Annual Retreat PROGRAM: Banff Centre April 30 - May 1
Friday, April 30
Arrival at the Banff Centre – proceed to the Max Bell Auditorium (room registration in afternoon only).
Note: Special invited or Trainee Award Presentations are shown in italics.
10:30 am – Noon
Session #1 (Max Bell Auditorium)
10:30 Welcome and Opening Comments. Jonathan Lytton
10:45 Investigating molecular events during S phase that lead to genomic instability and
cancer. Mireille Tittel-Elmer, Margaret Renaud-Young, Denise Bustard, Carin Pihl, Jennifer
Cobb.
11:05 Understanding the Mechanisms of the Tid1 Tumor Suppressor. Diane Trinh, Bo
Young Ahn, Byoungchun Lee, Adam Elwi, Sung-Woo Kim.
11:25 Starting a New Fly Lab: Bugs and Bouquets. Savraj Grewal.
Noon – 1:30 pm
Lunch (Vistas Dining Room – Sally Borden Bldg)
1:30 – 3:30 pm
Session #2 (Max Bell Auditorium)
1:30 Science to Enterprise: Creating a Biotech Start-up. Norman Wong.
2:10 Carol Braat Memorial Awardee - DiscoverING, UncoverING, and UpdatING: History of
the INGs. Sitar Shah, J Luo, JB Rattner, Paul Mains, Karl Riabowol.
2:30 Src can do a whole lot of things: New insights on an old oncogene product. Lisa Yu,
Donald J. Fujita, Jeffrey D. Bjorge, Arja Kaipainen, Mary T-H. Chou, Josephine Anthony, Andy
Pang, Rebecca Leong-Quong, Laura Robson.
2:50 Estrogen mediated transcription induces replication associated DNA. Laura
Williamson, Susan Lees-Miller (see also Poster #1)
3:10 Postdoctoral Prize Awardee - Telomere resolution and hematogenous dissemination
mechanisms of Borrelia spirochetes. Tara Moriarty, M. Ursula Norman, Woo-Yong Lee,
Yvonne Tourand, Ashley R. Dresser, Pina Colarusso, Brandie Millen, Jan Deneke, Connie H.
Wong, Hong Zhou, Troy Bankhead, Robert M. Strieter, Nico van Rooijen, Paul Kubes, George
Chaconas
3:30 – 4:00 pm
Coffee/Nutrition Break (Max Bell Foyer)
4:00 – 5:30 pm
Session #3 (Max Bell Auditorium)
4:00 Postdoctoral Prize Awardee - The NLRP3 inflammasome and the regulation of
intestinal homeostasis. Simon Hirota, Justin MacDonald
4:20 Understanding the Transferrin Binding Protein B – Porcine Transferrin interaction
and its Role in Iron Release and Transfer. Leslie P. Silva; Ronghua Yu; Anthony B.
Schryvers; David C. Schriemer (see also Poster #2)
4:40 Investigating Novel Molecular Events Involved in Ca2+-Calmodulin Activation of
Smooth Muscle Myosin Light Chain Kinase Using Hydrogen-Deuterium Exchange Mass
Spectrometry. Lily Choi, Michael Walsh, John Chik (see also Poster #3)
5:00 How did I ever end up working on worm guts? Jim McGhee
5:30 pm – 6:00 pm
Time to mount posters, register rooms, etc.
6:00 – 7:15 pm
Dining Hall)
Banquet and Departmental Awards (Donald Cameron
7:30 – 9:00 pm
Poster viewing and Bar (Max Bell Foyer)
9:00 pm – ?
Social event (Max Bell Foyer and Lounge)
Saturday, May 1
8:00 – 9:00 am
Breakfast (Vistas Dining Room – Sally Borden Bldg)
9:00 am – 10:30 am
Session #4 (Max Bell Auditorium)
9:00 ATM-deficiency Sensitizes Mantle Cell Lymphoma Cells to PARP-1 Inhibitors.
Williamson CT, Muzik H, Turhan AG, ZamÒ A, O’Connor MJ, Bebb DG, Lees-Miller SP (see
also Poster #4)
9:20 Generation of induced pluripotent stem cells in suspension culture. Mehdi Shafa,
Roman Krawetz, Derrick E. Rancourt (see also Poster #5)
9:40 Redox control of phagosomal function in macrophages. Joanna Rybicka, Dale Balce,
Regina Krohn, Robin Yates.
10:00 Women in Science: Building on Success. Susan Lees-Miller, Jennifer Cobb, Kamela
Patel, Ana Colina, Lauren Killip.
10:30 – 11:00 am
Coffee/ nutrition break (Max Bell Foyer)
11:00 – Noon
Session #5 (Max Bell Auditorium)
11:00 Drosophila as a model system to study the TOR pathway, growth control and
cancer. Lynne Marshall, Savraj Grewal.
11:20 Creating the Future: Departmental Vision. Jonathan Lytton
11:50 Student Poster and Presentation Awards. Jonathan Lytton
12:00 noon
Retreat ends
We are very pleased to acknowledge our Sponsors for this event:
Silver Medal Sponsors:
Bronze Medal Sponsors:
Abstracts for Oral or Oral&Poster Presentations
Investigating molecular events during S phase that lead to genomic instability and cancer.
Mireille Tittel-Elmer, Margaret Renaud-Young, Denise Bustard, Carin Pihl, and Jennifer Cobb.
One central focus of the Cobb laboratory investigates the genetic factors that initiate and/or drive
tumourigenesis. For life to continue a cell must accurately replicate and separate its genome.
The failure to perform these cellular processes results in genomic mutations, a feature of most if
not all cancer cells. A major focus of the Laboratory is how the cell protects its genome by
preventing DNA damage during normal cellular events such as DNA replication and upon
environmental insult. As well, when damage occurs what mechanisms are triggered to initiate
repair without losing critical genetic information. Understanding these events at the molecular
level will likely reveal novel therapeutic targets to battle cancer.
Understanding the Mechanisms of the Tid1 Tumor Suppressor. Diane Trinh, Bo Young Ahn,
Byoungchun Lee, Adam Elwi, Sung-Woo Kim.
Tid1 is a novel tumor suppressor belonging to the DnaJ family of molecular co-chaperones.
Studies have shown it to interact with a number of proteins and to be involved in a wide variety
of signaling pathways as well as apoptosis. It has been found to localize mainly to the
mitochondria; however, its molecular mechanism at the mitochondria is yet unknown. Our lab
has shown that Tid1 forms a complex with p53 and induces mitochondrial translocation of p53
followed by a mitochondrial apoptotic program under conditions of cellular stress such as
hypoxia. We are currently focusing on understanding the biological significance and the
molecular specificities of this interaction such as whether the interaction is direct/indirect,
domains important for this interaction, involvement of post-translational modifications
(ubiquitination), and if a correlation of expression and cellular localization exists in breast cancer
tissues. In addition to our Tid1/p53 story, we are also establishing the role that Tid1 plays in
mitochondrial structure and fragmentation, interactions with other apoptotic proteins of the
mitochondria, and studying the regulation of gene expression of and by Tid1. Overall, we hope
to generate a better understanding of the function/mechanism of this tumor suppressor as it may
be a novel therapeutic to promote apoptosis in a wide variety of cancers.
DiscoverING, UncoverING, and UpdatING: History of the INGs. Shah S., Luo J., Rattner, JB.,
Mains P.E., and Riabowol K.
The INhibitor of Growth (ING) family of proteins is involved in multiple cellular processes such as
growth regulation, DNA repair and apoptosis. Loss or downregulation of ING protein function
frequently occurs in tumours. ING proteins are activated by stress, such as ionizing radiation,
which also leads to the activation of p53 and subsequently apoptosis. Mammals have five ING
genes and three are found in C. elegans. My research focuses on the characterization of C.
elegans ing-3, the gene with the highest sequence identity to mammalian ING3. Previous
experiments from our lab have shown that ing-3 mutant worms exhibit a decrease in apoptosis
when exposed to radiation. Initially it was thought that ing-3 may function in concert with cep1/p53, yet my results indicate that ing-3 does not affect the downstream target of cep-1/p53, and
therefore the two genes function in a parallel apoptotic pathway.
Our lab has recently found other ing-3 phenotypes. ing-3 mutants have a weak kinker
phenotype. Under electron microscopy, a subset of neuronal cells in ing-3 mutants show an
increase in condensed heterochromatin, which might be linked to the kinker movement of ing-3.
Finally, the ing-3 mutant has a lifespan of 25% longer than wild type, and is dependent upon daf16, the main regulator of lifespan in C. elegans. Studying the functional role of INGs in C.
elegans development will increase our knowledge about the role(s) they play in cancers and
cancer therapy.
Src can do a whole lot of things: New insights on an old oncogene product. Lisa Yu, Donald J.
Fujita, Jeffrey D. Bjorge, Arja Kaipainen, Lisa Yu, Mary T-H. Chou, Josephine Anthony, Andy Pang,
Rebecca Leong-Quong, and Laura Robson.
Non-activated
Activated
Src is a cytoplasmic and membrane-bound tyrosine kinase that is a master regulator of
several important signalling events and pathways that are involved in cell proliferation, migration,
angiogenesis, adhesion, and other cellular processes in normal cells. If Src kinase activity is
constitutively elevated and activated in an unregulated manner, then Src can trigger and
promote uncontrolled cell proliferation, angiogenesis, and other events leading to tumor growth
and metastasis. This presentation will provide an overview of our lab’s research concerning the
regulation, activation, and function of Src in normal and cancer cells. Recent findings
concerning the newly discovered role of Src in inactivating the VHL (von Hippel Lindau) tumor
suppressor gene product*, and the use of siRNAs that can block tumor growth and metastasis
by targeting Src and other critical gene products will be highlighted.
Estrogen mediated transcription induces replication associated DNA. Laura Williamson, Susan
Lees-Miller (Poster #1)
Reduced expression and mutations in several DNA double strand break response proteins
confers an increased risk of breast cancer. This increased risk is likely due to an inability of cells
to efficiently manage DNA damage within the dynamic breast tissue. One possible source of this
damage is the presence of hormones, specifically estrogen. Estrogen has previously been
shown to result in an accumulation of oxidized bases and abasic sites mediated by its
metabolism. In this study we show that treatment of breast cancer cells with 17-estradiol results
in an accumulation of DNA repair foci that are representative of replication associated DNA
damage. However, in contrast to the previously demonstrated model, these foci are not
mediated by estrogen metabolites and are dependent on the estrogen receptor and
transcription. Furthermore, the foci are dependent on topoisomerase II , an enzyme involved in
transcription at estrogen inducible genes. Here we propose a novel model of replication
associated DNA damage that is mediated by topoisomerase II  induced double strand break
formation occurring in response to estrogen stimulation. Importantly, this endogenous source of
DNA damage may be common to other regulated transcription events mediated by different
transcription factors.
Telomere resolution and hematogenous dissemination mechanisms of Borrelia spirochetes.
Tara Moriarty, M. Ursula Norman, Woo-Yong Lee, Yvonne Tourand, Ashley R. Dresser, Pina
Colarusso, Brandie Millen, Jan Deneke, Connie H. Wong, Hong Zhou, Troy Bankhead, Robert M.
Strieter, Nico van Rooijen, Paul Kubes, George Chaconas
Pathogenic spirochetes are bacteria associated with human illnesses such as syphilis,
periodontal disease, leptospirosis, relapsing fever and Lyme disease. Lyme disease is the most
common vector-borne infection in the northern hemisphere and is caused by tick-transmitted
Borrelia burgdorferi. Hematogenous dissemination is an important phase of most spirochete
infections; however dissemination mechanisms are still very poorly understood for spirochetes
and other pathogens. Borrelia species are also genetically fascinating because they maintain
linear DNA replicons, which are capped by covalently closed hairpin ends generated by telomere
resolvase (ResT). ResTs constitute a novel class of enzymes which are known or predicted to
catalyze telomere resolution in a growing number of bacteria and phages. I work on two major
projects in the Chaconas lab: 1) investigation of telomere resolution mechanisms in Lyme
disease and relapsing fever Borrelia species; and 2) investigation of B. burgdorferi dissemination
mechanisms using real-time imaging in living hosts. In my presentation I will discuss some of the
approaches I have taken to study these important scientific problems.
The NLRP3 inflammasome and the regulation of intestinal homeostasis. Simon Hirota, Justin
MacDonald
No abstract provided. This will be a special video presentation.
Understanding the Transferrin Binding Protein B – Porcine Transferrin interaction and its Role
in Iron Release and Transfer. Leslie P. Silva; Ronghua Yu; Anthony B. Schryvers; David C.
Schriemer (Poster #2)
Actinobaccillus pleuropneumoniae (Ap) is a porcine pathogen, causing pneumonia in pigs. The
bacterial transferrin binding protein (Tbp) receptor complex, comprised of TbpA and TbpB, plays
an important role in facilitating iron acquisition from its hosts during infection. How TbpB binds
porcine transferrin (pTf) and facilitates iron release is unknown. Insight into the pTf-TbpB binding
interaction and iron release process is crucial for vaccine development, as TbpB is known to
induce a protective immune response. Hydrogen-deuterium exchange mass spectrometry
(H/DX-MS) can be used to explore the interfacial regions in the Tf-TbpB interaction, while
Stability of unpurified proteins from rates of H/D exchange (SUPREX) can be used to
understand the process of iron release. Methods: pTf was expressed in Pichia pastoris, while 3
ApTbpBs and 1 Actinobaccillus suis TbpB were expressed in E. coli C43 cells. All were
processed in their free and complexed states, using a bottom-up H/DX-MS strategy. Disulfide
bonds were reduced in a TCEP-quench solution. Deuterium incorporation was quantitated after
an online pepsin digestion, and separation by an LC gradient. Peptides were analyzed with a
QSTAR Pulsari Qq-TOF MS. Data was analyzed using “Hydra” software for deuterium
quantitation. SUPREX experiments were performed on intact pTf, and FbpA, to assess the
release and acquisition of iron, and analyzed by LC-TOF-MS. Complex results were mapped to
pdb structures of Rosetta- and computationally-docked models, and will be tested with the
HADDOCK data-directed docking program. Results: All H/DX-MS studies mentioned above
have been analyzed with “Hydra” software using 69% sequence coverage of pTf, and 50-60%
sequence coverage of the TbpBs. Deuteration ratios of the complex vs. free protein have been
calculated for pTf, indicating that binding of the TbpBs to pTf results only in protection of both
proteins. The potential binding interaction has been mapped onto crystal structures of both holo
pTf, and one ApTbpB receptor. Peptides demonstrating interaction and/or stabilization were
mapped onto pTf’s C-lobe only, specifically in the iron-binding cleft, and the C2 lobe. Similarly,
results were mapped onto TbpB’s N-lobe, specifically, the loops extending from the N-lobe βbarrel. While all four receptors demonstrate similar protection profiles on pTf, the strength of
binding for two of the ApTbpB receptors appears to be higher, indicating a stronger binding
affinity. This conclusion has been confirmed via surface Plasmon resonance experiments.
Mutational studies with pTf have confirmed that binding occurs on the C-lobe of pTf, and have
identified one critical residue for binding, Arg 509. Using the H/DX-MS results obtained from
interaction with the h049 receptor, four additional mutations are currently being attempted to
identify other residues critical for the pTf-TbpB interaction. As solely protection was seen in all
experiments above, this would indicate a stabilization of pTf upon complexation. This was tested
via SUPREX studies with FbpA, in order to assess the stabilized complex’s ability to transfer
iron. The SUPREX experiments have indicated an increase in stability in iron-loaded FbpA,
however, when iron-free FbpA was incubated with the pTf-TbpB complex, no increase in stability
was seen, indicating no transfer of iron. Collectively, these results have identified the binding
regions of four unique porcine pathogen receptors on pTf, which were previously unknown.
These results have also indicated that TbpB does not cause iron release from pTf to TbpB.
Investigating Novel Molecular Events Involved in Ca2+-Calmodulin Activation of Smooth Muscle
Myosin Light Chain Kinase Using Hydrogen-Deuterium Exchange Mass Spectrometry. Lily Choi,
Michael Walsh, John Chik (Poster #3)
It has been well documented in the literature that an increase in smooth muscle intracellular
calcium concentration triggers calcium-calmodulin (Ca2+-CaM) activation of myosin light chain
kinase (MLCK). MLCK phosphorylates myosin and leads to muscle contraction. While models of
Ca2+-CaM activation of MLCK have been proposed, critical molecular events involved in the
activation have not yet been elucidated. The objective of this study is to decipher the essential
elements required for Ca2+-CaM-MLCK activation using soybean CaM isoforms (SCaM). In
human, three CaM genes encode for the same protein, whereas in soybean, five CaM genes
give rise to four CaM isoforms. SCaM-1, 90% identical to human CaM (hCaM) activates MLCK,
whereas SCaM-4, only 76% identical to hCaM does not. Previous experiments have shown that
SCaM-4 is a competitive inhibitor of SCaM-1, suggesting that both isoforms bind the same
MLCK site. Thus, SCaM-4 acts as a ready-made SCaM-1 mutant. Using hydrogen and
deuterium exchange mass spectrometry, the conformational differences between SCaM1/MLCK and SCaM-4/MLCK can be identified to decipher the molecular events critical for Ca2+CaM-MLCK activation. This study will aid in the development of new treatments for smooth
muscle disorders, expand our knowledge of other CaM mediated processes, and develop a
novel method to explore protein complexes, which are often difficult to study via NMR or X-ray
crystallography.
ATM-deficiency Sensitizes Mantle Cell Lymphoma Cells to PARP-1 Inhibitors. Williamson CT,
Muzik H, Turhan AG, ZamÒ A, O’Connor MJ, Bebb DG and Lees-Miller SP (Poster #4)
Poly-ADP ribose polymerase-1 (PARP-1) inhibition is toxic to cells with mutations in the breast
and ovarian cancer susceptibility genes BRCA1 or BRCA2, a concept, termed synthetic lethality.
However, whether this approach is applicable to other human cancers with defects in other DNA
repair genes has yet to be determined. The Ataxia-Telangiectasia Mutated (ATM) gene is altered
in a number of human cancers including Mantle Cell Lymphoma (MCL). Here, we characterize a
panel of MCL cell lines for ATM status and function and investigate the potential for synthetic
lethality in the presence of small molecule inhibitors of PARP-1. We show that Granta-519 and
UPN2 cells have low levels of ATM protein, are defective in DNA damage-induced ATMdependent signaling, are radiation sensitive and have cell cycle checkpoint defects: all
characteristics of defective ATM function. Significantly, Granta-519 and UPN2 cells were more
sensitive to PARP-1 inhibition, than were the ATM-proficient MCL cell lines examined.
Furthermore, the PARP-1 inhibitor olaparib (previously known as AZD2281/KU-0059436)
significantly decreased tumour growth and increased overall survival in mice bearing
subcutaneous xenografts of ATM-deficient Granta-519 cells, while producing only a modest
effect on overall survival of mice bearing xenografts of the ATM-proficient cell line, Z138. Our
results suggest that PARP inhibitors have therapeutic potential in the treatment of MCL and that
the concept of synthetic lethality extends to human cancers with alterations in ATM.
Generation of induced pluripotent stem cells in suspension culture. Mehdi Shafa, Roman
Krawetz, Derrick E. Rancourt (Poster #5)
The therapeutic use of stem cells requires the availability of pluripotent cells that are not limited
by technical, ethical, or immunological considerations. Recently, embryonic stem (ES)-like cells
called “induced pluripotent stem” (iPS) cells have been reprogrammed from a variety of different
somatic cells. One drawback of the iPS cell generation is its slow rate and inefficiency which
requires several weeks for an cell line to be established. In this study we sought to examine
whether the suspension bioreactor, would present a selective advantage in iPS cell derivation.
In order to generate large numbers of iPS cells, we used suspension culture to derive iPS cell
from Mouse Embryonic Fibroblasts (MEFs). We transduced fibroblasts with retroviral vectors
bearing the four reprogramming factors: Oct4, Sox2, Klf4 and c-Myc. When transferred to
suspension culture following transfection, the MEFs form aggregates, which were
morphologically similar to ESC. Bioreactor-derived iPS (BiPS) cell aggregates expressed
Alkaline Phosphatase after only 5 days post-transfection. We observed the de novo expression
of major ES cell pluripotency markers in more than 90% of cells (i.e. Oct 4, Sox2, Nanog,
SSEA1). Our results showed that fibroblasts can be reprogrammed in suspension bioreactor
very efficiently, and in large quantities in less than one week. We suggest that suspension
culture provides a selective advantage to enhance iPS cell generation. This is an important step
toward future clinical application of iPS cell, which necessiates establishing an efficient method
capable of generating large numbers of reprogrammed cells.
Redox control of phagosomal function in macrophages. Joanna Rybicka, Dale Balce, Regina
Krohn, Robin Yates.
The phagosomal lumen in macrophages is the site of numerous interacting chemistries that
mediate microbial killing, macromolecular degradation and antigen processing. Using a nonhypothesis based screen to explore the interconnectivity of phagosomal functions, we found that
NADPH oxidase (NOX2) negatively regulates levels of proteolysis within the maturing
phagosome of macrophages. Unlike the NOX2 mechanism of proteolytic control reported in
dendritic cells, this phenomenon in macrophages is independent of changes to lumenal pH and
is also independent of hydrolase delivery to the phagosome. We found that NOX2 mediates the
inhibition of phagosomal proteolysis in macrophages through reversible oxidative-inactivation of
local cysteine cathepsins. We also show that NOX2 activity significantly compromises the
phagosome's ability to reduce disulfides. These findings indicate that NOX2 oxidatively
inactivates cysteine cathepsins, through sustained ablation of the reductive capacity of the
phagosomal lumen. This constitutes a unique mechanism of spatio-temporal control of
phagosomal chemistries through the modulation of the local redox environment. In addition, this
work further implicates the microbicidal effector NOX2 as a global modulator of phagosomal
physiologies, particularly of those pertinent to antigen processing.
Drosophila as a model system to study the TOR pathway, growth control and cancer. Lynne
Marshall, Savraj Grewal.
TOR is a highly conserved kinase that regulates cell growth in all eukaryotes. It is activated by
signaling through a number of oncogenes and tumor suppressors, such as PTEN, TSC1/2, PI3
Kinase and Ras. Probably as a result of this, elevated TOR activity is a common feature of many
tumors, and may drive the overgrowth characteristic of the disease. Control of protein synthesis
is widely proposed as a growth-regulatory function of the TOR pathway. Current models suggest
TOR mediates this function by regulating mRNA translation. However, the control of ribosome
synthesis is an additional, but less studied, mechanism. We use Drosophila larvae as our model
system as they provide an excellent in vivo system for understanding how TOR regulates
growth. During the four-day larval period, animals increase in mass 200-fold, which is nutrition
and TOR-dependent. As such, starvation represents a simple mechanism to inactivate the TOR
pathway in Drosophila.
The control of ribosome synthesis has emerged as a conserved TOR function. Studies in yeast
have provided insights into how TOR controls ribosome synthesis. However less is known about
the mechanisms that operate in multicellular animals. We found that TOR signaling is both
necessary and sufficient to control ribosome synthesis in all tissues during Drosophila
development. This control is exerted through regulation of transcriptional responses via RNA
polymerases (Pols) I, II and III. We have identified transcription factors and repressors involved
in transcription by each of these polymerases that are TOR-regulated, suggesting another
mechanism by which TOR regulates growth.
Abstracts for Poster Presentations
P6. A biochemical assay to study protein recruitment to DNA double-strand breaks. Tracey A.
Dobbs, Ruiqiong Ye, Susan P. Lees-Miller
DNA integrity is continually challenged by exposure to endogenous and exogenous DNA
damaging agents. A major form of damage is double-strand breaks (DSBs), which if mis- or unrepaired are mutagenic or cytotoxic. Radiation-induced DSBs are characterized by base
lesions, abasic sites or single-strand breaks in close proximity to DSB termini. The diverse type,
yield and spatial orientation of these breaks require intense co-ordination of repair.
Non-homologous end joining (NHEJ), the predominant DSB repair pathway in
mammalian cells, involves break recognition, repair factor recruitment, processing and ligation of
the DNA. Although the 'core' components of NHEJ have been widely studied, little is known
about the order of recruitment or the structural conformations these components adopt during
repair. Even less is known about the interplay between different repair pathways (e.g. base
excision repair) or the resolution/ dissociation of repair factors.
We have developed an in vitro assay to study the interaction of repair factors with
modeled radiation-induced DSBs. Synthetic biotinylated oligonucleotides, coupled to magnetic
beads, are incubated with extracts from unirradiated or irradiated cells. Protein recruitment is
detected by western blot and mass spectrometry to study the effect of DNA structure on protein
recruitment and the potential role of phosphorylation on repair. Through transient knock-down of
repair factors, the requirement for NHEJ factors can be assessed and insights gained into
possible hierarchies of protein recruitment, back-up repair pathways and interchangeability of
repair proteins in the absence of classic NHEJ.
P7. DNA damage response regulated by the spindle assembly factor TPX2.
Gernot Neumayer, Jian Wang, Su Shim, Hoa Le, Hong Tran, Mathieu Chansard, Camille Belzil, Oliver
Gruss, Susan Lees-Miller, Thomas Helleday, Minh Dang Nguyen.
The Microtubule-associated protein TPX2 is required for mitotic spindle assembly and cell cycle
progression. During interphase, TPX2 is actively imported into the nucleus. The function of TPX2
in the nucleus is unknown. We now report that TPX2 plays a key role in the DNA damage
response (DDR) to Ionizing radiation (IR). Organisms are constantly subjected to DNA damage.
Inability to amplify and regulate a proper DDR results in cancers. Molecular amplification of the
DDR is mediated by a complex composed of the Mediator of DNA damage Checkpoint 1
(MDC1) and Ataxia Telangiectasia Mutated (ATM) kinase, which triggers phosphorylation of
histone H2AX (γ-H2AX when phosphorylated). The amplification of γ-H2AX is essential to
promote cellular resistance to DNA damage. We found that TPX2 associates with the
MDC1/ATM-complex after IR and, critically, dampens the MDC1/ATM-dependent
phosphorylation of H2AX. Overexpression of TPX2 leads to decreased γ-H2AX signals and a
disrupted intra-S-phase checkpoint after IR whereas cells with decreased levels of TPX2 overamplify γ-H2AX signals and exhibit repair defects and increased apoptosis after IR. Thus, our
study identifies a first function for TPX2 in the nucleus and a novel regulator of DDR.
P8. SMTNL1 from smooth muscle associates with thin filament tropomyosin. Annegret UlkeLemÈe, Hiroaki Ishida, Meredith A. Borman, Hans J. Vogel and Justin A. MacDonald.
Hypertension is probably the leading cause of death in the world. Smooth muscle contraction is
a key property of the blood vessels and contributes to health; and when dysfunctional is
associated with hypertension causing many other disorders. The process of smooth muscle
contraction is highly dynamic and tightly regulated, but not well understood. Recently, we
discovered SMTNL1 (smoothelin-like 1), a novel smooth muscle protein which is associated with
improved contractile performance of muscle. SMTNL1 is phosphorylated on serine 301 in
response to relaxant agonists that activate protein kinase A/G-dependent pathways, and this
phosphorylation correlates with smooth muscle relaxation. PKG has been implicated in calcium
desensitization, a process which leads to smooth muscle relaxation without a drop in the
intracellular calcium concentration. Based on homology with the smoothelins, a family of actin
binding proteins, SMTNL1 contains a calponin-homology (CH) domain. The CH domain is
implicated in actin-binding; however, no actin-association of SMTNL1 was detected. Using pulldown studies and isothermal-calorimetrie, we could show that SMTNL1 can associate with
tropomyosin via its CH domain. Tropomyosin is tightly associated with actin and forms an
integral part of the thin filament in all muscles. Furthermore, in smooth muscle cells, SMTNL1
appears in filament-like structures while a mutant lacking the CH domain does not. It appears
SMTNL1 is responsible for increased contraction in vascular smooth muscle. For future work, I
hypothesize that SMTNL1 hinders vasodilatation and contributes to hypertensive vasculature
through its interaction with tropomyosin in smooth muscle which might be regulated by PKGdependent phosphorylation.
P9. Exploring erlin protein structure and function in mammalian cells and Caenorhabditis
elegans. Maja B. Hoegg, James D. McGhee, Stephen M. Robbins
The endoplasmic reticulum (ER) lipid Raft-associated proteins (erlins) are novel members
of the group of Stomatin/ Prohibitin/ Flotillin/ HflK/C (SPFH) domain-containing proteins, which
were discovered in our laboratory using an antibody screen against lipid raft proteins. Mammals
have two highly related erlin proteins (erlin-1 and -2) and recent studies on cell lines suggest that
they are involved in targeting activated IP3-receptors and potentially other proteins for ERassociated protein degradation (ERAD). We demonstrated that the erlins form homo- and
hetero-oligomers and are associated with high molecular (MW) weight complexes. These are
two distinct properties of the erlins, since they are mediated by different regions within these
proteins. Ectopic expression of multimerization-defective erlin mutants disrupts erlin complexes,
suggesting that they could act as dominant negative mutants.
The nematode C. elegans has one erlin protein, which shares strong similarity with mammalian
erlins. We examined whether C. elegans erlin functions in similar processes than its mammalian
counterparts. However, our data suggest that C. elegans erlin does not play a role in regulating
IP3-receptor dependent processes or in ERAD of other substrate proteins.
P10. The XLF C-terminal region is required for DNA binding and interaction with Ku70/80 in
vitro but not for repair of double-strand breaks in vivo. Brandi Mahaney, Yaping Yu, Shujuan
Fang, Susan P. Lees-Miller.
DNA double strand breaks (DSBs) are one of the most detrimental DNA lesions in the cell.
DSBs can be induced by ionizing radiation (IR) and in mammalian cells these DSBs are primarily
repaired by the non homologous end-joining pathway (NHEJ) which involves several core
proteins including the DNA-PK complex which is composed of the Ku70/80 heterodimer and the
DNA-PKcs catalytic subunit; the XRCC4-DNA ligase IV complex; and the XRCC4-like factor
(XLF) which stimulates XRCC4-DNA ligase IV mediated DNA end-joining. Here we show that
the C-terminal region of XLF, which is predicted to be unstructured, is required for the DNAdependent interaction between XLF and the DNA-PK complex in cell extracts and for interaction
with purified Ku70/80 in pulldown assays. Interestingly, the highly conserved penultimate amino
acid F298 is absolutely required for the interaction between XLF and Ku as well as for DNA
binding in electrophoretic mobility shift assays. However, C-terminal truncation of XLF or
mutation of F298 does not significantly alter the kinetics of gamma-H2AX foci resolution
following IR in vivo. These results suggest that although the C-terminal region of XLF is
important for DNA binding and interaction with Ku in vitro these interactions may not be
necessary for double-strand break repair in vivo.
P11. The effect of Clostridium difficile toxin on ecto-5’-nucleotidase (CD73) and epithelial
barrier function. Patrick Schenck, Simon Hirota, Paul Beck, Justin MacDonald.
Clostridium difficile (Cdif) is one of the most prevalent nosocomial infections in North America.
Cdif releases a toxin that can lead to the destruction of the intestinal epithelial barrier, leading to
diarrhea and in severe cases, toxic megacolon and removal of the colon. Recently, it has been
demonstrated that Cdif toxin stabilizes the transcription factor hypoxia-inducible factor-1 (HIF-1).
This stabilization offers an innate protective response to Cdif toxin. Our research has shown
that HIF-1 up-regulates the transcript for ecto-5’-nucleotidase (CD73), an enzyme that cleaves
neutrophil-released adenosine-5’-monophosphate (AMP) to adenosine, which signals to
adenosine receptors (AdoRs) to increase barrier function. We hypothesize that Cdif toxininduced elevations in CD73 transcript lead to an up-regulation of CD73 protein, which lead to
increased barrier function in cultured Caco-2 intestinal epithelial cells (IECs). Preliminary studies
reveal that Cdif toxin and CoCl2 (stabilizer of HIF-1) exposure result in increased CD73 transcript
and protein levels. Functionally, treating Caco-2 cells with AMP resulted in decreased
permeability when measured with a trans-epithelial resistance assay. Future experiments will
include the use of AdoR agonists and antagonists while inhibiting CD73 to further elucidate the
role of CD73 in barrier function during Cdif-associated colitis.
P12. A nutrition-dependent insulin/TOR pathway regulates growth via the transcription factor
Myc in Drosophila. Sabarish Nagarajan, Dr. Savraj Grewal.
Cellular and organismal growth are determined by nutrients and environmental factors
that regulate metabolism. We use Drosophila to study how growth factors alter metabolism to
promote growth. Many of the metabolic pathways important for growth regulation are conserved
between Drosophila and mammals. One important way by which growth is regulated across
eukaryotes is the Insulin/TOR kinase pathway. Nutrients activate the Insulin/TOR pathway to
promote growth in Drosophila. In contrast, dietary starvation leads to the suppression of the
Insulin/TOR pathway and leads to a growth arrest in Drosophila larvae. TOR kinase promotes
growth in part via changes in metabolic gene expression. Many of these genes are induced by
the transcription factor dMyc or Drosophila Myc – for example ribosome biosynthesis genes and
protein synthesis genes. Thus we hypothesize that Myc might be one of the mediators of
Nutrition/Insulin/TOR dependent gene expression and growth. My western blots show that dMyc
protein levels are downregulated upon starvation of larvae, even though the mRNA levels
remain relatively constant. Initial experiments by polysome gradient centrifugation suggest that
dMyc mRNA translation decreases upon starvation. Thus I hypothesize that Insulin/TOR
regulates dMyc protein at the level of its translation. dMyc translational regulation might be one
mechanism to maintain homeostasis and promote survival under starvation. I shall further
investigate the mechanisms of translational control by nutrition coupled Insulin/TOR signaling.
P13. TGF-β signalling regulates C. elegans body size in a cell non-autonomous manner. Aidan
Dineen, Jeb Gaudet
Body size in Caenorhabditis elegans is regulated by the Sma/Mab pathway, one of two
Transforming Growth Factor β (TGF-β) signalling cascades. Loss of function mutations in
components of this pathway result in a 50% reduction in size compared to wild type. Previous
study of the downstream receptor-regulated Smad SMA-3 has shown that targeted rescue in the
hypodermis is necessary and sufficient to rescue body size. Our lab studies development of the
C. elegans pharynx, and we wished to examine whether the SMA/TGF- β pathway might also be
involved in regulation of pharynx size, which had not been previously examined. We observed
that sma-3 mutants have reduced pharynx and body size in contrast to dpy mutants, which
exhibit a reduced body size but wild type pharynx size. We hypothesized that the reduced
pharynx size of sma-3 mutants could be rescued by pharynx specific expression of SMA-3.
Surprisingly, we found that targeted expression of SMA-3 in muscle and marginal cells of the
pharynx partially restored wild type pharynx and body size. In contrast to previous studies, our
data suggests a cell non-autonomous role for SMA/TGF- β signalling in body size regulation.
P14. The risk of oncogenic transformation of human induced pluripotent stem cells during
cartilage differentiation. Akihiro Yamashita, Guoliang Meng, Derrick E Rancourt
Induced pluripotent stem cells (iPSCs) are attractive cells for future use in regenerative
medicine. However, iPSCs have an increased risk of tumorigenicity. Considering this
propensity toward oncogenic transformation it is essential to find an assay for evaluating this
risk. Formation of cartilage may be one of the most attractive assays since functional cartilagelike tissue can be formed in vitro. In this study, we focused on chondrocyte differentiation and
cartilage tissue engineering of human embryonic stem cells (ESCs) and iPSCs.
Both human ESCs and iPSCs differentiated into chondrocytes lineage using micro-mass
culture. Following 6 weeks of culture, human ESCs showed the phenotypic hallmarks of
cartilage. Although human iPSCs could form cartilage, 35% of the aggregates were found to
include glandular epithelium consistent with columnar epithelium. The glandular epithelium
displayed the lack of cartilage matrix/proteoglycan and the presence of markers of
tumorigenicity. These results indicated that human iPSCs present a risk of oncogenic
transformation following prolonged culture/differentiation.
In conclusion, we demonstrate that cartilage tissue can be engineered using human ESCs and
iPSCs. Although ESCs could form cartilage safely, iPSCs showed a high risk of oncogenic
transformation. We suggest that cartilage tissue engineering can be useful as an indicator
and/or research tool for evaluating the risk of oncogenic transformation of human iPSCs.
P15. A pre-clinical evaluation of two oncolytic viruses against neuroblastoma. Nicole Redding,
Karen Blote, Loen Hansford, HongYuan Zhou, XueQing Lun, Donna Senger, John Bell, Grant
McFadden, David Kaplan, Peter Forsyth, Stephen Robbins, Paul Beaudry.
Neuroblastoma is the most common solid, extracranial tumor of childhood. High-risk patients
have a poor prognosis and little progress has been made in improving their outcome. A subpopulation of highly tumorigenic cells with stem cell properties, termed Tumor Initiating Cells
(TICs) has been identified in neuroblastoma, however their role in tumor behavior is not
understood and their susceptibility to novel therapeutics is unknown. Myxoma and vesicular
stomatitis virus (VSV) are two oncolytic viruses that can selectively kill cancerous cells. It is
currently unknown if these oncolytic viruses are effective therapies for NB. We have
demonstrated that both viruses kill panel of neuroblastoma established cells in vitro and
significantly reduce the tumor burden and increase the life span in mice carrying subcutaneous
neuroblastoma xenografts in vivo. While myxoma was able to kill neuroblastoma TICs in vitro,
VSV was not able to cause lysis and cell death, as measured by cell viability assays. The mode
of resistance of TICs to VSVs oncolytic effect is currently under investigation, along with their in
vivo susceptibility to both viruses.
P16. siRNA-mediated Targeting of Src and Downstream Signaling Pathways Inhibit Tumor
Growth and Metastasis. Jeffrey Bjorge; Melanie Funnell; Ke Yun Chen; Andy Pang; Roman Diaz;
Anthony Magliocco; Donald Fujita
Elevated Src activity and activation of pathways downstream of Src contribute to the
malignant phenotype of several human cancers. We are characterizing several of these
pathways by utilizing siRNAs (small-interfering RNAs) to downregulate specific gene products. In
NOD/SCID mice, human breast cancer cells were either pretransfected with siRNA, or activelygrowing breast tumors were treated by intratumoral injection of siRNAs. Simultaneous siRNAmediated knockdown of Src and the downstream Src-target Stat3 inhibited primary tumor growth
of MDA-MB-435 cells as well as inhibiting tumor metastasis. In MDA-MB-231, Stat3 siRNA by
itself had a substantial inhibitory effect on primary tumor formation. These results suggest that it
should be possible to determine optimal patterns of gene knockdown for the inhibition of tumor
growth and metastasis of breast cancer subgroups. To enhance the biological activity of these
siRNAs, we examined the effects of replacing unmodified with 2’-O-methyl-modified nucleotides.
We found that this had varying effects on siRNA stability and activity, depending upon the
location and extent of modification. Several effective modified siRNAs have been identified and
are being studied further. We are currently examining various methods of “in vivo” delivery to
enable the systemic administration of these siRNA.
These approaches of gene targeting may further clarify the roles of Src, downstream, and
parallel signaling pathways and facilitate the development of safer and more effective
therapeutic drugs.
P17. The Regulation of RNA Polymerase III and Cell Growth by Drosophila Maf1 and the Targetof-Rapamycin (TOR) Pathway. Elizabeth J. Rideout and Savraj S. Grewal
The Target-of-Rapamycin (TOR) pathway is a conserved regulator of cell and organismal growth
in eukaryotes. We use Drosophila larvae as a model to examine how TOR regulates cell and
organismal growth. In Drosophila, the TOR pathway couples nutrition with growth. Although
many inputs to the TOR pathway are known, the downstream effectors used by TOR to regulate
cell growth and metabolism remain largely unknown. Recent evidence suggests TOR controls
growth by regulating tRNA synthesis by RNA Polymerase III (Pol III). In Drosophila, we have
demonstrated that reduced TOR signaling leads to decreased levels of tRNA synthesis. In my
study, I will focus on a conserved repressor of Pol III transcription, Maf1. In yeast and mammals,
Maf1 represses Pol III transcription in response to growth-inhibitory cues. My data suggests that
Drosophila Maf1 (dMaf1) functions similarly to repress Pol III transcription. When levels of dMaf1
were decreased by RNA interference (RNAi), tRNA levels were significantly elevated. Even in
starved larvae, where TOR signaling is reduced, levels of tRNA synthesis remained high if
dMaf1 levels were low. This suggests that dMaf1 inactivation is required for TOR-induced growth
and tRNA synthesis. Interestingly, decreased levels of dMaf1 lead to increased growth and
accelerated larval development. Preliminary data suggests that in addition to their accelerated
development, individuals with decreased dMaf1 are starvation-sensitive and have reduced
survival in response to starvation. Thus in Drosophila dMaf1 plays an important role in
controlling growth by regulating Pol III transcription. Future studies will address how dMaf1 is
regulated by TOR, and whether this regulation is important for mediating organismal responses
to stress.
P18. Probing Tid1 suppressor function in modulating Bax and PUMA pro-apoptotic function.
Adam Elwi, Byoungchun Lee, Diane Trinh, Bo Young Ahn, Sung-Woo Kim.
Loss of Tid1 tumor suppressor protein function induces stress resistance in response to
cellular stresses, including genotoxic and hypoxic stresses. Mounting evidence points to the
existence of both a delayed p53 transcription-dependent apoptotic program and a rapid p53
transcription-independent mitochondrial apoptotic pathway. Recent studies in our laboratory
have shown that tumor suppressor Tid1 forms a complex with p53 under hypoxic stress and
mediates mitochondrial translocation of p53 and initiation of a transcription-independent
mitochondrial apoptotic pathway in breast cancer cells. Here we investigated whether Tid1
modulates function of pro-apoptotic proteins Bax and PUMA in rapid first wave induction of a
transcription-independent mitochondrial apoptotic pathway and in delayed induction of a
transcription-dependent apoptotic program. Preliminary evidence suggests that suppression of
Tid1 modulates translocation of pro-apoptotic protein Bax after short-term genotoxic and hypoxic
stresses and modulates expression of pro-apoptotic proteins Bax and PUMA after long-term
cellular stresses. Our results suggest Tid1 may have a role in modulating expression,
translocation, and assembly of pro-apoptotic proteins involved in mitochondrial apoptotic
pathway.
P19. Zyxin translocation from focal adhesions and stress fibers to the nucleus on Human
Umbilical Vein Endothelial Cells. Juan Voutssas, Kamala D. Patel, Hong Zang, Ritu Sharma
Zyxin is an 83 kD protein isolated from chicken smooth muscle in 1991 and preserved in many
species. It localizes at focal adhesion and stress fibers and it resembles to the protein ActA of
Listeria monocytogenes. On its NH2 terminus, this protein presents an actin binding site and four
proline rich repeats; on its COOH terminus, zyxin presents a non classical nuclear exportation
sequence and three LIM domains. It has been shown previously that zyxin is essential for actin
cytoskeleton rearrangements and the proper localization of other proteins such as ENA/VASP,
p130Cas and h-warts/LATS so it is strongly related to cell motility and mitosis progression
regulation. Zyxin shuttles from focal adhesion and accumulates into the nucleus in
cardiomyocites treated with atrial natiruretic peptide (ANP) by PI3K/Akt signaling leading to cell
survival. Our lab is the first one to shown that zyxin leaves focal adhesions and accumulate in
the nucleus in activated endothelial cells when neutrophils (PMNs) transmigrate but the
mechanism of translocation and the function of zyxin in the nucleus in this cells remains
unknown. So far, our results show that zyxin leaves focal adhesion and stress fibers from TNF-a
stimulated endothelial cells within 10-15 minutes after PMNs and a radical tubulin degradation
occurs. This events does not depend on Ca2+ or PI3K/Akt cascade signaling and possibly, these
events are triggered by nitric oxide (NO).
P20. TID1 may be a bridge between mitochondrial apoptosis and mitochondrial fragmentation.
Byoungchun Lee, Adam Elwi, Bo Young Ahn, Diane Trinh, Sung-Woo Kim.
TID1 has been discovered as a tumour suppressor firstly, and some later findings suggested
that Tid1 overexpression sensitized cells to stresses and drugs. However, the role of TID1
during the sensitization of cells remains unclear. Recently, some interesting findings showed that
fragmented mitochondria induced by DRP1 activation and/or BAX/BAK activation are more
sensitive to stress conditions, leading to apoptosis. Therefore, we hypothesized that Tid1
overexpression sensitizes cells to stresses through mitochondrial fragmentation. To address this
hypothesis, we tried Tid1 overexpression and suppression, and we found that Tid1
overexpression induced mitochondrial fragmentation, but not the suppression of TID1 did affect
at a significant level in HeLa cells. In addition, fragmented mitochondria didn’t show the release
of cytochrome c to the cytosol, suggesting that fragmented mitochondria induced by Tid1
overexpression were not apoptotic, which was similar to the phenotype from DRP1 activation.
Now, we are testing some possible pathways such as DRP1 activation, MFN1/MFN2 inhition,
and BAX/BAK activation. Taken together, overexpressed TID1 fragmented mitochondria, which
are not apoptotic, and the mechanisms behind this event are being investigated. We hope this
research to elucidate the bridge between mitochondrial apoptosis and mitochondrial
fragmentation, and can provide a clue to developing a new treatment of cancers and
neurodegenerative diseases.
P21. Environmental cues regulate Ribosome Synthesis in Drosophila. Abhishek Ghosh, Savraj
Grewal.
Cell growth is essential for the regulation of organ and organism size. Cell growth is regulated by
the conserved insulin/TOR pathway in metazoans. Studies have revealed several signaling
inputs to this pathway. However, less is known about the growth regulatory outputs of TOR. Our
lab uses Drosophila as a model system to study ribosome synthesis. TOR regulates ribosome
synthesis to drive cell growth. We previously found that the conserved Target-of-Rapamycin
(TOR) kinase pathway modulates ribosomal RNA (rRNA) synthesis via the transcription factor,
TIFIA. We propose that TIFIA is a growth-regulatory target of TOR. I will examine the molecular
mechanisms by which TOR regulates TIFIA. In contrast to growth cues, I hypothesize that stress
cues inhibit ribosome synthesis to promote cell survival and homeostasis. My preliminary data
has shown that stress cues - starvation and oxidative stress inhibit rRNA synthesis. I will
examine whether inhibition of TIFIA is a downstream target of these stress cues and explore
how stress-dependent signaling pathways regulate TIFIA function. Together these studies will
outline how growth and stress cues regulate cell behavior by modulating TIFIA function and
ribosome synthesis. Understanding ribosome synthesis will enhance our knowledge on
mammalian cell growth regulation in the context of development and diseases, e.g. cancer.
P22. ING2 as a potential novel regulator of cell differentiation. Shawn Eapen, Stuart Netherton,
Krishna Sarker, Angela Chan, Lili Deng, and Shirin Bonni.
Members of the Inhibitor of Growth (ING) family of chromatin modifiers regulate gene expression
and consequent cellular responses, eg cell proliferation and apoptosis. Whether the INGs
control cell differentiation remained unexplored until recently. We examined the role of the ING
member, ING2, in cell differentiation. We used the C2C12 myotube formation as a model for cell
differentiation. We employed cell-based and transcriptional-based approaches to follow
myogenic differentiation of C2C12 myoblasts. Interestingly, RNAi-triggered knockdown of ING2
led to a drastic reduction in the ability of C2C12 myoblasts to undergo differentiation-induced
myogenesis. Thus, the ING2 loss of function data suggested that ING2 may promote muscle
differentiation. We next carried out structural-functional studies to determine the regions within
ING2 that regulate the ability of ING2 to control myotube formation. These analyses showed
that the N-terminal “conserved region 1” and C-terminal “plant homeodomain” regions may
regulate positively and negatively, respectively, the ability of ING2 to promote muscle
differentiation. Further research is being carried out to elucidate the molecular mechanisms by
which ING2 regulates this differentiation process. In summary, our findings reveal that ING2 may
regulate cell differentiation. In addition, our results help to map out the domains involved in the
ability of ING2 to modulate this cellular response.
P23. Smooth muscle myosin light chain phosphatase regulation by cyclic nucleotide induced
phosphorylation of MYPT1 at serine 852. Mike Grassie, Shannon Brown, Annegret Ulke-Lemee,
Mona Chappellaz, and Justin MacDonald.
Smooth muscle contraction occurs when the 20-KDa regulatory light chain of myosin II
becomes phosphorylated, a process that is regulated by myosin light chain kinase (MLCK) and
myosin light chain phosphatase (MLCP). MLCP is composed of three subunits; a PP1c catalytic
subunit, a myosin phosphatase targeting subunit 1 (MYPT1) and M20. MYPT1 acts as the
primary mechanism of MLCP regulation via different phosphorylation sites. Rho-kinase and
ZIPK have been shown to phosphorylate two inhibitory threonine sites, Thr697 and Thr853
(numbering of the human isoform), leading to MLCP inhibition. Phosphorylation of the Ser696
residue in response to cyclic nucleotides, causes a decrease in Thr697 phosphorylation, leading
to relaxation. Our first goal is to map the specific sites phosphorylated by PKA and PKG,
hypothesizing that Ser852 will also be an important site. Secondly, the effects of Ser852
phosphorylation on the subsequent phosphorylation of Thr853 by ROK will be examined. We
predict the phosphorylation of Ser852 will inhibit phosphorylation of Thr853. If correct, we plan
to develop phospho-specific antibodies and test the physiological effects of Ser852
phosphorylation in vitro and in vivo. Thus far we have developed and expressed the MYPT1
proteins needed. Moreover, we have demonstrated that PKA phosphorylates wild-type MYPT1
with a time course increase in phosphorylation, leading to saturation with a stoichiometric factor
of 3 mol phosphate / mol MYPT1.
P24. Interaction of Quantum Dots in Zebrafish. Sean Jiang, Sarah Childs
Nanoscale particles, or nanoparticles, are particles 10-500nm in diameter. They are currently
used in the cosmetic industry and have been proposed for use in other applications such as drug
delivery, diagnostic imaging and tissue engineering. The increase in nanoparticle use will
inevitably lead to greater environmental and human exposure, and thus necessitating further
investigation of the potential detrimental effects of nanoparticles. We are interested in embryonic
exposure to nanoparticles and its effect on angiogenesis. For initial experiments we used
fluorescent microspheres 20-100nm in diameter which have been previously shown to be taken
up by reticular cells of the tail vein. After injection of FluoSpheres at 1-cell stage, 20nm particles
were confined to the yolk, but some larger 1000nm particles were observed in tissue after
24hours suggesting a size preference for interaction with embryonic tissue. Injection of
FluoSpheres by angiography directly into the circulation of 30-48 hour embryos resulted in
formation of a punctate pattern of FluoSpheres on vessel walls 1hr post-injection. Quantum dots,
10nm in diameter, made of a CdSe core, ZnS shell, and PEG polymer coating were injected into
the circulation of 48hr embryos through angiography. QDs appeared to adhere to the wall of the
blood vessels shortly after injection, similar to FluoSpheres. 18 hours after injecting QDs, the
particles were dispersed and no longer lined the wall of vessels. Rather, they appeared to be
randomly scattered in the caudal tail plexus in a punctate pattern.
P25. Effects of nitric oxide on HIF-1α and FIH-1 following Clostridium difficile toxin exposure.
Joshua Lee, Simon Hirota, Danya Traboulsi, Paul Beck, Justin MacDonald
Clostridium difficile (C.diff) is a leading cause of infectious colitis in humans. C.diff toxins disrupt
the intestinal epithelial barrier and induce the release of proinflammatory mediators. The
hypoxia-inducible factor-1 (HIF-1α) is a stress-induced transcription factor that mediates
mucosal and barrier protective genes. Nitric oxide (NO) and the factor-inhibiting HIF-1α (FIH-1)
modulate HIF-1α expression and signaling, respectively. We hypothesize that NO produced
during C.diff toxin-induced intestinal injury is an important signal to activate HIF-1a-mediated
protection against C.diff toxin-induced damage.
The in vitro studies analyzed HIF-1α and FIH-1 levels during toxin exposure of Caco2 cells.
Cells were challenged with both toxin and nitric oxide synthase (NOS) inhibitors to study NO
effects on HIF-1α/FIH-1 expression. In vivo studies assessed ileal loop tissue from toxin-treated
wild-type (WT) and inducible NOS knockout (iNOS KO) mice.
We have identified that HIF-1α levels increase at least two-fold in toxin-treated cells. FIH-1
levels also show a 35% decrease in response to toxin. This increase in HIF-1α protein was
suppressed with NOS inhibition. In vivo, serum NO levels increased with toxin exposure; a
response which was abrogated in iNOS KO mice.
Further study is needed to demonstrate a direct effect of NO on HIF-1α levels in response to
C.diff toxin in vivo.
P26. PI3K/TOR-Dependent Control of Ribosome Synthesis and Growth in Drosophila
melanogaster. Lauren Killip, Savraj Grewal.
The conserved PI3K/Target-of-Rapamycin (TOR) pathway regulates cell growth in eukaryotes.
Mutations that lead to over-activation of this pathway are often found in cancer. Although
upstream pathway components are well studied, less is known about downstream mechanisms
that control cellular metabolism and growth. Defining these outputs will provide critical
information on normal and tumour cell growth as well as novel therapeutic targets. We use
Drosophila larvae to study ribosome synthesis as a growth-regulatory mechanism. Reduced
PI3K/TOR signaling leads to decreased expression of the ribosome biogenesis (RiBi) genes in
larvae. This study examines the regulation of RiBi genes by the conserved transcription factor
DREF. We find that loss of DREF leads to decreased RiBi gene expression. We also show that
homozygous DREF mutants exhibit larval growth arrest. This effect is likely due to reduced
cellular growth, since mosaic expression of a DREF RNAi transgene reduces larval cell size.
Interestingly, these growth defects become exacerbated in conditions of reduced PI3K/TOR
activity. This suggests DREF may be a target of the PI3K/TOR pathway in the control of
ribosome synthesis and cell growth. We are currently examining genetic interactions between
DREF and the PI3K/TOR pathway and determining whether DREF is required for PI3K/TORdependent growth.
P27. Signaling pathways required for retrograde extension - formation of long cellular
projections by migration of C. elegans pharyngeal glands. Jay Kormish, Patricia Rohs and Jeb
Gaudet
During pharynx/foregut development in Caenorhabditis elegans, three gland cells are born at the
site of duct formation, where they attach to the pharyngeal lumen. In subsequent development,
the glands migrate towards the posterior of the pharynx resulting in the “drawing out” of a long
cellular projection. This process, termed “retrograde extension”, has only recently been
described and the underlying mechanisms are largely unknown. We have used a genetic
candidate approach in C. elegans in order to define the molecular pathways governing gland cell
migration. Of the many guidance pathways tested, Wnt/ROR and FGFR display antagonistic
roles. The Wnt/ROR pathway appears to function in attraction: in cwn-2/Wnt and cam-1/ROR
mutants, one of the gland cells fails to migrate to its normal position. In contrast, FGFR appears
to function in repulsive signaling: in let-756/FGF and egl-15/FGFR mutants, glands migrate
beyond their normal positions. FGFR has multiple downstream effectors that affect gene
expression, protein stability, cell adhesion and/or cytoskeletion dynamics. Integrins maybe
mediating the ability of EGL-15/FGFR to prevent gland cell over migration since mutants in the
α-integrin ina-1 result in an over migration defect similar to egl-15. Wnt signaling via the
receptor tyrosine kinase CAM-1/ROR can function via multiple downstream effectors. Further
mutant analysis will identify the molecules that are mediating the effect of FGFR and ROR and
will determine how these two pathways are integrated by the gland cells to reach their final
positions.
P28. Simultaneous siRNA-mediated inactivation of Src and other signaling elements in
blocking colon cancer growth and metastasis. Yu L, Robson L, Pang A, Klimowicz A, Bjorge JD,
Bathe OF, Magliocco AM, Fujita DJ.
Metastasis is the major cause of mortality in colon cancer patients. Various studies have
demonstrated that elevated levels of the oncogenic protein Src are present in a high percentage
of colon cancer cell lines and tumors. The critical roles that Src and its signaling pathways play
in cancer led us to study how Src and its signaling pathways contribute to colon cancer
development and metastasis.
We used a mouse tail-vein metastasis model to study the effect of knock-down of Src,
Stat3, and the Src and Stat3 combination on colon cancer metastasis. We found that inactivation
of these genes caused a substantial decrease in metastasis to the lung. Colony formation, cell
migration, and apoptosis assays were used to reveal pathways that mediate the effect of Src
and Stat3 siRNAs on colon cancer metastasis.
Cancer metastasis is a complex process that arises from a combination of events in tumor
cells and microenvironments. Our goal is to determine suitable combinations of siRNAs that are
effective in blocking tumor growth and metastasis in colon cancer.
P29. Loss of NLRP3 signaling enhances intestinal inflammation in mouse models of colitis in
an IL-15-dependent manner. Simon A. Hirota, Mireille S. Potentier, Patrick L. Schenck, Jeffrey Ng,
Yan Li, Victor Lam, Jurg Tschopp, Daniel A. Muruve, Paul L. Beck, Justin A. MacDonald.
The inflammatory bowel diseases (IBD) are characterized by chronic mucosal inflammation with
an unknown specific etiology involving genetic and environmental factors. Although IBD
pathogenesis is multifaceted, several lines of recent evidence suggest that IBD may result from
deficiencies in the innate immune system. We have chosen the DSS model of colitis in mice to
investigate the effect loss of NLRP3 signaling within the innate immune system has on intestinal
homeostasis, and to elucidate some of the factors downstream of NLRP3 that are responsible
for the altered phenotype observed in NLRP3-/- mice. The NLRP3-/- mice were more
susceptible to colitis when compared to wild type animals. They exhibited increased weight loss,
increased MPO levels, and decreased hematocrit levels. Furthermore, these animals had altered
levels of colonic cytokines. Compared to wild type animals, NLRP3-/- animals exhibited
decreased levels of IL-1b, decreased levels of both IL-10 and TGF-B, and increased IL-15
levels. Assessment of colonic lamina propria cells uncovered increased levels of CD3-/NK1.1+
cells, whose proliferation is driven by IL-15. NLRP3-/- bone-marrow-derived dendritic cells
expressed elevated IL-15 and were able to drive the proliferation of Tregs in T-cell co-culture
experiments. NLRP3-/- mice treated with a neutralizing anti-IL-15 antibody in the DSS model of
colitis showed reversion back to the wild type phenotype, with decreased MPO levels, increased
hematocrit levels, and decreased numbers of Tregs compared to NLRP3-/- mice. These data
show that IL-15 plays a key role in effecting the susceptibility to DSS colitis in the absence of
NLRP3 signalling.
P30. Combinatorial transcriptional control of vein specification. Chang-Yi Wu, Ryan Lamont,
Ryan Sobering, Wendy Vu, Aly Carter and Sarah Childs
Genetic programs promoting an arterial identity have been well described, but the specification
of a venous identity is much less well understood. Here, we identify that Islet2 (Isl2), a LIMhomeodomain transcription factor is required for specification of the vein. Isl2 mRNA and protein
are expressed in vein as early as the 15-16 somite stage of zebrafish, and knockdown of isl2
expression by morpholino results in decreased numbers of vein cells and reduced expression of
the vein specific markers, the VEGF-C receptor (flt4), consistent with an effect on cell
specification. We confirmed the effect of isl2 knockdown is autonomous to endothelial cells using
cell transplantation. We show that Isl2 expression is negatively regulated by Notch signaling.
When Notch signaling is repressed using an Rbpsuh morpholino or DAPT treatment, Isl2
expression is upregulated, and there are larger numbers of Isl2 positive cells in the vein. Only
one other transcription factor, CoupTFII, an orphan nuclear receptor, has been shown to be
involved in vein, formation, most likely in differentiation. Here we show that Isl2 and CoupTF act
together in vein specification. The combinatorial downstream pathways regulated by Isl2 and
CoupTF are currently being investigated by a microarray approach.
P31. Characterizing the role of the smooth muscle protein Smoothelin-like 1 in mesenteric
arteriole contractility. Sara R. Turner, Justin A. MacDonald
The small arterioles of the mesenteric vascular tree are a major site of resistance contributing to
regulation of central blood pressure, and increased contractility in the smooth muscle cells of
these vessels is known to play a role in development of chronic hypertension. Significantly, the
smoothelin-like 1 (SMTNL1) protein was recently demonstrated to promote an exaggerated
contractile phenotype. Our preliminary RT-PCR studies show that SMTNL1 transcript is present
in all smooth muscle beds examined thus far. Therefore, we hypothesize that SMTNL1 plays a
key role in mediating the contractile phenotype of these resistance vessels. We will complete
experiments with a global smtnl1 knockout mouse that is available for study. Using pressure
myography, we have carried out a comparison of the contractile physiology of 3rd order
mesenteric arterioles from the wild type and smtnl1-/- mice. When held at a constant pressure
(80mm Hg), 3rd order mesenteric arterioles of the smtnl1-/- show decreased response to the
contractile agonist phenylephrine as well as increased response to the relaxant agonist NO.
Future studies will include an examination of the expression levels of SMTNL1 mRNA and
protein throughout the mesenteric vascular tree, the role that SMTNL1 plays in development and
maintenance of myogenic tone, as well as the phosphorylation state of SMTNL1 during
contraction and relaxation of the mesenteric vascular smooth muscle.
P32. The MRX complex regulates sister chromatin cohesion during replication stress. Mireille
Tittel-Elmer, Marcel Hohl, John Petrini, Jennifer A. Cobb
The Mre11-Rad50-Xrs2 (MRX) complex is important for the maintenance of genomic integrity
and is recruited to forks during pauses in replication. Moreover, one essential function of the
MRX complex is to provide architectural support to DNA during repair, meiotic recombination
and telomere maintenance. The structure of the MRX complex indicates it can bridge two
strands of duplexed DNA and serve as a long-range tether, becoming ‘critical glue’ between
sister chromatids. Cohesion between sister chromatids is coupled to fork progression and the
cohesin complex holds replicated chromosomes together from their synthesis until the onset of
anaphase. Here we show that deficiency in the cohesin complex in combination with the
absence of Mre11 during replication stress leads to an additive loss of cohesion between newly
synthesized daughter strands and correlates with a dramatic loss of cell viability. This function is
further characterized using a structurally defective Rad50 allele. In all, our data indicate that the
MRX and cohesin complexes both contribute to the maintenance of sister chromatid cohesion
(SCC) at stalled replication forks but independently of each other. One critical role for the MRX
complex in preserving chromosome integrity involves supporting the architecture of newly
synthesized daughter strands during pauses in replication, preventing fork-associated damage.
P33. Upregulation of myocardin expression by IL-1β and TNFα in vascular smooth muscle
cells. Pavneet Singh, Xi-long Zheng
In vascular smooth muscle cells (VSMC), cytokines such as IL-1β and TNFα regulate a number
of processes which play important roles in various vascular diseases like atherosclerosis,
hypertension and restenosis after angioplasty. Cytokines have been shown to up-regulate NF-κβ
during inflammation and also inhibit Serum Response Factor (SRF) activity. Myocardin, a
cofactor of SRF, is a major regulator in maintaining smooth muscle phenotype and has also
been shown to inhibit proliferation by inhibiting NF-κβ pathway in cardiac and smooth muscle
cells. Thus, it is possible that these cytokines may in fact regulate the expression and/or activity
of myocardin and thus play a role in regulating the phenotype of VSMC. Our data have shown
that IL-1β and TNFα increased the expression of Myocardin in VSMCs, but there was not a
significant corresponding increase in the expression of contractile marker proteins. Moreover,
nitric oxide (NO), which is also induced by cytokines through induction of iNOS, was shown to
increase the expression of Myocardin without any corresponding increase in the expression of
contractile marker proteins. The exact mechanisms of this regulation are still under investigation.
However, our results have suggested that cytokine effects on VSMCs may be modulated by
upregulation of Myocardin.
P34. Glycogen synthase kinase-3β regulates the expression and activity of myocardin in
human vascular smooth muscle cells. Yi-xia Zhou, Hao Yin, Yu Gui and Xi-long Zheng
Activation of Glycogen Synthase Kinase -3β (GSK-3β), a serine/threonine kinase, is involved in
injury-induced neointima formation, systematic vascular remodeling and airway smooth muscle
hypertrophy. Myocardin is a muscle-specific transcriptional co-activator of serum response factor
(SRF) and induces transcription of genes encoding contractile proteins (SM alpha-actin, SM22
and calponin). It has been reported that GSK-3β inhibits myocardin-dependent transcription and
induction of hypertrophy in cardiomyocytes through direct phosphorylation of myocardin.
However, it is not known how GSK3-β regulates myocardin and phenotype of vascular SMCs.
Therefore, we investigated whether inhibition of GSK-3β was sufficient to modulate myocardin
activity. In this study, we used AR-A014418, GSK-3β Inhibitor, to treat SMCs with and without
harboring T-REX inducible system for myocardin expression. We first established the mRNA and
protein levels of myocardin and myocardin-induced smooth muscle markers using quantitative
PCR and Western blot, respectively. We found that AR-A014418 treatment inhibited the
expression of endogenous myocardin and smooth muscle marker genes (SM α-actin, SM22 and
calponin). Importantly, AR-A014418 treatment inhibited the induction of smooth muscle marker
genes in response to myocardin overexpression through T-Rex system. However, inhibition of
GSK33β did not significantly reduce the expression of myocardin protein in SMCs overexpressing myocardin. Therefore, our data suggest that GSK-3β stimulates myocardin activities
through induction of myocardin gene expression and stimulatory phosphorylation. GSK-3β may
become a therapeutic target of cardiovascular diseases, such as atherosclerosis, hypertension
and restenosis after angioplasty.
P35. Overexpression of myocardin does not cause hypertrophy in vascular smooth muscle
cells. Sarvan Kumar, Xi-long Zheng
Hypertension is one of the major causes of cardiovascular related mortality worldwide. Even
though the exact cause of primary hypertension is not fully understood yet, but it has been
shown that hypertension in animal models as well as in human hypertensive patients is
associated with vascular smooth muscle cell (VSMC) hypertrophy. Myocardin, a transcription
cofactor, interacts with serum response factor and mediates the expression of smooth muscle
contractile proteins like α-actin, myosin heavy chain etc. Recent studies have shown that
myocardin induces the expression of smooth muscle-enriched microRNA 143/145 in CArG box
dependent manner. These microRNAs were shown to be involved in regulating the cytoskeletal
dynamics in VSMC. It was also shown that microRNA 143/145-knockout mice exhibited thinning
of aorta and hypotension. It was shown that forced expression of myocardin can induce
cardiomyocyte hypertrophy, which could be attenuated by glycogen synthase kinase-3β. Based
on these observations we hypothesised that overexpression of myocardin and microRNA
143/145 could be involved in VSM hypertrophy and hypertension. Using a T-REx tetracyclineregulated system for myocardin overexpression in human aortic SMCs, we observed that
overexpression of myocardin did increase the expression level of microRNA 143/145 upto two
fold in quantitative PCR. However, myocardin overexpression did not lead to an increase in
protein synthesis rate as measured in leucine incorporation assay. Based on these results we
propose that myocardin and myocardin-dependent miR-143/145 expression may not be involved
in VSMC hypertrophy.
P36. Potent Oncolytic Activity of Reovirus Against Human Gastric Cancer With Chemotherapy.
Andreea Roxana Fratiloiu,Yoshinori Mori, Sandi Nishikawa, Lara Daisley, Mio Tsutsui, Hiromi
Kataoka, Takashi Joh, Randal Johnston
Patients with stage IV gastric cancer have a dismal chance of survival and novel therapeutic
regimens are needed to improve the outcome. Reovirus normally replicates in the
gastrointestinal system and is an effective oncolytic agent against many colon cancers, but it has
not previously been evaluated with gastric cancer. Here we describe our studies of oncolytic
reovirus in combination with currently used chemotherapeutic drugs (irinotecan, paclitaxel and
docetaxel) with gastric cancer cell lines in vitro and in vivo. We measured the in vitro impact of
reovirus plus chemotherapy on cell viability (WST-1 assays), and the expression of reovirus
protein and caspase activity by flow cytometry. Studies in vivo were with xenografts in athymic
mice that received intratumoral injections of reovirus in combination with irinotecan and
paclitaxel while we monitored the sizes of the tumours. The results indicate that a combination of
reovirus and chemotherapy show a synergistic cytopathic effect in vitro, as well as enhanced
reovirus replication and apoptosis. In vivo experiments show that reovirus alone and in
combination with chemotherapeutic agents repress tumour growth. In conclusion, oncolytic
reovirus therapy is effective against gastric cancer. Moreover, the combination of reovirus and
chemotherapeutic agents synergistically enhance cytotoxicity in human gastric cancer cell lines
in vitro and in vivo suggesting that the reovirus/chemotherapy combination may be useful in
future clinical trials in the treatment of stage IV gastric cancer.
P37. A Role of Focal Adhesion Kinase (FAK) in neutrophil recruitment. Ritu Sharma, Hong Zhang
and Kamala Patel
Inflammation is a complex biological response to harmful stimuli, such as pathogens, damaged
cells, or irritants. Various cell types are critically involved in the initiation and maintenance of
inflammation including leukocytes and endothelial cells. In the body, both cell types are exposed
to mechanical shear stress created by the flow of blood through the blood vessels. Endothelial
cells respond to this shear stress by activating many signaling pathways through
mechanotransduction. Focal adhesions are cellular structures important for
mechanotransduction and they also play an important role in leukocytes recruitment. Studies in
our lab have shown that many focal adhesion proteins, including zyxin, paxillin and FAK are
critical for optimal leukocyte recruitment. FAK is a protein tyrosine kinase that plays a critical role
in many cellular events including adhesion, proliferation, migration and survival. Studies from our
lab have shown that there is remodeling of paxillin and zyxin during leukocyte recruitment. Here
using in vitro assays we illustrate that FAK re-distributes away from the site of transmigrating
neutrophils. The redistribution of FAK is unaffected by rolling and adhesion but occurs during
transmigration. Down-regulation of FAK by siRNA resulted in a decrease in transmigration.
These data suggest a role for FAK in neutrophil transmigration.
P38. Roscovitine, a selective inhibitor of cyclin-dependent kinase 5, inhibits myocardininduced expression of differentiation markers in human vascular smooth muscle cells. Mostafa
Ghozlan, Hao Yin, Xi-Long Zheng.
Cyclin-dependent kinase 5 (Cdk5) is a critical regulator of differentiation, growth and
homeostasis of neuron and skeletal muscle. However, the potential roles of Cdk5 in the
differentiation and proliferation of smooth muscle cells (SMCs) remains unknown. Myocardin, a
transcription co-factor for serum response factor, is a master regulator of differentiation and
contractile phenotype of SMCs. It can drive the transcription of smooth muscle-specific
differentiation markers, including smooth muscle α-actin, SM22, calponin, smooth muscle
myosin heavy chain, etc. In the current study, using T-REx tetracycline-regulated system for
myocardin overexpression in human aortic SMCs, we investigated the effects of roscovitine, a
selective of Cdk5 and a trial anti-cancer drug, on the myocardin-dependent expression of
smooth muscle differentiation markers. Using quantitative PCR assay and western blotting
analysis, we found that, in concentration-dependent manner (1~ 10 µM), roscovitine treatment
for 24 h blocked the myocardin-induced expression of smooth muscle differentiation markers,
including SM22, calponin and smooth muscle α-actin. Furthermore, the spindle-shaped
morphology of SMCs induced by myocardin overexpression was compromised by roscovitine.
Considering roscovitine as a selective inhibitor of Cdk5, our data suggested that Cdk5 could
serve as a positive regulator of differentiation and contractile phenotype of human vascular
SMCs.
P39. Characterization of Dpb11TopBP1 Localization and its Effects During the Replication
Checkpoint and DNA Double-Strand-Breaks. Margaret Renaud-Young, Jennifer Cobb.
Dpb11TopBP1 is a protein that performs a dual function: it promotes the assembly of the DNA
replication complex, and it enables full activation of Mec1-Ddc2ATR-ATRIP kinase in response to the
replication checkpoint and DNA Double-Strand-Breaks (DSBs) in the budding yeast
Saccharomyces cerevisiae. One question in the field is how Dpb11 is recruited to either forks or
sites of DNA damage as well as how it contributes to downstream checkpoint activation events.
Using Chromosomal Immunoprecipitation (ChIP) and Co-Immunoprecipitation (CoIP), I plan to
track the changing localization and interaction partners of Dpb11TopBP1 as the replication
checkpoint is activated by Hydroxyurea (HU), as well as if these interaction partners differ from
when the cell is challenged with an irrepairable DSB. I have performed some mutagenesis of
tagged dpb11 alleles to determine the regions responsible for replication fork recruitment. I plan
to perform further mutations to better characterize this region. I also plan to compare the
kinetics of its recruitment in Wt cells and dpb11 alleles in cells with alleles of the replication
checkpoint and DNA damage response pathway to determine the hierarchy of Dpb11, Mec1Ddc2 and the checkpoint clamp 9-1-1’s recruitment to stalled forks and damaged sites. These
experiments should provide a comprehensive understanding of the role of Dpb11TopBP1 in its
checkpoint function under different stimuli.
P40. Development and Function of the C. elegans pharyngeal glands. Vikas Ghai, Jeb Gaudet
Organ development (organogenesis) is a complex process, and how it occurs is an
important biological question. A simple model to study organogenesis is the pharynx (foregut) of
C. elegans. Recently, work in our lab has been focused on the development and function of the
pharyngeal gland cells. We have shown that a transcription factor, HLH-6 is required for gland
development and for proper feeding.
However, hlh-6 is not required for specification of the gland fate. To identify upstream
components we identified and characterized several critical regulatory elements in the hlh-6
promoter. Factors that bind these elements act to regulate gland expression of hlh-6 in a
combinatorial manner, and contribute to gland specification. We have identified one of the transacting factors (LAG-1/CSL), a critical component of Notch signaling, and are currently trying to
identify other factors involved.
In addition to development/transcriptional regulation we are also interested in
understanding gland morphogenesis and migration. Gland migration is controlled by retrograde
extension, a process that the underlying mechanisms largely unknown. Through a candidatebased approach, we have identified two pathways that play opposing roles in gland migration: A
Wnt/ROR dependant attractive signal, and a FGF dependant repulsive signal. Further
investigation will characterize how these pathways are integrated for proper gland migration.
Additionally, we have shown that the gland cells are required for proper migration of a
pharyngeal motor neuron, suggesting at least two unique roles for the glands in pharyngeal
function.