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Poster Full Abstracts - Cell Biology and Signal Transduction
Poster board number is above title. The first author is the presenter
181
intercalation events occurs without obvious defects. In order to precisely monitor the MT requirement leading to branch break, we have developed a live
imaging approach in which MT are depolymerized by over expressing the MT severing protein Spastin. We revealed that breaks induced in tracheal
branches appear in a progressive instead of abrupt manner, with cells that overstretch, as seen in DE-Cadherin hypomorph mutant embryos. This
overstretched phenotype occurs after intercalation when cells have to elongate their apical part. As tracheal cells fail to maintain tension during apical
extension, we are currently investigating a link between MT and myosin II whose activity has already been involved as a tension driving force.
178A
Both Capulet and Slingshot restrict actin polymerization through regulating Twinstar activity during Drosophila eye morphogenesis.
Chiao-Ming
Lin, Pei-Yi Lin, Yu-Chiao Li, Yu-Huei Ho, Jui-Chou Hsu. Institute of Molecular Medicine, Department of Life Science, National Tsing Hua University,
Hsinchu, Taiwan 30034, Republic of China.
In response to extracellular signaling cues, the cortical actin cytoskeleton within a cell reorganizes to regulate various cell activities, including
morphogenesis, cell movement, and cell division. Dynamic turnover provides cells with the plasticity necessary to remodel actin networks rapidly and
replenish the pool of ATP-bound actin monomers available for new growth. A striking example of a transient shape change is the morphogenetic furrow
(MF) that progresses across the eye disc from posterior to anterior during Drosophila eye morphogenesis. CAP/Capulet (Capt), Slingshot (Ssh) and
Cofilin/Twinstar (Tsr) are all involved in restriction of actin polymerization. Loss of capt, ssh and tsr in the Drosophila eye epithelia similarly cause
accumulation of F-actin and enlarged apical area. By comparing their phenotypes, at single cell resolution, in eye epithelia, we found that capt and ssh, but
not tsr, mutant cells within and posterior to the morphogenetic furrow (MF) shared similar phenotypes. These include capt/ssh mutant cells possessed (1)
largely complementary accumulation of excessive F-actin and phosphorylated myosin light chain (p-MLC) at apical cortex, (2) hexagonal cell packing with
discontinuous adherens junctions (AJs) and (3) increased Ci155 accumulation in the eye epithelial cells. Similar phenotypes could be observed in capt/ssh
mutant cells anterior to the MF, upon activation of Hh pathway. We further found that the accumulation of F-actin, but not p-MLC, depended on the removal
of Ci75. Conversely, Capt/Ssh negatively regulated Ci155 levels within the MF, at a step upstream of Protein kinase A (PKA)-mediated Ci155 proteolysis.
Significantly, overexpressing a constitutively active form of cofilin and N-terminal region of Capt that recycle cofilin respectively rescued the ssh and capt
mutant phenotypes. Together, we conclude that Capt and Ssh act at distinct steps to modulate cofilin-mediated F-actin remodeling during eye
morphogenesis.
179B
Dissecting the domains of APC2 required for cortical actin association.
Olivia Molinar
1
, Molly Berntsen
2,3
, Paige Davison
2,3
, Terrence Wong
2,3
, Ezgi
Kunttas-Tatli
1
, Gordon Rule
1,2
, Brooke McCartney
1
. 1) Biological Sciences, Carnegie Mellon University, Pittsburgh, PA; 2) HHMI-Summer Research
Institute; 3) equal contribution.
Adenomatous polyposis coli (APC) proteins regulate the actin and microtubule cytoskeletons, and negatively regulate the Wnt signaling pathway. While
APC’s role in Wnt signaling and microtubule function are well studied, the mechanisms by which APC proteins interact with and regulate the actin
cytoskeleton are not well understood. APC2 localizes to actin in the Drosophila syncytial embryo, where together with the formin Diaphanous, it is required
for the extension of actin pseudocleavage furrows that form during metaphase. To understand how APC2 promotes actin furrow extension, we are
investigating the mechanisms that affect the localization of APC2 to the actin cortex. Previously we reported that the N-terminal Armadillo (Arm) repeats
and the C-terminal 30 amino acids (C30) together are necessary and sufficient for the cortical localization of APC2. Deletion of C30 alone results in loss of
cortical localization in the syncytial embryo and defects in furrow extension. Within C30 is a 15 amino acid sequence (amino acids 1048-1063) that is highly
conserved within Drosophila species suggesting functional significance. Computational structural analysis of this conserved region predicts a coiled-coil. We
found that the 15 amino acid conserved region together with the Arm repeats are necessary and sufficient for cortical localization of APC2 in S2 cells. A
point mutation targeting one of the key hydrophobic residues in the predicted coil (V1050T) abolished cortical localization, strongly suggesting that the coil
is necessary for APC2 localization to the actin cortex. Because the Arm repeats of APC2 promote its self-association and cortical localization, we predict
that oligomerization of APC2 via the Arm repeats may be necessary for cortical localization. We are currently testing whether the self-association of APC2
is required for C30 function.
180C
Does
htsN4
RNA localization matter for developing oocytes?
Nancy J. Pokrywka, Lita Sacks, Huadi Zhang, Kathleen M. Raley-Susman. Dept. of
Biology, Vassar College, Poughkeepsie, NY.
The cytoskeleton plays a key role in the establishment of cell polarity, a process that is crucial for such biological processes as cell motility, neuronal
function and embryonic patterning. A prime model of cell polarity is the localization of mRNAs during oogenesis.
Swallow (swa)
is required for the proper
localization of several RNAs, including the N4 splicing variant of
hu li tai shao
(
htsN4
). The significance of
htsN4
mRNA localization during late oogenesis
and early embryogenesis is unclear, but it encodes an adducin-like protein, and like mammalian adducins, may play a role in the regulation of the actin-
spectrin cytoskeleton. This is of interest because
swa
mutations, in addition to disrupting
htsN4
RNA localization, also result in actin defects during mid-to-
late oogenesis. However, recent evidence suggests that Swa protein may itself be membrane associated and interact with actin filaments. Thus, we were
interested in distinguishing between two models for
swa
function; one that posits a direct role for
swa
in organizing actin structures, and an alternate model
that argues
swa
affects actin organization indirectly by localizing
htsN4
RNA (and presumably Hts protein). We tested these models in several ways. First,
we looked for actin defects under other conditions that interfere with
htsN4
RNA localization, such as microtubule disruption, to see if loss of
htsN4
RNA
localization correlates with actin defects. In order to determine if
htsN4
RNA localization defects are sufficient to induce actin aberrations, we also designed
variants of
htsN4
that lack some or all of the sequences necessary for correct RNA localization. We find that actin defects are often a consequence of
microtubule disruption, but are not necessarily induced by a loss of
htsN4
RNA localization. Finally, we will also present evidence that the distribution of
Ovhts-RC protein in mid- and late-stage oocytes is independent of
htsN4
RNA localization, suggesting that
htsN4
RNA localization may be unnecessary for
oocyte organization.
181A