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Poster Full Abstracts - Cell Biology and Signal Transduction
Poster board number is above title. The first author is the presenter
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dynamics of active GTPases in this response. Surprisingly, we find that Rho GTPases utilize specific effectors to mediate their signals: our data shows that
different Rho GTPases and their effectors are locally recruited in response to wounding. This complex spatial-temporal array may also involve crosstalk
among the different GTPases and their signaling modules. Importantly, by genetic and pharmacological assays we also find that Rho, Rac and Cdc42 are
required for proper wound repair, and each of them make specific contributions to the assembly and organization of the actomyosin array.
241A
Septate junctions play an unexpected role in the cell division of polarized epithelia.
Vanessa J. Auld, Kristi Charish. Dept Zoology, Life Sciences
Institute, Univ British Columbia, Vancouver, BC, Canada.
Septate junctions (SJs) are found basal to adherens junctions and form permeability barriers in Drosophila epithelia similar to tight junctions in vertebrates.
SJs are generated by a large complex of highly conserved proteins, which have recently been shown to function beyond the formation of permeability
barriers. Along these lines, in the columnar epithelia of the Drosophila imaginal disc, the plane of cell division occurs exclusively at the SJ. Null mutations
in SJ genes are cell lethal when somatic clones are generated in the imaginal disc of Drosophila and thus we wanted to test if the loss of SJ proteins resulted
in a disruption of cell division. Using RNAi approaches, we specifically knocked-down SJ components in the wing imaginal disc and then assayed for
changes in cell division. In addition using an array of cellular markers we characterized each step of mitosis in the context of the SJ domain. We show that
SJs play a critical role in determining the plane of cell division. Loss of the SJ domain results in the basal spread of the plane of cell division away from the
apical domain. This spreads correlates with the displacement of the SJ associated protein Gliotactin and can be phenocopied by loss of Gliotactin. In addition
we show that the final stage of cell division, cytokinesis, occurs exclusively within the SJ domain. As the cleavage and ingression furrows develop, a SJ
tunnel is created that encases the contractile ring and midbody during cytokinesis. Knock down of SJ proteins leads to basal mislocalization of the plane of
cell division, dislodgement of the contractile ring and a failure to form the ingression furrow. We propose a model for cytokinesis within differentiated
epithelia that requires the presence of the SJs to successfully complete cell division perhaps as a means to maintain the paracellular barrier across epithelia.
242B
Distinct subcellular distributions of three CTP synthase isoforms in
Drosophila
.
Ghows Azzam, Ji-Long Liu. MRC Functional Genomics Unit,
Department of Physiology, Anatomy and Genetics, University of Oxford, Oxford, Oxfordshire, United Kingdom.
The cytoophidium is a novel filamentous organelle that contains CTP synthase, a critical metabolic enzyme. Cytoophidia have been observed in a wide
range of organisms and different cell types. However, the function of the cytoophidium and how it forms is still elusive.
Drosophila
CTP synthase, one of
the main components of cytoophidia, has three isoforms. We have made transgenic flies expressing Venus fused with each individual isoform. To our
surprise, these three isoforms exhibit distinct intracellular localizations. It appears that only isoform C assembles into the cytoophidium, while isoform B is
dispersed in the cytoplasm and isoform A forms punctate in the nucleus. The aim of this study is to understand which region is important to form filaments
and whether the non-filament forming isoforms are functional. Dissecting isoform C and studying CTP synthase mutants could shed light on understanding
how the cytoophidia is formed and how the cytoophidium functions.
243C
Dissection of the NR box-dependent interaction between the bHLH-PAS paralogs MET and GCE and the FTZ-F1 nuclear receptor.
Travis J.
Bernardo, Edward B. Dubrovsky. Fordham University, Bronx, NY.
Juvenile hormone (JH) has been implicated in many developmental processes in holometabolous insects, but its mechanism of signaling remains
controversial. We previously found that in
Drosophila
S2 cells the orphan nuclear receptor FTZ-F1 is required for the activation of
E75A
by JH. It binds to
enhancers upstream of the
E75A
promoter and interacts with two JH receptor candidates, the bHLH-PAS paralogs MET and GCE. Here, we investigated the
molecular basis of FTZ-F1 involvement in JH signaling. In quantitative two-hybrid assays we observed that FTZ-F1 interacts with MET and GCE in a JH-
dependent manner. These interactions are severely reduced when helix 12 of the FTZ-F1 LBD is removed, implicating AF2 as a potential interacting site. To
gain insight into the physical interaction between these proteins we used a homology-modeling approach and found that MET and GCE possess an α-helix
C-terminal to the second PAS fold that contains the conserved motif LIxxL. This motif resembles an NR box and is essential for the interactions with FTZ-
F1. Docking simulations supported by two-hybrid experiments reveal that the FTZ-F1 AF2 provides a distinct interacting surface for MET and GCE, which
resembles that of a typical NR box/AF2 interaction but does not require charge clamp residues. Our findings suggest that a novel NR box enables MET and
GCE to interact JH-dependently with the AF2 of FTZ-F1.
244A
Regulation of autophagy by the Atg1/Ulk family of protein kinases in
Drosophila melanogaster
.
Christopher R Braden, Thomas P Neufeld. University
of Minnesota, Minneapolis, MN.
Macroautophagy (hereafter “autophagy”) is a process involving the sequestration of cytoplasm for lysosomal degradation. The level of autophagic activity
varies under the control of the Target of Rapamycin (TOR) pathway. In D. melanogaster models TOR exerts its influence through a complex involving the
Ser/Thr protein kinase Atg1. Defects in these regulatory pathways have been implicated in human afflictions such as cancer and neurodegenerative diseases,
perhaps due to the role of autophagy in cellular response to nutritional, oxidative, or ER stress. Atg1 is a member of the Unc51-like kinase (Ulk) family.
While several Ulk family proteins are described in mammals, only two exist in D. melanogaster: Atg1 and Ulk3 (CG8866). Ulk3 induces autophagy by
overexpression in mammalian cell culture, but remains largely unstudied. We show that overexpression of Ulk3 induces autophagy in the fat body, and that
Ulk3 suppresses Atg1 activity in overexpression studies. Generation of a large deletion in Ulk3 does not obviously inhibit starvation-induced autophagy, but
apparently reduces oxidative stress survival in a manner dependent on Atg1 gene dosage. Together, our data suggest that autophagy is regulated by distinct
Ulk-family kinases in response to different upstream cues, with Atg1 required for nutrient responses and Atg1 and Ulk3 behaving antagonistically in
oxidative stress signaling.
245B
The effects of PKA in Drosophila fat body.
Yu-Yun Chang, Thomas Neufeld. Gen & Cell Development, Univ Minnesota, Minneapolis, MN.