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Poster Full Abstracts - Cell Biology and Signal Transduction
Poster board number is above title. The first author is the presenter
188
phosphorylation and activation, and that Fu regulates both the activator and repressor forms of Ci.
205A
A dsRNA Based Screen Identifies Novel Proteins Involved in Drosophila Hedgehog Signaling Pathway.
Maggie Sledd, Ryan Hurtado, Robert
Holmgren. Molecular Biosciences, Northwestern University, Evanston, IL.
A screen has been performed identifying potential novel members of the Hedgehog (Hh) signaling pathway. The Hh signaling pathway is responsible for
the patterning of many different organs during development. In mammals, aberrant Hh signaling is associated with a number of different cancers, including
basal cell carcinoma and prostate cancer, and therefore, a further understanding of Hh could lead to new treatments. While many proteins involved in the Hh
signaling pathway have been discovered, there are still many that remain unknown. In
Drosophila melanogaster
, the Hh signaling pathway is responsible for
the patterning of the wing, so a UAS-Gal4 RNAi screen was devised using the MS1096-Gal4 driver to specifically knock down gene expression in the wing.
The screen was performed in a sensitized
fused (fu
1
)
mutant background, looking for both suppressors and enhancers of the
fu
1
phenotype. By observing
wing vein patterning, it was possible to determine whether loss of a particular gene had a significant effect on Hh signaling. Thus far, the screen has covered
11% of the
Drosophila
genome. Out of 1590 genes knocked down, 29 were strong enhancers and 11 were suppressors of the
fu
1
phenotype. Several of these
hits were followed up using RNAi in larval wing discs and visualizing gene expression with immunofluoresence. One gene of particular interest,
megator
(
mtor
), was a strong enhancer of the
fu
1
phenotype. Knockdown of
mtor
with RNAi resulted in a decrease in expression of the Hh target genes
decapentapleigic
and
collier
. Future research will include testing for genetic interactions with a mutation in the endogenous mtor gene to validate its
involvement, and elucidating the function of Mtor in the Hh signaling pathway.
206B
USP8 promotes Smoothened signaling by preventing its ubiquitination and changing its subcellular localization.
Ruohan Xia, Hongge Jia, Junkai Fan,
Yajuan Liu, Jianhang Jia. Department of Molecular and Cellular Biochemistry, Markey Cancer Center,University of Kentucky, Lexington, KY 40536, USA.
The seven transmembrane protein Smoothened (Smo) is a critical component of the Hedgehog (Hh) signaling pathway and is regulated by
phosphorylation, dimerization, and cell-surface accumulation upon Hh stimulation. However, it is not clear how Hh regulates Smo accumulation on the cell
surface or how Hh regulates the intracellular trafficking of Smo. In addition, little is known about whether ubiquitination is involved in Smo regulation. In
this study, we demonstrate that Smo is multi-monoubiquitinated and that Smo ubiquitination is inhibited by Hh and by hosphorylation. Using an in vivo
RNAi screen, we identified ubiquitin-specific protease 8 (USP8) as a deubiquitinase that downregulates Smo ubiquitination. Inactivation of USP8 increases
Smo ubiquitination and attenuates Hh-induced Smo accumulation, leading to decreased Hh signaling activity. Moreover, overexpression of USP8 prevents
Smo ubiquitination and elevates Smo accumulation, leading to increased Hh signaling activity. Mechanistically, we show that Hh promotes the interaction of
USP8 with Smo aa625-753, which covers the three PKA and CK1 phosphorylation clusters. Finally, USP8 promotes the accumulation of Smo at the cell
surface and prevents localization to the early endosomes, presumably by deubiquitinating Smo. Our studies identify USP8 as a positive regulator in Hh
signaling by downregulating Smo ubiquitination and thereby mediating Smo intracellular trafficking.
207C
Rab GTPase mediated Golgi trafficking as a potential major orchestrator of Autophagy in
Drosophila melanogaster
.
Carlos I Ayala-Navarro, Thomas
P Neufeld. Genetics, Cell Biology and Development, University of Minnesota, Minneapolis, MN.
Autophagy is a catabolic housekeeping mechanism employed by cells to achieve quality control of organelles and proteins, cope with stress and combat
starvation. The process occurs by sequestration of cytoplasmic material into autophagosomes to ultimately fuse with lysosomes to degrade cargo, replenish
nutrients and maintain cellular health. Vesicular traffic has been shown to be a major player in this process from the beginning to the end stages. Among the
latter, a small number of Rab GTPases have been identified as important regulators of autophagy. Nonetheless, our understanding of the role of traffic is still
incompletely understood in regards to autophagy and autophagosomal dynamics. It should be highlighted that most of the focus and research has been
exhaustively researched in mammal and yeast models. Therefore, we decided to perform a screen of the ~30 Rab GTPases in the genetic workhorse
Drosophila melanogaster to elucidate their role in autophagy. The screen consisted of knockdown (dsRNA) and over-expression of constitutively active,
dominant negative and wild-type versions of each Rab protein. Our results highlight the value of Drosophila as powerful biological model in the study of
autophagy and reveal novel Rab GTPases involved in the regulation of this process. Of special interest is a selected group of Rab proteins, 2, 6, 19 and 39,
involved in traffic towards, within and from the Golgi network. The fact that these GTPases were detected as candidates regulating golgi trafficking
prompted us to develop an interest for dAtg9, a golgi localized transmembrane protein in mammals, which has been proposed to deliver membrane to
nascent autophagosomes. However, traffic regulation of Atg9 is incompletely understood and exhaustively being researched. Whether our Rab candidates
regulate traffic of Atg9 under Fed or starvation induced autophagy will be explored.
208A
Investigating the Role of PI4P in Lysosome-related Organelle Biogenesis in the
Drosophila
Eye.
Lauren M. Del Bel
1,2
, Ronit Wilk
2
, Jason Burgess
1,2
,
Gordon Polevoy
2
, Ho-Chun Wei
2
, Julie A. Brill
1,2
. 1) Molecular Genetics, University of Toronto, Toronto, Ontario; 2) Cell Biology Program, Hospital for
Sick Children, Toronto, Ontario.
Phosphatidylinositol (PI) phosphates (PIPs) are membrane lipids that play essential roles within the cell. PIPs, such as PI 4-phosphate (PI4P), are strictly
regulated by various kinases and phosphatases. PI4P localizes to the Golgi where it recruits important regulators of intracellular trafficking. In addition, PI4P
has also been implicated in the biogenesis of lysosome-related organelles (LROs). LROs are a class of organelles that include pigment granules (required for
Drosophila
eye pigmentation), melanosomes, and dense and alpha granules in platelets. A collection of heritable diseases that disrupt LRO function lead to
hypopigmentation, prolonged bleeding, neurodegeneration, immunodeficiency and lung fibrosis. These diseases are often a result of mutations in genes
encoding trafficking complexes, such as the clathrin adaptor protein complex AP-3 and the homotypic fusion and vacuole protein sorting complex (HOPS);
however, the cellular mechanisms underlying LRO biogenesis are not well understood. By manipulating PI4P levels in the cell using
Drosophila
mutants for
the type II PI 4-kinase (PI4KII) and the PI4P phosphatase Sac1, our lab has identified PI4P as a critical player in LRO formation.
PI4KII
and
sac1
mutants
have defects in red and brown eye pigment levels due to an altered number and distribution of eye pigment granules. Additionally, we have shown that