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Poster Full Abstracts - Cell Biology and Signal Transduction
Poster board number is above title. The first author is the presenter
182
Genetic interactions between Clic, Cdc42 and Cdc42 effectors in filopodia formation.
Regan Price
1
, Soichi Tanda
1
, Mark Berryman
2
. 1) Biological
Sciences, Ohio University, Athens, OH; 2) Biomedical Sciences, Ohio University, Athens, OH.
Cell motility is driven by membrane protrusion, which begins with the projection of filopodia and lamellipodia. Actin dynamics at the cell cortex are
regulated by the small GTPase Cell Division Cycle 42 (Cdc42) and its downstream effectors, the actin nucleators Diaphanous (Dia) and Wiskott-Aldrich
syndrome protein (WASp). We seek to understand how Chloride intracellular channels (Clics) contribute to cell motility. To study Clic’s role in filopodia
formation, gene dosage and protein activity of Cdc42, Dia, and WASp were altered in the presence and absence of Clic function. Larval hemocytes were
stained with the F-actin marker phalloidin, and scored for filopodia formation. We found that a constitutively active form of Cdc42 (Cdc42-CA), stimulates
filopodia formation in larval hemocytes, consistent with previous studies. When Cdc42-CA was expressed in a Clic null mutant background, filopodia
formation was suppressed, suggesting that Clic functions downstream of Cdc42. Therefore, we tested for genetic interactions between Clic and Dia, another
protein important in filopodia formation. Overexpression of wild-type Dia resulted in an increase in the number of cells forming filopodia. This phenotype
was dependent on Clic, as the number significantly decreased when wild-type Dia was expressed in the Clic mutant background. When a constitutively
active form of Dia was overexpressed, a greater increase in the number of cells forming filopodia was observed as compared to wild-type Dia. However, this
phenotype was not altered in the Clic mutant background. Next, we tested for interactions between Clic and WASp. Overexpression of wild-type WASp in
hemoctyes led to an increase in the number of cells forming filopodia. This phenotype was unchanged when WASp was overexpressed in the Clic mutant
background, suggesting that Clic does not interact with this protein in filopodia formation. Together these results suggest that Clic functions downstream of
Cdc42, and that it interacts with the autoinhibited form of wild-type Dia, possibly promoting its activation during filopodia formation.
182B
Drosophila nurse cell dumping reveals a novel interaction between prostaglandin signaling and Fascin.
Tina Tootle, Christopher Groen, Andrew
Spracklen, Tiffany Fagan. Anatomy and Cell Biology, University of Iowa, Iowa City, IA.
While actin cytoskeletal dynamics are known to be regulated by prostaglandins, lipid signals produced downstream of cyclooxygenase (COX) enzymes,
the mechanisms by which PGs mediate this remain unknown. Drosophila oogenesis provides a model for studying how prostaglandin signaling affects actin
remodeling. During oogenesis, a process called nurse cell dumping occurs. This process requires active remodeling of the actin cytoskeleton to allow the
nurse cells to squeeze their cytoplasmic contents into the growing oocyte. Using this model, we have previously shown that prostaglandins are required for
actin remodeling during dumping, and that Pxt is the Drosophila COX-like enzyme. A screen utilizing our in vitro follicle maturation assay (Spracklen,
Meyer, and Tootle, unpublished data) identified Fascin (
singed
), an actin bundling protein, as a downstream target of prostaglandin signaling. Here, we show
that fascin and pxt mutants display similar actin remodeling defects in nurse cells. Reduced Fascin levels enhance the dumping, and thus actin remodeling,
defects of both COX inhibition and reduced Pxt levels. Additionally, over-expression of Fascin in the germline, using the UAS-Gal4 system, suppresses the
effects of COX inhibition. Importantly, Fascin levels, both mRNA and protein, are not affected by alterations in prostaglandin signaling. Additionally,
prostaglandin signaling does not appear to globally affect actin bundling, as another actin bundling protein Villin (
quail
) fails to interact with Pxt or COX
inhibition. These data indicate that one role of prostaglandin signaling in regulating actin remodeling is to specifically modulate Fascin activity. Current
efforts are focused on determining the mechanism by which prostaglandins regulate Fascin. This is the first link between prostaglandins and Fascin, and is
particularly intriguing as both are implicated in mediating cancer progression and metastasis.
183C
Polymerization, Metabolic Regulation, and the Origins of the Cytoskeleton.
James E. Wilhelm, Chalongrat Noree, Dane Samilo, Risa Broyer, Brian
Sato. Section on Cell and Developmental Biology, Univ California San Diego, La Jolla, CA.
In eukaryotes, four major classes of filament forming proteins are known to play a role in cellular organization and function: septins, tubulin, actin, and
intermediate filament proteins. However, while no new filaments have been discovered in over 20 years, it has been unclear whether all of the filaments that
comprise the cytoskeleton have been found. In order to address this question, we have conducted visual screens of both yeast and Drosophila GFP strain
collections to identify proteins that form novel intracellular filaments. This screen identified a large number of intracellular structures including four novel
filament systems comprised of glutamate synthase, GDP-mannose pyrophosphorylase, CTP synthase, or subunits of the eIF2/2B translation factor complex.
Given the novelty of these structures, we have focused our efforts on characterizing CTP synthase filaments. By combining structure function analysis with a
novel in vitro polymerization assay, we have found that regulation of enzyme activity is intimately connected with the regulation of polymerization. CTP
synthase filaments are also present in all species examined from bacteria to humans. Interestingly, CTP synthase filaments are restricted to axons in neurons.
This spatial regulation suggests that these filaments have additional functions separate from the regulation of enzyme activity. The identification of four
novel filaments doubles the number of known intracellular filament networks and has broad implications for our understanding of how cells organize
biochemical activities in the cytoplasm. This work also has implications for the evolution of the cytoskeleton since it suggests that the classic cytoskeleton
may have evolved from metabolic enzymes that used polymerization as a mechanism for regulating enzyme activity.
184A
The role of CTP Synthase during CNS development.
Omur Y. Tastan, M. Ghows Azzam, Kemian Gou, Mayte Siswick, Ji-Long Liu. MRC Functional
Genomics Unit Department of Physiology, Anatomy and Genetics, University of Oxford, United Kingdom.
CTP Synthase is an enzyme that is involved in pyrimidine biosynthesis which converts UTP to CTP. When expressed at high levels, CTP Synthase forms
filaments called cytoophidia. CTP Synthase has been shown to be enriched in certain cancer types. In addition, several inhibitors of CTP Synthase, e.g. DON
(6-diazo-5-oxo-L-norleucine), has shown promising potential as therapeutic drugs. Recent studies show that cytoophidium is found in many cell types.
Expression analysis showed that CTP Synthase can be found in the cytoplasm, nucleus and also in the cytoophidium at different cell types and at different
stages of development in various tissues. Mutant analysis showed that CTP Synthase mutations result in larval lethality with pupation defects which is fully
rescuable with a CTP Synthase transgene. In an effort to identify CTP Synthase interactors we performed a yeast two hybrid screen and recovered three
potential CTP Synthase interactors among 20 hits. The verification of interactions by co-immunoprecipitation experiments is ongoing. Expression analysis
showed that all three proteins form filaments in DON treated human cell lines. We are in the process of generating transgenic animals and antibodies to
characterize these genes further in vivo.