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Poster Full Abstracts - Cell Biology and Signal Transduction
Poster board number is above title. The first author is the presenter
206
Characterisation of the trafficking route taken by Wingless in secreting cells.
Lucy Palmer, Cyrille Alexandre, Karen Beckett, Jean-Paul Vincent.
Developmental Biology, NIMR, London, United Kingdom.
Wingless (Wg) is the major
Drosophila
Wnt and is important for patterning, growth and cell survival during development. Wg is produced in a stripe of
cells in the
Drosophila
wing imaginal disc and spreads from these secreting cells to form a gradient. Total staining (intra and extracellular) shows that
Wingless is localized at the apical surface of secreting cells, suggesting that Wingless could be secreted on the apical surface. However, extracellular
staining reveals that the Wingless gradient forms at the basal surface of receiving cell. There is as yet no direct proof that Wingless is indeed secreted on the
apical surface and if it is, as expected, the mechanism underlying apical to basal transfer remains unknown. It is also not known how Wingless spreads along
the disc surface, despite carrying lipid modifications. We are focusing on Wingless trafficking within secreting cells to address these questions. Within
secreting cells Wg associates with Evi/Wntless which is required for its secretion. It is thought that Wg joins Evi in the Golgi and that Evi is required to
transport Wg to the plasma membrane. The route Wg and Evi take in Wg-producing cells is not fully understood and we plan to characterize by high-
resolution microscopy. To this end we have constructed Bacterial Artificial Chromosomes (BACs) that express tagged forms of Wingless and Evi at
endogenous levels. These BACs rescue wg or evi mutants. In addition, we have designed a method to produce a step of Wg within secreting cells. By fixing
at defined time points we will be able to visualize its movement through the secretory pathway, and determine where and when it joins and separates from
Evi. Taken together these approaches will allow us to define the secretory route that Wg takes out of the cell to form the gradient in the wing imaginal discs.
278B
The microRNA-310/13 cluster antagonizes β-catenin function in
Drosophila
.
Felix Peng
1
, Raluca Pancratov
1
, Peter Smibert
2
, Jr-Shiuan Yang
2
, Emily R
Olson
1
, Ciaran Guha-Gilford
1
, Eric C Lai
2
, Ramanuj DasGupta
1
. 1) New York University School of Medicine, Department of Pharmacology and the NYU
Cancer Institute; 522 First Ave., SRB #1211, New York NY 10016; 2) Sloan-Kettering Institute, Department of Developmental Biology, 1275 York Ave
Box 252, New York NY 10065.
microRNAs (miRs) are important regulators of global gene expression, and function in regulating a broad range of biological processes. We identified the
miR-310/13 cluster as part of a comprehensive cell-based screen for Drosophila microRNAs involved in regulating the activity of the evolutionarily
conserved Wnt/wingless (wg) signalling pathway. We demonstrate that this evolutionarily conserved cluster can directly target the 3'UTR of β-
catenin/Armadillo (arm) and dTCF in Drosophila cells. Overexpression of miR-310/13 phenocopies a loss of Wg signalling in Drosophila imaginal discs.
We observed reduced fertility among males lacking miR310/13. Subsequent examination of mutant testes revealed an abnormal clustering of germ cells.
This mutant phenotype can be rescued by reducing Arm activity in both the germ and somatic lineage of the Drosophila testis, thus implicating a previously
unrecognised function for Wg/Arm in stem cell regulation in the Drosophila testis.
279C
Screening the
Drosophila
kinome and phosphatome
in vivo
to identify novel regulators of the Wnt/Wg signaling pathway.
Tirthadipa Pradhan, Sharan
Swarup, Esther Verheyen. SSB7152,MBB, Simon Fraser Unversity, Burnaby, BC.
The Wnt/Wg is an evolutionary conserved signalling pathway in metazoans, which regulates cell proliferation and cell fate specification. The key step in
the pathway is the regulation of the levels of cytoplasmic β-catenin. β-catenin acts as a transcriptional regulator, which upon pathway activation accumulates
in the cytoplasm and subsequently translocates to the nucleus where it interacts with the Tcf/Lef family of transcription factors to direct target gene
expression. In the absence of the Wnt, the levels of β-catenin are kept low in the cytoplasm through constitutive degradation via a protein destruction
complex composed of Axin, Adenomatous Polyposis Coli (APC), Glycogen synthase kinase-3 (GSK-3), Casein kinase1 (CK1). Phosphorylation events are
known to regulate multiple steps of the Wnt pathway. The key components such as Armadillo, Dishevelled, Arrow, APC, Axin and TCF are phosphorylated
in the pathway. The ubiquitous kinases GSK-3β and CKIα regulate multiple steps of the pathway by distinct and opposing mechanisms. Other kinases such
as Nemo, Hipk and lipid kinases (PI4KIIα, PIP5K1β) and phosphatases such as PP1 and PP2 were also found to regulate different aspects of this pathway.
Although the Wnt pathway is regulated by numerous phosphorylation events, the significance of most of these events is not well understood. To fill the gap
in our knowledge we are in the process of performing an
in vivo
kinome and phosphatome RNAi screen in the
Drosophila
third instar wing disc to identify
novel regulators of the Wnt pathway. Our primary screen has yielded a number of potential novel regulators of the Wnt pathway. Preliminary
characterization of the one of the phosphatases by loss of function and O/A analysis revealed its role in Wnt secretion. We are in the process of performing
further genetic interaction studies with the members of Wnt secretion machinery. Further characterization of the hits fond in our screen will help us to
understand the Wnt/Wg pathway as a whole and how this pathway regulates different aspects of development and diseases in different organisms.
280A
The destruction complex in the Wnt pathway: APC’s mechanism of action in βcatenin degradation.
Mira I. Pronobis
1
, David M. Roberts
2
, John S.
Poulton
1
, Mark Peifer
1
. 1) Biology, UNC, Chapel Hill, NC; 2) Biology, F&M, Lancaster, PA.
The canonical Wnt signaling pathway controls cell proliferation and cell fate choices, and is regulated by the protein levels of βcatenin, a transcriptional
co-activator. The tumor suppressor APC acts in the destruction complex together with Axin, GSK3 and casein kinase to target βcatenin for degradation in
the proteasome. However, APC’s role in the destruction complex remains unclear. We tested APC’s mechanism of action using both Drosophila embryos
and colon cancer cells. Current models suggest that APC’s high-affinity βcatenin-binding sites are essential for βcatenin degradation. However, our findings
show that high-affinity βcatenin-binding sites are fully dispensable for down regulation of βcatenin in both colon cancer cells and Drosophila, and that
multiple binding sites act additively to fine tune Wnt signaling via βcatenin sequestration. The SAMP motifs of APC were defined as the binding sites for
Axin. However, our study identified a novel SAMP-independent Axin interaction site, which we mapped to APC’s Armadillo repeats (Arm rpts). We also
found 2 additional sites in APC, 20-amino-acid repeat 2 (20R2) and conserved region B, which are essential for βcatenin degradation in colon cancer cells
and Drosophila. Our data suggest they help to release APC’s Arm rpts from Axin and that this mechanism of disassembly is critical to target βcatenin for
degradation, as part of an essential catalytic cycle. We hypothesize that the Arm rpts and 20R2/conserved region B of APC act as binding sites for yet
unknown proteins. Using mass spec analysis, we are screening for candidates that are involved in this catalytic cycle of the destruction complex. Since colon
tumors invariably express truncated APC proteins that retain the Arm rpts, we hypothesize that truncated APCs can influence the destruction complex by
acting on Axin. To test this, we are performing live cell imaging and FRAP analysis to elucidate dynamic features of the destruction complex. Our study will