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Poster Full Abstracts - Cell Biology and Signal Transduction
Poster board number is above title. The first author is the presenter
196
Notch ligands and suggests that the NHR1 and NHR2 domains together may regulate Neuralized activity and, by extension, Notch signal transduction.
237C
Importin-α3 mediates nuclear import of Notch and it displays synergistic effect with Notch activation on cell proliferation.
Nalani Sachan, Abhinava
Mishra, Mousumi Mutsuddi, Ashim Mukherjee. Department of Molecular & Human Genetics, Banaras Hindu University, Varanasi, India.
The Notch pathway is an evolutionarily conserved signaling system which has been shown to play major role in cell fate determination, differentiation,
proliferation and apoptotic events as well as self-renewal processes of different tissues. The same pathway can be deployed in numerous cellular contexts to
play varied and critical roles for the development of an organism. The versatility of this pathway to influence different aspects of development comes from
its multiple levels of regulation. In an effort to identify novel components involved in Notch signaling and its regulation, a yeast two-hybrid screen was
carried out using intracellular domain of Notch receptor (Notch-ICD) as bait and we identified Drosophila Importin-α3 as binding partner of Notch. Further,
GST-pull down experiments confirmed the interaction between Notch and Importin-α3. Immunocytochemical analysis revealed that Importin-α3 and Notch-
ICD indeed co-localized in cell nuclei. Different alleles of importin-α3 also showed strong genetic interactions with Notch pathway components in
transheterozygous combinations. Somatic clonal analysis of importin-α3 using FLP-FRT system showed that loss of importin-α3 function results in
cytoplasmic accumulation of the Notch receptor. Using MARCM (Mosaic Analysis with a Repressible Cell Marker) technique, we demonstrate that
Importin-α3 is required for nuclear localization of Notch receptor. These results clearly show that the nuclear transport of Notch-ICD is mediated by the
canonical Importin-α3/Importin-β transport pathway. In addition, co-expression of both Notch-ICD and Importin-α3 displays synergistic effects on cell
proliferation. Taken together, our results suggest that nuclear import of Notch-ICD may play important role in regulation of Notch signaling.
238A
Mutual bi-directional Notch activation represses fusion competence in swarming adult Drosophila myoblasts.
Eyal D. Schejter, Boaz Gildor, Ben-
Zion Shilo. Dept. Molecular Genetics, Weizmann Institute of Science, Rehovot, Israel.
Establishment of the adult Drosophila flight musculature involves hundreds of cell-cell fusions between nascent myotubes, and a migrating “swarm” of
wing-disc derived myoblasts. Our analysis suggests that these fusion events require functional contributions from the Ig super-family cell adhesion proteins
Dumbfounded/Kirre, which is expressed on the myotubes, and the myoblast specific element Sticks-and-stones (Sns). This molecular paradigm resembles
the program of myoblast fusion underlying embryonic muscle fiber formation. However, Sns expression by adult myoblasts is tightly regulated, and is
restricted to myoblasts in the immediate vicinity of the myotubes, just prior to fusion. We find that during their migratory phase, myoblasts are maintained in
a semi-differentiated state by continuous activation of Notch signaling, where each myoblast provides the source of ligand, Delta, to its neighbors. This
unique form of bi-directional Notch activation is achieved by finely tuning the levels of the ligand and the Notch receptor. Activation of Notch signaling in
myoblasts represses expression of key fusion elements such as Sns. Only upon reaching the vicinity of the myotubes does Notch signaling decay, leading to
terminal differentiation of the myoblasts. The ensuing induction of fusion-related proteins enables formation of actin-rich attachment sites between
myoblasts and myotubes, leading to cell-cell fusion and muscle fiber growth.
239B
Function of a neurogenic gene,
pecanex
in Notch signaling.
Tomoko Yamakawa
1
, Kenta Yamada
1
, Takeshi Sasamura
1
, Naotaka Nakazawa
1
, Maiko
Kanai
1
, Emiko Suzuki
2
, Mark E. Fortini
3
, Kenji Matsuno
1
. 1) Dept of Biol Sci/Tec, Tokyo Univ of Sci, Chiba, Japan; 2) Gene Network Lab, National
Institute of Genetics, Mishima, Japan; 3) Dept of Biochem and Mol Biol, Kimmel Cancer Center, Thomas Jefferson Univ, Philadelphia, PA, USA.
Notch (N) signaling is an evolutionarily conserved mechanism that regulates a broad spectrum of cell-specification through local cell-cell interaction. The
homozygous mutant flies of
pecanex
(
pcx
) are viable, but
pcx
homozygous females mated with the
pcx
mutant males produce embryos that show a
N
-like
neurogenic phenotype, suggesting that Pcx is a component of N signaling. Pcx is a multi-pass membrane protein. However, its biochemical functions are still
unknown.
Here we established that Pcx is a component of the N-signaling pathway. Pcx was required upstream of activated form of N, probably in N-signal-receiving
cells, suggesting that
pcx
is required prior to or during the activation of N. We found that Pcx protein was specifically localized to the endoplasmic reticulum
(ER). In addition, ER was enlarged in the embryos homozygous for
pcx
lacking its maternal contribution. However, such ER enlargement was not observed
in embryos homozygous for
N
or in embryos homozygous for
Presenilin
and lacking its maternal contribution. These results suggest that the ER
enlargement is not due to the disruption of N signaling.
Hyper-induction of the unfolded protein response (UPR), by the expression of activated
Xbp1
or dominant-negative
Heat-shock cognate 70-3
, suppressed
the neurogenic phenotype and ER enlargement caused by the absence of
pcx
. A similar suppression of these phenotypes was increased by the overexpression
of
O
-fucosyltransferase 1, an N-specific chaperon. Taking these results together, we speculate that the reduction in N signaling in embryos lacking
pcx
function might be attributable to defective ER functions, which are compensated for by up-regulation of the UPR and possibly by enhancing N folding.
240C
Dynamics of the Rho family small GTPases in Single Cell Wound Repair.
Maria Teresa Abreu-Blanco, Susan M Parkhurst. Basic Sciences Division,
Fred Hutchinson Cancer Research Center, Seattle, WA.
Rho GTPases are important regulators of cytoskeleton dynamics in a variety of biological events including cell division, cell migration, vesicle trafficking
and gene expression. Rho GTPases are highly regulated in space and time, key features for their function as molecular switches that transmit environmental
cues to intracellular signaling pathways. Moreover, Rho GTPases are themselves regulated by input from the cytoskeleton, coordinating the multiple
dynamic responses required by the cell. One biological process that requires precise spatial and temporal coordination of membrane and cytoskeletal
components is cell wound repair. Single cell wounds heal by rapidly plugging the plasma membrane disruption with a vesicle patch, and requires the
assembly of contractile actomyosin ring and microtubules reorganization. To assess the role of Rho GTPases as regulators of the cell wound repair response,
we laser wounded embryos carrying fluorescently-tagged GTPases, then followed their repair in vivo by 3D microscopy. In our single cell model, Rho, Rac
(Rac1 and Rac2) and Cdc42 rapidly accumulate around the wound, and segregate into dynamic zones that move inward (basally) as healing progress. We
also developed biosensor probes for each GTPase, using the Rho binding domains of different downstream effectors, to determine the spatial and temporal