveals a major contribution of the helicase to RNA binding and a synergy between the helicase and

RD in the recognition of blunt-ended dsRNA. Helicase-RD organizes into a ring with the helicase

utilizing previously uncharacterized motifs to specifically recognize dsRNA. Additional biophysical

and biochemical results demonstrate that RIG-I, in absence of RNA is flexible and becomes more

compact upon RNA binding. These results provide a greater understanding of the cellular response

and immune activation to viral infection. However, the role of ATPase/helicase function of RIG-I

remains elusive. The RIG-I helicase-RD represents the first structure of an RNA helicase bound to

dsRNA and provides a new perspective in understanding how other homologous RNA helicases may

engage their targets.

T6. Functional Characterization of the Selenocysteine-specific Domain in the Translation Elongation Factor eEFSec Jonathan N. Gonzalez-Flores, Nirupama Gupta, Louise DeMong, Paul R. Copeland. Department of Molecular Genetics, Microbiology, and Immunology, UMDNJ-RWJMS The co-translational insertion of the 21st amino acid, selenocysteine (Sec), into 25 human proteins

provides the bulk of a cell’s catalytic activity against oxidative stress, which is causative in several

pathological conditions such as cancer, cardiomyophathy, osteoarthritis, and neurodegeneration.

Selenocysteine is incorporated at in-frame UGA codons only when several cis and trans-acting

factors are present. The Sec-specific elongation factor, eEFSec, binds and delivers the

selenocysteine-tRNA (Sec-tRNA) during translation elongation of a nascent peptide. A Sec Insertion

Sequence (SECIS) element found in the 3’ UTR of all selenoprotein mRNAs and the SECIS binding

protein 2 (SBP2) are also required for Sec incorporation. Multiple studies have highlighted the

importance of SBP2 binding the SECIS and forming a complex, but is not clear how this regulates

eEFSec during Sec incorporation. Protein sequence alignment analysis between the canonical

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translation elongation factor eEF1A and eEFSec shows an unique C-terminal extension in eEFSec

called the Domain IV. Our working hypothesis is that SBP2 and the SECIS activate eEFSec for Sec

incorporation by functional interactions at this Sec-specific domain. To test whether Domain IV of

eEFSec is required for Sec incorporation we created and purified penta-alanine mutants at

conserved regions and an eEFSec without Domain IV. Wild-type and mutant versions of eEFSec

were analyzed for Sec incorporation activity in an eEFSec dependent translation extract with a

Luciferase-SECIS mRNA construct that contained an UGA in the coding region. The results of this

assay showed that Domain IV is essential for Sec incorporation. Binding data from native gel shift

assay demonstrated that SBP2 and the SECIS interact at Domain IV, specifically at its C-terminal

region. The Domain IV is also essential for Sec-tRNA binding according to our filter binding studies

using 75Se radiolabel Sec-tRNA. These analyses have revealed that the Domain IV in eEFSec has an

novel role in Sec incorporation by being involved in Sec-tRNA delivery and activation by the SBP2-

SECIS complex. This research is supported by NIH Biotechnology Training Program, NSF IGERT

Biointerface and the NIGMS.

T7. Cdx1 Regulates Neural Fold Fusion Through Retinoic Acid (RA) And Wnt Signaling Bo Li1,2, Paul G. Matteson1, Alejandro Q. Nato, Jr3., Tara C. Matise3, Vikas Nanda1,4 and James H. Millonig1,2,3 1CABM; 2Department of Neuroscience and Cell Biology, UMDNJ-RWJMS; 3Department of Genetics, Rutgers University; 4Department of Biochemistry, UMDNJ-RWJMS.

To understand the molecular basis of neurulation, we study the vacuolated lens (vl) mutation that

arose on the C3H background. The mutation causes Neural Tube Defects (NTDs) and lethality, and

are due to a mutation in the orphan G-protein coupled receptor (Gpr161). Crossing vlC3H mice to the

MOLF strain rescues the vl defects. QTL analysis mapped 3 modifiers (Modvl: Modifier of vl). The

one being studied is Modvl5 (LOD=5.0; Chr18). Modvl5MOLF congenic was generated to study

whether this locus is sufficient to rescue the vl phenotypes. Analyses on congenic embryos

determined that Modvl5C/M rescues the lethality (P=0.004) and partially rescues the NTDs (p=0.03).

Bioinformatics analysis determined the transcription factor, Cdx1, is the only gene within the

Modvl5 95% CI co-expressed with Gpr161 in the neural folds. Re-sequencing Cdx1 identified a poly-

Q polymorphism (5Q-C3H, 7Q-MOLF) predicted to affect protein structure. EMSAs demonstrated

the 7Q binds better than 5Q allele (p<0.001) to the promoters of RA and Wnt signaling genes. QRT-

PCR indicated that the expression of these genes is altered by vl and rescued by Modvl5C/M. These

results place Gpr161 upstream of RA and Wnt signaling to regulate neural fold fusion and indicate

that Cdx1MOLF bypasses the vl mutation by up-regulating the downstream RA and Wnt genes.

This research is supported by The New Jersey Commission on Spinal Cord Research.

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T8. A Role of TRPM7 in Trace Metal Homeostasis Omayra González-Pagán and Loren W. Runnels

Department of Pharmacology, UMDNJ-RWJMS TRPM7 is a ubiquitously expressed channel-kinase widely permeable to a range of divalent metal

ions, with a permeability sequence of Zn2+~Ni2+>>Ba2+>Co2+>Mg2+> Mn2+> Sr2+ Cd2+ Ca2+. TRPM7’s

unusual permeability profile raises the possibility that the channel may be regulating the cellular

homeostasis of more than one of these essential metal ions. Study of TRPM7 has linked the

channel to a number of physiological roles, including cell proliferation and cellular Mg2+

homeostasis. However, TRPM7’s role in Mg2+ homeostasis as well as the mechanism(s) by which

the channel-kinase influences cell proliferation remains controversial, as conditional knockout of

TRPM7 in neural stem cells, thymocytes, and T cells failed to produce any changes in cell

proliferation or in Mg2+ homeostasis. To better understand the role of the channel in metal ion

homeostasis we’ve generated recombinant adenoviruses that express TRPM7 channel-dead and

TRPM7 channel/kinase-dead mutants. Expression of these mutants in mouse embryonic fibroblasts

(MEFS) dramatically inhibits cell proliferation, arresting the cells in the S- and G2- phases of the cell

cycle. In addition, expression of the mutants lowers cellular free Mg2+ and decreases Zn2+ uptake.

Consistent with a role for TRPM7 in Mg2+ and Zn2+ homeostasis, overexpression of the Mg2+

transporter SLC41A2 or supplementation of the growth media with high Mg2+ or Zn2+, partially

rescues the growth of cells expressing the TRPM7 mutants. We conclude that TRPM7 plays a critical

role in both Mg2+ and Zn2+ homeostasis and that its regulation of the metal ions is vital to the

channel’s control of cell proliferation.

T9. A Novel Strategy for the Expression of Hepatitis C Virus Envelope Protein Using Mammaliam Cell Culture Jillian Whidby1, Abdul Khan1, John Shires2, and Joseph Marcotrigiano1

1CABM, Rutgers University; 2Emory University School of Medicine Within the last few years, structural biology has grown ever more limited by the inability to

produce highly complex proteins in typical expression systems (e.g. bacteria, yeast, insect

cells). Significant technological barriers must be overcome to expand the field, compelling us to

explore mammalian cell culture for the expression of two especially challenging hepatitis C virus

(HCV) structural proteins, envelope 1 (E1) and envelope 2 (E2). E2 is a type I transmembrane

protein with an amino-terminal ectodomain and a carboxy-terminal membrane-associating

segment, separated by a stem region that is proposed to be an amphipathic helix. It is highly

modified post-translationally with numerous N-linkage and O-linkage glycosylation consensus sites

and may have as many as nine disulfide bonds from 18 absolutely conserved cysteines. Described

here is a method to produce >20mg/L quantities of eE2 in human cells, optimized using the

combination of i) a novel bioreactor, ii) lentiviral gene delivery, and iii) a mannosidase inhibitor.

Purified eE2 constitutes an exceptional tool for probing envelope protein function and may

facilitate in the design of an entry inhibitor or an HCV vaccine. Furthermore, we hope that the

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combination of technologies utilized will open up new opportunities for studying other complex

proteins of biological and pharmacological interest.

T10. Disruption of the mTORC2 Complex Results in Increased Insulin-like Growth Factor-1 Receptor Expression and Decreased Insulin Receptor Expression Nicole M. Vega-Cotto, Won Jun Oh, Sung Jin Kim, Michael DeStefan and, Estela Jacinto1

Department of Physiology and Biophysics, UMDNJ-RWJMS

The insulin receptor (IR) and the insulin-like growth factor 1 receptor (IGF1R) are tetrameric

tyrosine kinase receptors that become activated upon binding of insulin or IGF1. Activation of both

receptors can regulate cell differentiation, growth, and metabolism, through the mammalian target

of rapamycin complex 2 (mTORC2), leading to increased PI3K/Akt activity. Recently, we found that

mTORC2 disruption leads to increased expression of the insulin receptor substrate 1 (IRS-1). Since

IRS-1 is the adaptor protein that couples signals from IR or IGFR to downstream signaling molecules

such as PI3K, we sought to determine if the expression of IR and IGFR is also altered in mTORC2-

disrupted cells. Interestingly, we found that total levels of IR are decreased whereas IGF1R levels

are increased in these cells. Our findings suggest that mTORC2 can distinctly regulate IR and IGFR

expression. We will address possible mechanisms of how mTORC2 can control IR versus IGFR

signaling. This research is supported by the NIH and the American Cancer Society.

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Poster Presentations

P1. Morphogenesis of the C. elegans Intestine Depends on the Ability of Arp2/3 to Promote Membrane Association of Apical Proteins Yelena Bernadskaya, Falshruti Patel, Hiao-Ting Hsu, and Martha C. Soto Department of Pathology, UMDNJ-RWJMS The establishment of the apical domain of C. elegans intestinal epithelia requires the apical

enrichment of actin and formation of junctions at the apicolateral domain. The contribution of

different actin regulators to junction formation is poorly understood. We have found that Arp2/3, a

regulator of branched actin formation, is required for normal intestinal morphogenesis. Loss of

Arp2/3 or of its activating WAVE/SCAR complex leads to reduced levels of apical actin in the

intestine during development along with a widened intestinal lumen. Interestingly, overall polarity

of the tissue is initiated, suggesting a more subtle regulation of epithelial integrity. It has previously

been proposed that Arp2/3 is only required for protrusion formation during the establishment of

adherens junction but plays no role in junctional maintenance. However, we have observed similar

apical defects when Arp2/3 or WAVE/SCAR are depleted from adult worms, suggesting that Arp2/3

is required continuously throughout the life of the animal to maintain the apical domain. Loss of

Arp2/3 in embryos results in reduced apical accumulation of DLG-1, a component of the apical

adherens junction. During embryonic development, the DLG-1/AJM-1 complex rather than the

cadherin/catenin complex is required for enrichment of apical F-actin. The role of Arp2/3 in

promoting apical enrichment of F-actin and DLG-1 is supported by ERM-1, a protein that connects

F-actin to membranes. Loss of Arp2/3 shifts both ERM-1 and DLG-1 to a subcellular fraction

enriched in recycling endosome proteins and loss of a regulator of recycling endosomes shifts ERM-

1 and DLG-1 back toward pellet fractions enriched in plasma membrane. Taken together, these

data support a model in which Arp2/3 is recruited to the apical region of the intestine by ERM-1

where it promotes and maintains junction formation by regulating the membrane association of

proteins that establish and maintain the apical domain.

P2.

Cohesion of Silenced and Non-Silenced chromatin in Saccharomyces cerevisiae John Campor and Marc Gartenberg Department of Pharmacology, UMDNJ-RWJMS Newly replicated chromatids are held to one another by cohesin, a ring-shaped protein complex.

Defects in the pathway, termed sister chromatid cohesion, lead to several human developmental

diseases, recently coined cohesinopathies. Whether pathological consequences are related to

cohesion defects or some other role for cohesin (e.g. transcription) is not known. Roberts

Syndrome is characterized cytologically by loss of cohesion in heterochromatin. The disease is

caused by mutations of Esco2, a protein acetyltransferase that activates cohesin by modifying

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subunits of the complex. We examined the impact of one Roberts Syndrome mutation

(eco1W216G in yeast) on cohesion by converting specific loci into GFP-tagged, chromatin circles in

vivo. Whereas cohesion of chromosomes generally persisted (also see Gard and Gerton, 2009), we

discovered specific cases where cohesion of specific heterochromatic and euchromatic domains

was abolished. Our results indicate that Roberts Syndrome mutations can cause localized cohesion

defects, and that these defects can be masked in assays for global chromatidcohesion.

P3. Elucidating Robo2 Function During Drosophila melanogaster Heart Tube Formation Judith J. Canabal Alvear and Sunita G. Kramer Pathology Department, UMDNJ-RWJMS Biological tubes are required for the development of complex organisms given that they distribute

fluids and other important molecules to different parts of the organism. In this study, we

investigate a previously unknown function for the transmembrane receptor Roundabout2 (Robo2)

during lumen formation of the Drosophila dorsal vessel, a simple linear tube required to pump

hemolymph throughout the embryo. Two major steps are required for dorsal vessel formation.

First, specified cardioblasts (CBs) migrate in rows toward the dorsal midline of the embryo and

second, the CBs undergo a series of cell shape changes to form a linear tube with a central lumen.

The two rows of CBs are flanked on either side by two rows of non-muscle pericardial cells (PCs).

The role of the PCs in lumen formation is not well understood. CBs express a single Roundabout

receptor (Robo1), while PCs express both Robo1 and Robo2. Our lab has shown that loss of Robo1

results in defects in lumen formation, however the role for Robo2 in this process remains unclear.

The present work investigates the role for Robo2 in lumen formation through loss of function and

gain of function studies. Our results show that ectopic expression of Robo2 at low levels in the CBs

results in a robo loss of function phenotype, suggesting that Robo2 may function to antagonize

Robo1. However, when Robo2 is expressed at high levels in the CBs, it can mimic Robo, resulting in

a Robo gain of function phenotype. Furthermore, we found that the ability of Robo2 to antagonize

Robo 1 is conferred by its IG1+IG2 extracellular domains. We are in the process of elucidating this

biphasic nature of Robo2 function and determining its normal role during during lumen formation.

P4. Functional Dissection of Toxin-Antitoxin Systems in Uropathogenic E. coli Jonathan W. Cruz*, Jennifer M. Hurley*, Lauren A. De Stefano and Nancy A. Woychik *Authors contributed equally Department of Molecular Genetics, Microbiology, & Immunology and Department of Biochemistry, UMDNJ-RWJMS Uropathogenic strains of Escherichia coli (UPEC), including the well-characterized CFT073 strain, are

the most common etiologic agents of nonhospital-acquired urinary tract infections. These

infections can lead to acute cystitis or pyelonephritis. E. coli CFT073 has developed a variety of

traits that allow it to colonize the urinary tract despite harsh environmental conditions and host

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defenses. Recurring infections are common, even after proper antibiotic treatment. Toxin-antitoxin

(TA) systems have not only been linked to survival during stress conditions, persistence, and biofilm

formation, they also represent a novel target for antimicrobials. TA systems are composed of a

stable toxin and a labile antitoxin. In E. coli, stress leads to a decrease in the amount of antitoxin,

thus freeing up toxin to act on its intracellular target. Curiously, E. coli CFT073 possesses three

HigBA TA systems: HigBA-cft1, -cft2 and -cft3. The function of HigB from the Proteus vulgaris Rts1

plasmid has been recently elucidated. It is a ribosome-associated, sequence-specific (i.e. AAA)

endoribonuclease whose action leads to a rapid block in translation followed by growth arrest.

E. coli CFT073 HigB expression led to growth arrest, but only after 4 hrs (vs. 20 min for Rts1 HigB)

and only marginally reduced translation. This toxin associated with the ribosome and led to mRNA

cleavage with only mild sequence specificity. HigB-cft1 expression also led to an increase in

persister formation.

P5.

Identification of Novel Notch Targets in Non-Small Cell Lung Cancer Kathleen M. Capaccione 1, 2, Gregory Miles1, Sohrab Amiri1, Sharon R. Pine 1,2 1 The Cancer Institute of New Jersey, UMDNJ 2 Department of Medicine, UMDNJ-RWJMS Lung cancer is the leading cause of cancer death worldwide and there is urgent need to elucidate

its molecular pathogenesis and identify novel druggable targets. The Notch pathway governs

embryonic development and is associated with lung carcinogenesis. Notch signaling is correlated

with poor outcomes and has been implicated in maintaining stem cell-like characteristics in cancer.

Gamma secretase inhibitors that block Notch activation are in clinical trials, but are associated with

significant side effects. We seek to identify novel Notch targets responsible for malignant

characteristics. We analyzed four published lung cancer microarray GEO data sets and one

unpublished set and correlated genes with Notch-1 and Hes-1 expression, a known Notch target

gene. We then identified genes with RPB-JK sites, the canonical Notch binding cis-element. We are

testing lung cancer cell lines by inducing and inhibiting the Notch pathway and assessing changes in

target gene expression. We will assess if activated Notch-1 binds to the target genes’ promoters by

CHIP and examine if these genes participate in therapy resistance, cell survival, proliferation,

migration, and ability to form tumors in mice. The results may enhance our understanding of the

contribution of Notch signaling and its products to the malignant character of lung cancer cells, and

may provide novel targets for therapy. This research is supported by NCI-K22 CA140719 (SRP).

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P6. Promoter Recognition in Yeast Mitochondria by Transcription Factor Induced DNA Bending. Deshpande A.P., Tang G.Q. and Patel S.S. Department of Biochemistry, UMDNJ-RWJMS Promoter recognition is the first key step during gene expression. In yeast mitochondria,

transcription is carried out by a nuclear encoded two-component system, the core RNA polymerase

Rpo41 and the accessory transcription factor Mtf1. To dissect the mechanistic roles of the core RNA

polymerase and the transcription factor in promoter recognition, we have quantified the

interactions of Rpo41 alone and Rpo41-Mtf1 holoenzyme with the promoter and non-promoter

DNA. Our studies show that the core enzyme does not differentiate between the promoter and

non-promoter DNA, but requires the presence of Mtf1 for this function. Although Rpo41-Mtf1

binds the promoter and non-promoter DNA with different affinities, this differential binding is not

the major mechanism of distinction. Instead Rpo41-Mtf1 induces selective bending in the promoter

and unbending in the non-promoter. This induced-fit mechanism of promoter recognition results in

a severe bend in the promoter DNA leading to the formation of a catalytically competent open

complex. On the other hand, unbending of the non-promoter DNA regions ensures a

conformational state for translocation of the RNA polymerase to scan for sites, where it can carry

out specific transcription.

P7. Defining Mechanisms by which Neurotherapeutic Valproic Acid (VPA) Stimulates Cell Cycle Machinery During Embryonic Rat Cerebral Cortex Development Robert Connacher 1,2, Xiaofeng Zhou1, Emanuel DiCicco-Bloom1 1 Department of Neuroscience and Cell Biology, UMDNJ 2 Graduate Program in Neuroscience VPA is used to treat epilepsy, migraine and depression, and induces brain malformations, though

mechanisms are undefined. In previous culture studies, VPA stimulated embryonic day 14.5

precursor DNA synthesis, cell division ad cell numbers, acting via increases in G1 cyclins D3 and E.

Further, VPA increased protein levels of acetylated histone 3, consistent with the proposal VPA is a

histone deacetylase (HDAC) inhibitor. In vivo, a single maternal injection of VPA elicited increased

G1 cyclins in the fetus as early as 1.5 hr with minimal second messenger activation, potentially

suggesting VPA regulates gene transcription via epigenetic mechanisms. To begin defining

mechanisms, we are characterizing cerebral cortex expression of potential target HDACs using RT-

PCR and western analyses. Preliminary studies conducted on E14.5, E16.5 and adult cerebral cortex

indicate that class I, II, and IV HDACs are expressed most strongly during development, and exhibit

region specific patterns. Future studies will explore whether VPA inhibits specific HDACs, and

whether HDAC are required for its developmental effects. This research is supported by PO1

HD23315-23

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P8. Follow-up Linkage and Association Analyses of a Nonverbal Motor Speech Phenotype Identified in the AGRE Data Set A. Hare1, M. Azaro1, R. Zimmerman1, J. Flax1, J. Burian2, V. Vieland2 and L. Brzustowicz1 1Department of Genetics, Rutgers University, 2Battelle Center for Mathematical Medicine , The Research Institute at Nationwide Children’s Hospital & The Ohio State University, Columbus, OH Using behavioral and genetic information from the Autism Genetics Resource Exchange (AGRE)

data set we developed a non-verbal motor speech phenotype (NVMSD:ALL) and investigated

linkage and association for individuals with and without Autism Spectrum Disorders (ASD). We

previously identified several linkage peaks using the PPL (Posterior Probability of Linkage)

framework to assess the strength of evidence for or against trait-marker linkage and linkage

disequilibrium across the genome using Affymetrix 5.0 genotype data. As evidence for linkage

disequilibrium was limited, Ingenuity Pathway Analysis (IPA) was then utilized to identify potential

genes for further investigation. We fine mapped our genes of interest and used the PPL framework

to assess evidence for association. We also present the sequential update of our initial PPL analysis

with the addition of 88 new families from the AGRE data set who met criteria for the NVMSD:ALL

phenotype. Additional individuals were genotyped in linkage regions identified in the initial

analysis using SNPstream technology. Overall, evidence for linkage disequilibrium was limited in

our candidate genes of interest, with the highest signals in TRPV2 (8%) and LMX1A (7%). The

additional 88 families from AGRE added power to our PPL analysis with increasing evidence for

linkage in several regions. This research is supported by RO1MH76435

P9. Identifying Mutations in the Chromosome Passenger Complex (CPC) and Associated Regulators of Spindle Assembly in Drosophila. Arunika Das1, Shital J. Shah2, Kim S. McKim1 1Department of Genetics, Rutgers University; 2University of Medicine and Dentistry, Newark Accurate segregation of chromosomes during meiosis or mitosis is facilitated by the formation of a

bipolar array of microtubules. In mitotic spindle assembly, the centrosomes define the two poles

and organize the microtubules. In the oocytes of many animals, however, the centrosomes are

absent and consequently it is poorly understood what organizes the bipolar spindle and directs

biorientation of chromosomes. Previous studies have suggested that the CPC is the master

regulator of spindle assembly. The CPC is composed of four proteins, Incenp, Aurora B kinase,

Survivin and Borealin. Unlike mitotic cells, where the CPC localizes to centromeres, during meiotic

metaphase it localizes in a ring around the chromosomes. I am testing the hypothesis that this

novel localization pattern is critical for the mechanism of forming a bipolar spindle and orienting

chromosomes by identifying proteins that interact with the CPC during formation of the

acentrosomal spindle. Although my studies are in meiotic cells, I have also used an unbiased

screen-based approach to uncover novel regulators of CPC activity. This screen was based on

synthetic lethality with subito mutations. Subito is a member of the kinesin-6 family, which

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associate with and regulate microtubules. I have mapped two mutations from the screen to

transcription factor snail and RacGAP50C and am currently characterizing their roles in meiosis.

P10. Improving Photohydrogen Performance in Chlamydomonas reinhardtii: Metabolic Engineering of the PSII D1 Isoforms Anagha Krishnan, David J. Vinyard, Nicholas J. Skizim, Gennady Ananyev, and G. Charles Dismukes Waksman Institute of Microbiology, Department of Chemistry & Chemical Biology, Rutgers University Herein we investigate the influence of two natural isoforms of the PSII reaction center protein, D1,

on the capacity to photo-generate hydrogen by the native [FeFe]-hydrogenasein the unicellular

green alga Chlamydomonas reinhardtii. Water oxidation by PSII not only provides electrons and

protons needed for photosynthetic reductantgeneration (including hydrogen), but also generates

oxygen that strongly inhibits [FeFe]-hydrogenase. Cyanobacteria utilize a few D1 isoforms to adapt

to environmental conditions; for example, Synechococcuselongatus contains both low light (D1:1)

and high light (D1:2) isoforms. Under high light intensities when the photosynthetic electron

transport chain is light saturated and aerobic conditions, the D1:2 isoform is more efficient at water

oxidation compared to D1:2. Dissolved H2 measurements demonstrate that the total (integrated)

yield of hydrogen production is unchanged in the two mutants, while the steady state rate of

hydrogen evolution is higher in the D1:1 mutant. H2-evolution inhibition by O2 is also faster in the

D1:1 mutant, consistent with the interpretation that the overall photo-hydrogen yield is limited by

O2 poisoning. In both the strains, the steady state rate of oxygen evolution is established after a

transient lag phase as a response to the onset of illumination. However, D1:2 has delayed oxygen

production with a lag phase twice as long as the D1:1 mutant. In chlorophyll-a fluorescence studies,

D1:1 has a photochemical quenching (qP) coefficient double that of the D1:2 mutant. qP reflects

the number of open and closed PSII reaction centers which is governed by steady state electron

transport rate downstream of the PSII plastoquinone pool. These results indicate that a faster rate

of H2 production is achieved by the D1:1 mutant, but this cannot be fully realized owing to the

faster inactivation rate by the O2 coproduct. Thus, future studies should focus on elimination of the

O2 sensitivity of the hydrogenase.

P11. Developmental Vulnerability of Rat Hippocampal Neural Stem Cells to the Neurotoxicant Methylmercury (MeHg) Poku, M.1, Sokolowski, K.2, and DiCicco-Bloom, E.1 1 Department of Neuroscience and Cell Biology, UMDNJ-RWJMS; 2 Department of Toxicology, UMDNJ-RWJMS Methylmercury (MeHg) is an environmental teratogen that is known to cause cognitive deficits in

children who are exposed pre- or perinatally. Humans are exposed via consumption of

contaminated fish and shellfish. MeHg is known to harm the developing brain. One affected region

is the hippocampus – a structure that modulates learning and memory formation. It contains a

small but essential population of proliferating neural stem cells (NSCs) in the dentate gyrus (DG),

19

which produce new neurons throughout life. Our lab has shown that at an age when precursor

proliferation peaks (postnatal day 7 – P7), a single MeHg injection that approximates human

exposures (0.6 ug/gbw) causes cell cycle arrest and apoptosis in NSCs of the DG within 24 hours,

resulting in later deficits of hippocampal size (P21) and hippocampal-dependent behaviors (P35).

Preliminary studies suggest that this is caused by a lasting deficit in the NSC pool, after a 5 ug/gbw

MeHg exposure at P7 led to a 35% reduction of mitotic cells at P21. Ongoing studies of markers of

apoptosis and the cell cycle at P14 and P21 will determine the temporal schedule of NSC

vulnerability to MeHg, as well as the effects of early exposure on later neurogenesis. In turn, we

will gain insights into vulnerable periods in hippocampal neurogenesis that may inform guidelines

for fish and shellfish consumption before puberty. This research is supported by NS062591,

ES05022, NSF DGE 0801620

P12. Regulation of TRPM7 By 80K-H and Wnt Signaling During Early Embryonic Development Jeffrey D. Overton1, Courtney Mezzacappa2, Yuko Komiya2, Raymond Habas2, and Loren W. Runnels1 1Department of Pharmacology, UMDNJ-RWJMS 2Department of Biology, Temple University, Philadelphia, PA TRPM7 is a bifunctional protein that contains both ion channel and kinase domains. Our studies

have identified TRPM7 as an essential component of the non-canonical Wnt pathway regulating

gastrulation, a pivotal process during early embryonic life that executes the emergence of the body

plan and closure of the neural tube. The mechanism by which TRPM7 is regulated during early

development is unknown. To gain insight into how the channel-kinase is controlled we conducted

a yeast two hybrid screen, which revealed 80K-H as a potential binding partner. Pulldown

purification assays and immunoprecipitation experiments confirm 80K-H as a bona fide TRPM7-

interacting protein. Surprisingly, the strength of the interaction between 80K-H and TRPM7 is

dependent upon serum as well as on the channel’s own kinase domain. Knockdown of 80K-H

decreases TRPM7 protein expression, which can be prevented by inhibiting proteosomal

degradation. Depletion of 80-H in Xenopus laevis embryos produces a gastrulation defect, which

can be made more severe by simultaneous depletion of 80K-H and TRPM7. These results uncover a

novel role for 80K-H in early development and suggest a model in which 80-H regulates TRPM7

during early embryonic development, by regulating the protein’s turnover. This research is

supported by the NIH, NIGMS (1R01GM080753) to LWR, NIH, NIGMS (GM078172) to RH, and the

Foundation of UMDNJ to JDO

P13. A Novel Isoform of Sox5 is Expressed in TRAF3-Deficient Mouse B lymphomas Shanique K.E. Edwards, Anand Desai, Carissa R. Moore, Yan Liu, Ronald P. Hart and Ping Xie Department of Cell Biology and Neuroscience, Rutgers University TRAF3 is a novel tumor suppressor identified in human non-Hodgkin lymphoma and multiple

myeloma. We recently reported that TRAF3 deletion causes vastly prolonged survival of mature B

cells, which eventually leads to B lymphoma development in mice. The long latency of B lymphoma

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development observed in B-TRAF3-/- mice suggest that additional oncogenic pathways are required

for B lymphomagenesis. To delineate such oncogenic pathways in TRAF3-/- B lymphomas, we

performed microarray analyses and identified Sox5 as a gene recurrently up-regulated in B

lymphomas spontaneously developed in different individual B-TRAF3-/- mice. We confirmed the

striking up-regulation of Sox5 expression in TRAF3-/- B lymphomas at both the mRNA and protein

levels by quantitative real time PCR and Western blot analyses, respectively. We further cloned the

full-length cDNA of Sox5 from B lymphomas derived from 4 different individual B-TRAF3-/- mice.

Surprisingly, we found that the sequence of Sox5 expressed in TRAF3-/- B lymphomas represents a

novel isoform of Sox5, which has not been previously reported. This new isoform of Sox5 contains a

35 aa deletion in the N-terminal region in front of the leucine zipper domain. When transduced into

human multiple myeloma cells, the cloned Sox5 cDNA was expressed into a protein of 80 kDa, a

size identical to that detected in TRAF3-/- B lymphomas. Taken together, our findings identified a

novel isoform of Sox5 as a candidate oncogene in B lymphoma. Our ongoing experiments will

further elucidate the roles and mechanisms of Sox5 in TRAF3 deficiency-initiated B cell malignant

transformation. This research is supported by a seed grant from the New Jersey Commission on

Cancer Research (10-1066-CCR-EO, P. Xie), a Faculty Research Grant (P. Xie), and the Arthur

Herrmann Endowed Cancer Research Fund (P. Xie); supported in part by a grant from NCI (R. Hart)

and an Aresty Research Grant (A. Desai).

P14. Downregulation of Translation at the Initiation Step through Defects in the Organization of the Actin Cytoskeleton by Elongation Factor 1A Winder B. Perez and Terri Goss Kinzy Department of Molecular Genetics, Microbiology and Immunology,UMDNJ-RWJMS During translation elongation, eukaryotic translation elongation factor 1A (eEF1A) delivers

aminoacylated-tRNA into the A site of the ribosome. Aside from its canonical function, eEF1A has

been shown to interact with the actin cytoskeleton. Mutational analysis of eEF1A identified several

mutations that reduce its ability to bind and bundle actin as well as reduce total translation. The

defects in translation are not at the level of elongation, as would be expected for an elongation

factor, but rather at the initiation step. Several actin and actin binding protein mutant strains also

exhibited blocks in initiation providing further evidence that regulation of the actin cytoskeleton by

eEF1A is crucial for efficient protein synthesis. We are currently investigating the mechanism

behind the translation initiation defect in eEF1A actin bundling mutant strains. These strains

exhibit increased levels of phosphorylated eIF2, suggesting the involvement of the Gcn2p protein

kinase. Gcn2p responds to increases in uncharged tRNA levels in the cell and causes

downregulation of total protein synthesis. Deletion of GCN2 partially suppresses the growth and

cytoskeletal defects associated with eEF1A F308L and S405P strains. Rhodamine-phalloidin staining

of the eEF1A actin mutant strains lacking GCN2 is similar to the staining pattern of wild-type

strains, exhibiting a decrease in cell size. Phosphorylation of eIF2 and deletion of GCN2 in strains

harboring mutations in actin binding protein Tpm1p and actin itself is also being analyzed to

determine if the effect is specific to eEF1A. Differences in aminoacylation levels of all tRNAs in

wild-type and eEF1A actin bundling mutant strains are also being assessed.

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P15. Following a Cuprizone Lesion, ACPD, a Metabotropic Agonist, May Regulate MBP Through the Mediation of BDNF Fulmer C.G., VonDran M.W., Honeywell J.Z., Lercher L.L., Huang Y., Dreyfus C.F. Department of Neuroscience and Cell Biology, UMDNJ-RWJMS Previous studies indicate that BDNF impacts the development of oligodendrocyte (OLG) lineage

cells and may impact demyelination. Culture work determined that BDNF promotes synthesis of

DNA and differentiation of basal forebrain (BF) OLGs (Van’t Veer 2009, Du 2006). In vivo, BDNF +/-

mice exhibit deficits in NG2+ BF progenitor cells and decreased levels of myelin proteins

(VonDran 2010) and after treatment with cuprizone, BDNF +/- mice exhibit blunted increases in

NG2 and decreases in myelin basic protein (MBP) levels in the corpus callosum compared to BDNF

+/+ mice. Moreover, injection of exogenous BDNF into a cuprizone lesioned corpus callosum

increases MBP, suggesting that BDNF may act to reverse some of the deficits associated with a

demyelinating lesion. To increase endogenous levels of BDNF following demyelination, the current

work is examining the in vivo effects of the metabotropic glutamate receptor (mGluR) agonist

ACPD, known to upregulate BDNF protein and promote the release of BDNF from glial cells in

vitro (Bagayogo 2009, Jean 2008). ACPD (0.5uM) was injected into the corpus callosum of mice fed

control or cuprizone (0.2%) feed for 4 weeks. While mice fed cuprizone exhibit decreased levels of

BDNF in the corpus callosum, mice fed cuprizone and injected with ACPD exhibit increased BDNF

compared to mice injected with vehicle. Furthermore, deficits in MBP and MAG were also reversed

by ACPD. Coadiministration of TrkB-fc eliminated this effect, suggesting that the effect of ACPD is

mediated by BDNF. To explore the relevant receptors and cells, Group I (mGluR1 and 5) and Group

II (mGluR2/3) metabotropic glutamate receptors were evaluated. Western blot analysis indicated

that both Group I and Group II receptors are elevated following cuprizone. Group I receptors are

found on GFAP+ astrocytes and CD11B+ microglia, but not CC1+ OLGs, NG2 progenitor cells or Nf-l+

axons. Group II receptors are not found on oligodendrocytes or astrocytes, but may be present on

NF-l+ axons. These studies suggest that callosal astrocytes and microglia that

areimmunoreactive for mGluR1 and 5, and possibly mGluR2/3 positive axons, may be target cells

for ACPD after a demyelinating lesion. This research is supported by NS036647 and the NMSS.

P16. The Role of Chromodomain in eukaryotic Elongation Factor 3 (eEF3)-Ribosome Interaction Arjun N. Sasikumar and Terri G. Kinzy Department of Molecular Genetics, Microbiology and Immunology, UMDNJ-RWJMS Translation elongation in eukaryotes is a conserved process catalyzed by eukaryotic Elongation

Factor 1A (eEF1A) which delivers aminoacylated- tRNA to the ribosome and the ribosomal

translocase eukaryotic Elongation Factor 2 (eEF2). In addition to these two G-proteins, fungal

translation is characterized by its unique and absolute requirement for an ATPase factor called

eukaryotic Elongation Factor 3 (eEF3). eEF3 has two ATP binding cassette domains the second of

which has a chromodomain insertion, predicted to play a key role in its function.

Using Saccharomyces cerevisiae, we are characterizing the functional relevance of this domain in its

22

interaction with the ribosomes. We have made a series of chromodomain mutants that adversely

affect the translation elongation and the growth rate of the cells. The mutants also have severely

compromised intrinsic as well as ribosome stimulated ATPase activity and exhibit increased binding

to the ribosomes. Our work demonstrates that the chromodomain plays a significant role in the

functioning of eEF3 as a translation elongation factor in fungi. Because it is an essential protein

found across all fungal species, the characterization of its ribosomal interactions may help in

utilizing eEF3 as a target for the development of a broad spectrum anti-fungal with low host

toxicity. This research is supported by the National Institutes of Health.

P17. The Role of Enabled in Drosophila Heart Tube Formation Tiffany King and Sunita G. Kramer Department of Cell and Developmental Biology, UMDNJ-RWJMS The Drosophila heart is comprised of two rows of cardiac progenitor cells, or cardioblasts (CBs) that

migrate toward the dorsal midline, and align making dorsal and ventral attachments with their

contralateral partners in a way that creates a central lumen. Such discrete adhesion and guidance

of migrating cells must be carefully maintained for proper lumen formation to occur. Slit and its

receptor Roundabout (Robo) are required for lumen formation in the heart by negatively regulating

CB cell adhesion, however the mechanism by which this occurs is still unclear. Furthermore,

Netrins and their receptors are known axon guidance molecules involved in repulsion and

attraction, and unpublished data from the Kramer lab suggests that Netrin and its receptor

Frazzled/DCC (Fra) also play a role in CB alignment and attachment. The actin regulatory protein

Enabled (Ena) could be working downstream of both the Slit/Robo and Netrin/Fra pathways in the

heart, to regulate heart tube formation. Ena modulates the actin cytoskeleton downstream of both

the Netrin/Fra and Slit/Robo pathways during axon guidance and cell motility. Ena has also been

recently shown to localize to adherens junctions suggesting that it also plays a role in cell adhesion.

We show that Ena is expressed by the CBs in the heart tube and that in ena mutants, CB alignment

and shape are defective, suggesting that Ena may play a role in regulating CB cell shape changes

during lumen formation. In addition, the cell adhesion molecule E-Cadherin (E-Cad) is mislocalized

in ena mutants, suggesting that Ena may also play a novel role in regulating cell adhesion between

adjacent CBs. Furthermore, heart tube assembly appears to be delayed in ena mutants, suggesting

that CB migration to the dorsal midline is impaired. There also appears to be gaps in the heart

section of a combined maternal and zygotic loss of ena function mutant, which are not resolved in

late-stage embryos. It is not certain whether this is due to a delay in migration, or a loss of

adhesion between these opposing CBs. This is being further investigated through live-imaging

analysis. In addition, we are using genetic interaction studies to test whether Ena functions

downstream of Slit/Robo and or Netrin/Fra signaling. Together, these data have led us to

hypothesize that Ena has a role in CB motility, alignment and/or adhesion and may function

downstream of the Slit/Robo and/or Netrin/Fra pathways during heart tube formation.

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P18. Metabotropic Glutamate Receptor 1 Disrupts Mammary Acinar Architecture and Initiates Malignant Transformation of Mammary Epithelial Cells Jessica L. Teh1*, Ning Chen2*, Sandy Price2, Vassiliki Karantza2, Suzie Chen1 *These authors contributed equally to this work 1Susan Lehman Cullman Laboratory for Cancer Research, Ernest Mario School of Pharmacy, Rutgers University; 2The Cancer Institute of New Jersey, UMDNJ-RWJMS Metabotropic glutamate receptor 1 (mGluR1) is a member of the G-protein coupled receptor

superfamilyknown to participate in synaptic transmission and neuronal excitability. We previously

described the oncogenic properties of Grm1 in melanoma development in vivo. TG-3, a transgenic

mouse line developed spontaneous melanoma with 100% penetrance in the absence of any known

stimuli. Additionally, a recent report implicated mGluR1 as a potential therapeutic target in breast

cancer. As most human cancers are of epithelial origin, we use immortalized mouse mammary

epithelial cells (iMMECs) as a model system to study the transformative properties ofmGluR1. We

transfectediMMECswith mGluR1 and several stable mGluR1-expressing clones were characterized

for changes in theacinararchitecture using three-dimensional (3D)morphogenesisassays. In this

study, we found that mGluR1 expression in iMMECsled to delayed lumen formation (through

decreased central acinar cell death) and disrupted cell polarity. We also observed a dramatic

increase in the activation of the MAPKinase pathway in mGluR-1-expressing clones. Furthermore,

orthotopic implantation of these stable mGluR1-iMMECs into mammary fat pads of

immunodeficient nude mice was sufficient to induce mammary tumorigenesis. Taken together, our

findingsdemonstrate that mGluR1 may also be oncogenic in mammary epithelial cells thus,

highlighting its possible role in breast cancer.

P19. Investigating the Role of Endocytosis and Recycling in Mediating Cardioblast Cell Shape Changes During Drosophila Heart Tube Formation Frank David Macabenta and Sunita G. Kramer Department of Cell and Developmental Biology, UMDNJ-RWJMS Blood vessel assembly is a conserved process that involves the formation of a linear tube with a

central lumen. In the fruit fly Drosophila melanogaster, this involves the coordinated migration of

progenitor cells, called cardioblasts (CBs), towards the dorsal midline, which eventually undergo

morphological changes to form a linear tube. Opposing CBs first establish contact at their dorsal-

most surfaces; they then undergo highly stereotyped cell shape changes to enable attachment at

their ventral surfaces, while remaining unattached in between, allowing for the formation of a

lumen. The Kramer lab has shown that Slit protein and its corresponding receptor, Roundabout

(Robo), facilitate repulsion of opposing CB membranes by negatively regulating E-cadherin

mediated cell adhesion between these surfaces. In addition, the Netrin/Frazzled pathway has been

shown to be involved in facilitating E-cadherin-mediated CB alignment and attachment. It is known

that E-cadherin is removed from the membrane via an endocytic pathway that allows it to be either

recycled back to specific areas of the cell membrane or targeted for degradation. The exact nature

of this pathway, as well as the means by which the factors involved in facilitating adhesion and de-

24

adhesion at the CB cell surface (i.e. Slit, Robo, Netrin, and Frazzled) are localized to discrete

domains remains to be elucidated. Rab11 is one of a large family of monomeric G proteins that

plays a necessary role in vesicle-mediated recycling; it has previously been shown to be important

for proper nuclear translocation in developing Drosophila eye neurons. We hypothesize that

Rab11-mediated recycling is a necessary mechanism involved in lumen formation and maintenance

by mediating E-cadherin localization and recycling in conjunction with the Slit-Robo and Netrin-

Frazzled pathways, allowing for discrete receptor localization and site-specific adhesion and de-

adhesion. By crossing a line that overexpresses a dominant-negative form of Rab11 (UAS-

Rab11N124I) with one of several GAL4 driver lines that allow for specific overexpression in the dorsal

vessel (Tin(CΔ4)GAL4 and Mef2GAL4), we attempted to impair Rab11 function in the heart; the

resulting embryos were subsequently fixed, subjected to immunofluorescence techniques, and

observed under a confocal microscope. Preliminary data indicates defects in CB shape that vary in

severity, which suggests a correlation between Rab11-mediated endocytosis and proper dorsal

vessel assembly. Future studies will be aimed at visualizing the effect of ablating Rab11 function in

real time, as well as determining possible additional effectors that help mediate endocytosis and

recycling in CBs by examining the effect of impairing specific steps in vesicle trafficking.

P20.

Profiling Topographic Modulation of Stem Cell Nuclear Proteins on Fibrous Scaffolds via High Content Imaging and Modeling Sebastián L. Vega1, Parth J Patel2, Maria Qadri2, Prabhas V. Moghe1,2 1Department of Chemical and Biochemical Engineering, 2Department of Biomedical Engineering, Rutgers University In order to efficiently design implantable materials, methodologies that can effectively determine

how stem cells respond to biomaterial properties are needed to identify 1) stem cell phenotypes in

heterogeneous populations and 2) biomaterial properties that assist in lineage commitment.

However, current methods that evaluate stem cell responses are low-throughput and population-

based. In this study, we showcase a high content imaging-based profiling platform to evaluate

single-cell responses to changes in substrate topography by quantifying morphometric footprints

distinct to stem cell culture conditions. Nuclear features from confocal images were acquired from

human mesenchymal stem cells (hMSCs) that were cultured on electrospun fibrous scaffolds

exhibiting varying degrees of fiber diameter, porosity, and alignment. Using dimensionality

reduction and binary classification techniques, combinations of nuclear features were isolated,

which allowed identification of morphological signatures unique to the cell's microenvironment.

These early morphological signatures can be used to link substrate properties to long-term stem

cell differentiation, establishing a novel way of profiling biomaterial-driven stem cell lineage

commitment.

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P21. Disruptions to the miRNA Regulatory Pathway May Cause an Increased Rate of Schizophrenia in Individuals with 22q11.2 DS. William Manley1, Myka Ababon2, Paul Matteson2, Jim Millonig2, and Linda Brzustowicz1 1 CABM, Rutgers University 2Department of Neuroscience & Cell Biology, UMDNJ-RWJMS Schizophrenia is a complex and poorly understood disease caused by the interplay of many

environmental and genetic factors. The 22q11.2 deletion syndrome is caused by the microdeletion

of part of chromosome 22 leading to a 25-fold greater chance of developing schizophrenia in

affected individuals versus the general population. The 22q11.2 region contains the Dgcr8 gene,

which is required for the initial step of miRNA biogenesis. The deletion itself is not directly the

cause of schizophrenia, since 75% of individuals with this deletion do not develop the disease. We

hypothesize that the deletion increases the risk of schizophrenia through alterations in miRNA

regulatory networks via depletion of several miRNAs which may serve as a protective buffer against

the accumulation of deleterious mutations. Currently, we are working with human cellular models

to study the disruptions to the miRNA regulatory network caused as a result of DGCR8 deficiency.

siRNA specific for DGCR8 is being used in multiple neuronal cell lines to mimic 22q11.2

DS. Additionally, we have begun to characterize disruption to the miRNA regulatory network in cell

lines taken from individuals with the deletion.

P22. mTORC2 Modulates the Processing and Surface Expression of Membrane Receptors Chang-Chih Wu1, Po-Chien Chou1, Guy Werlen2, and Estela Jacinto1 1Department of Physiology and Biophysics, UMDNJ-RWJMS; 2Department of Cell Biology and Neuroscience, Rutgers University The mechanistic target of rapamycin (mTOR), an atypical protein kinase, is the catalytic subunit of

two structural and functional distinct protein complexes, mTOR complex 1 (mTORC1) and mTOR

complex 2 (mTORC2). These two protein complexes play essential roles in cell growth and

metabolism in response to extracellular signals such as nutrients, energy, growth factors and

hormones. Previously our laboratory has revealed a novel function of mTORC2 in regulating the

folding and stability of nascent Akt polypeptides co-translationally. Here, we report that mTORC2

promotes the processing of surface receptors. Through flow cytometry, we observed that the

deletion of the mTORC2 component rictor at early stage of T-lymphocyte development led to

diminished surface expression of receptors, including T-cell receptor (TCR), CD4, CD8, and CD147.

Furthermore, defective surface expression and N-linked glycosylation of CD147 was detected in

mTORC2-disrupted mouse embryonic fibroblasts via western blotting. Thus, we uncover a novel

function for mTORC2 that provides rationale to target this complex in disorders caused by

deregulated expression of surface receptors.

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P23. The Arp2/3 Activator WAVE/SCAR Promotes Clathrin Mediated Endocytosis in the Polarized C. elegans Intestinal Epithelia. Falshruti B. Patel and Martha C. Soto Department of Pathology, UMDNJ-RWJMS

Studies in yeast and mammalian cells have suggested that Arp2/3, the only known activator of

branched actin nucleation, is required during multiple steps of clathrin-mediated endocytosis

(CME) including membrane invagination, vesicle scission and vesicle motility away from the

membrane. One important question is how branched actin assembly is regulated during these

steps in multicellular organisms. One of the major regulators of branched actin is the WAVE/SCAR

complex, a nucleation-promoting factor (NPF) that activates Arp2/3. During C.elegans

embryogenesis, loss of WAVE/SCAR leads to arrests in cell migration and morphogenesis failure.

We have shown that loss of the WAVE complex components lead to progressive defects in

intestinal lumen morphogenesis and altered distribution of Apical Junction proteins, which

suggested a role for WAVE in maintenance of polarity. Analysis of clathrin-dependent cargo in

animals depleted of the WAVE pathway showed altered accumulation. Loss of any component of

the WAVE complex causes increased accumulation of clathrin heavy chain and reduced

accumulation of early endosome (RAB-5) and recycling endosomes (RAB-11) proteins at the apical

intestinal epithelium. Consistent with WAVE/SCAR’s role in CME, one of the components of WAVE

complex, GEX-3 has been shown to interact with dynamin in yeast-two hybrid studies. F-BAR

domain proteins are proposed to connect the actin cytoskeleton to trafficking events. The TOCA/F-

BAR proteins biochemically interact with the WAVE/SCAR complex in mammals and C. elegans.

Similar to C.elegans TOCA and WAVE mutants, loss of proteins involved in CME exhibit embryonic

morphogenesis defects, along with intestinal lumen expansion that correlates with drop in apical F-

actin levels in the intestine. Since WAVE complex components and proteins involved in CME are

mutually dependent for proper enrichment at the apical region of the C. elegansintestine, we

propose that WAVE-Arp2/3 dependent actin nucleation promotes CME at the apical intestinal

epithelium and that altered CME contributes to the apical morphogenesis defects of WAVE

mutants.

P24.

The Roles of Fbxl15 in Mammalian Circadian Cycle Qili Yu1, Kevin Huang1, Mantu Bhaumik2 & Renping Zhou1 1Department of Chemical Biology, Ernest Mario School of Pharmacy, Rutgers University; 2Department of Pediatrics, UMDNJ-RWJMS. Organisms ranging from bacteria to humans synchronize their circadian clocks to daily cycles of

light and dark. It has been reported that in Drosophila, JET, an F-box protein with Leucine-rich

repeats (LRRs), targets clock proteins TIMELESS (TIM) and Cryptochrome (CRY) for degradation in a

light dependant manner. A mutation in JET results in reduced phase shifts in response to light

pulse. In this study, we have generated a mouse strain with a complete knock out of the F-box and

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leucine-rich repeat protein 15 (Fbxl15), a close mammalian homologue of JET. A detailed analysis of

these KO mice would help understand the role and function of Fbxl15 in the regulation of

mammalian circadian rhythm. Our preliminary data shows that although Fbxl15 KO mice can be

entrained to light:dark (LD) cycles, they exhibit anomalous activity patterns in constant dark (DD)

with impaired activity phase shifting compared to wild type.

P25. GenSpec, a Genome-based Taxonomy Mapper for Bacteria and Archaea Zhu,C. and Bromberg,Y. Department of Biochemistry and Microbiology, Rutgers University The discovery of 16S rRNA has shaped the tree of life into three primary kingdoms: bacteria,

archaea and eukarya. 16S rRNA is the most popular molecular marker for bacteria and archaea due

to its ubiquitous presence, slow evolution rate and both conservative and variable region make-up.

It is commonly assumed that organisms with less than 97% 16S rRNA similarity belong to different

species. This threshold allows using 16S rRNA to study organism diversity in environmental

microbiology, clinical microbiology and metagenomic studies. Sequencing the 16S rRNA from a bio-

sample with subsequent alignment searches (e.g. BLAST) in online databases is now a routine for

access to the phylogeny of the unculturable strains. However, phylogeny reconstructed from a

single gene is encumbered by problems such as multiple copies of the gene and horizontal gene

transfer. With more and more genome data available, is it time to start looking at microbial

speciation on phylogenomic, or genome-wide scale?

Based on full proteome data of 1,491 bacteria and archaea downloaded from NCBI we show that

16S rRNA mapping fails to make correct taxonomy annotations; even when sequence pair identity

is computed from an extensively curated multiple sequence alignment. We compared the 16S rRNA

similarity between organisms to proteome similarity. Here we suggest that a new taxonomy

annotation criterion based on whole proteome comparisons or on a subset of proteins chosen by

feature selection algorithms will offer better accuracy than 16S rRNA alone. Preliminary results

from organisms of the same genus show that using 97% 16S rRNA sequence similarity cutoff to find

the organisms of the same species correctly retains 61.6% of all organisms at 54.2% accuracy. At

the same level of coverage, using the full prtoeome is 9.4% more accurate.

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The JMBGSA would like to thank the

following sponsors: