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Poster Full Abstracts - Cell Biology and Signal Transduction
Poster board number is above title. The first author is the presenter
178
Congenital Amaurosis (LCA) and Retinitis Pigmentosa type 12 (RP12). Work in vertebrate models suggests that a loss of epithelial integrity precedes PRCs
degeneration in Crb compromised retinas. Over 40 distinct disease-causing missense mutations that map to the extracellular region have been reported for
human CRB1. To gain insight into how these missense mutations affect CRB1 function and to elucidate the function of the large extracellular region of
Crb/CRB1 we have recreated four disease-causing missense mutations that affect conserved amino acids in
Drosophila
Crb. Our analysis of these Crb
mutant isoforms in both wildtype and
crb
mutant PRCs identified specific residues/domains in Crb that are required for the normal localization of Crb at the
stalk membrane. Moreover, each of the four missense mutations tested so far shows a unique cell biological profile including mutations that cause
mislocalization of Crb to the rhabdomere, which leads to a displacement of Rhodopsin. Loss of Rhodopsin function is a known cause of RP, thus suggesting
a potential disease mechanism that may be independent from the role of Crumbs in supporting polarity for these particular mutant alleles. In contrast to
expression of normal Crb in a
crb
mutant background, none of the four Crb missense mutants can prevent PRC degeneration.
166A
Crumbs supports apical delivery in the developing photoreceptor.
Rhian F. Walther, Franck Pichaud. MRC LMCB, University College London,
London, United Kingdom.
Epithelial polarity remodeling is key for allowing epithelial-derived organ formation. Typically it involves a conserved set of factors, including the apically
localized Par6-DaPKC, Crumbs (Crb), Sdt and PATJ, the adherens junction associated Bazooka (Baz) as well as the laterally localized Lethal giant larvae
(Lgl), the Serine-Threonine kinase Par1 and the recently identified Yurt/Coracle pathway. However, the molecular nature underlying the genetic interactions
between these factors remains for the most part elusive. In order to tackle this question, we are making use of a striking step of apico-basal polarity
remodeling occurring during photoreceptor morphogenesis. In this context, we find that Par1 localizes at the lateral membrane of the cell where it limits
lateral clustering of Baz. In this cell, par1 function is required to limit Baz-dependent apical membrane morphogenesis. In parallel, Lgl is required to prevent
basal, but not lateral accumulation of the apical factor Crb. In this situation, Crb is sufficient to support the Rab11-MyoV apical trafficking route required to
build the apical organelle, called the rhabdomere, but it is also sufficient to redirect apical secretion of the luminal factor eyes-shut toward the basal side of
the cell. Our work therefore presents Crb as a main effector of directed apical trafficking during apico-basal epithelial polarity remodeling.
167B
An shRNA screen for genes involved in epithelial polarity identifies a novel member of the Par polarity complex.
Frederik Wirtz-Peitz
1
, Dong Yan
1
,
Takashi Nishimura
2
, Norbert Perrimon
1
. 1) Department of Genetics, Harvard Medical School, Boston, MA; 2) RIKEN Center for Developmental Biology,
Kobe, Japan.
In the Drosophila embryo, epithelial cells are polarized towards the end of cellularization by the concerted activity of a conserved set of polarity proteins.
How these proteins interact to establish and maintain distinct membrane domains is poorly understood. We screened for novel polarity genes using a library
of shRNA constructs, which permit the analysis of nearly protein-null embryos by knockdown of a gene’s maternal and zygotic expression. Thus, we
identified a previously uncharacterized protein, which, when knocked down, causes a defect similar to mutants in the Par complex. This polarity complex,
consisting of atypical protein kinase C together with its regulators Par-6 and Bazooka, localizes to the apical cell cortex where it stabilizes the apical domain
and inhibits determinants of the basolateral domain. Our data reveal that the protein identified in our screen participates in the Par complex by virtue of a
direct interaction with Bazooka, and we present an initial characterization of its molecular role in epithelial polarity.
168C
Determining the mechanisms of CTP synthase filament (cytoophidia) formation.
Gabriel N Aughey, Ji-long Liu. MRC Functional Genomics Unit,
University of Oxford, Oxford, United Kingdom.
Cytidine triphosphate synthase (CTPS) is the rate limiting enzyme for the de novo synthesis of cytidine triphosphate (CTP), a fundamental component of
DNA and RNA, as well as a precursor to fatty acid synthesis. It has recently been observed that CTP synthase (CTPS) can be compartmentalised into
discrete filamentous cytoplasmic structures that have been termed cytoophidia. This feature of CTPS is highly conserved throughout evolution and has been
reported in single celled organisms (the bacteria C. cerescentus, and budding yeast, S. cerevisiae) as well as complex eukaryotes including Drosophila and
humans, suggesting that the compartmentation of CTPS in this way is a fundamental feature of all cells. The rate limiting enzyme for GTP biosynthesis,
inosine monophosphate (IMPDH), also colocalises to this structure in Drosophila melanogaster and has been shown to form filaments in human cells.
Expression of GFP tagged CTPS in Drosophila S2 cells results in increased abundance of cytoophidia. This system will be used for the development of a
high throughput, genome-wide RNAi screen with Drosophila S2 cells in order to identify factors that regulate CTPS compartmentalisation.
169A
Coordination between stable and dynamic microtubule networks determines and maintains Drosophila bristle shape.
Amir Bitan, Uri Abdu.
Department of life sciences, Ben Gurion University, Beer Sheva, Israel.
Within interphase cells, microtubules (MT) are organized in a cell-specific manner to support cell shape and function. Here, we report that coordination
between stable and dynamic MTs determine and maintain the highly elongated bristle cell shape. By following MT-decorating hooks and by tracking EB1,
we identified two MT populations within bristles, namely a stable MT population polarized minus-ends-distal toward the bristle tip and a dynamic MT
population that exhibits mixed polarity. Manipulating MT dynamics by klp10A down-regulation demonstrates that MTs can initiate new shaft extensions,
thus possessing the ability to determine growth direction. Actin-filament bundling subsequently supports the newly-formed shaft extensions. Established by
elongation defects in the Drosophila ikk-epsilon homologue, ik2 mutant bristles, we report that stable and dynamic MT orientation and polarized
organization are important for proper bristle elongation. Thus, we demonstrate for the first time that coordination between stable and dynamic MT sets that
are both axially-organized but differently polarized, drives cell elongation.
170B
JNK signaling regulates the actin-binding protein Profilin in
Drosophila
larval wound closure.
Amanda R. Brock
1,2
, Yan Wang
1
, Susanne Berger
3
,
Violet C. Han
1
, Yujane Wu
1,2
, Renate Renkawitz-Pohl
3
, Michael J. Galko
1,2
. 1) Biochem & Molec Bio, UT MD Anderson Cancer Ctr, Houston, TX; 2)