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Poster Full Abstracts - Cell Biology and Signal Transduction
Poster board number is above title. The first author is the presenter
185
193A
A transmembrane RING ubiquitin E3 ligase, Godzilla, regulates endosomal trafficking.
Yasuo Yamazaki, Gaurav Varshney, Christina Schönherr,
Murat Dogru, Bengt Hallberg, Ruth Palmer. Department of Molecular Biology, Umeå University, Umeå, Sweden.
Endocytosis is a common cellular process, regulating cell signaling mediated by various types of membrane receptors. Recent studies have emerged that
endocytosis is crucial for several developmental processes such as the regulation of proper cell division and the formation and interpretation of morphogen
gradient. Here we characterize two transmembrane-type RING ubiquitin E3 ligases, Goliath and Godzilla, on endosomal trafficking. Both Goliath and
Godzilla is specifically localized on early endosomal membranes and when manipulated causes enlargement of endosome vesicles, a phenotype typically
observed upon disruption of endosomal trafficking. Mutation of the RING domain, abrogates ligase activity and results in a loss of the enlarged endosome
phenotype; suggesting that endosome enlargement dependent upon ubiquitin ligase activity. Moreover, endogenous Godzilla protein is localized on
endosomes and mutants display an enlarged endosome phenotype
in vivo
. This enlarged endosome phenotype can be rescued by a
godzilla
genomic
transgene. Our data strongly suggests that this family of E3 ubiquitin ligases are critical regulators of endosomal trafficking in
Drosophila
.
194B
Expression and Function of Glutactin in Drosophila Larvae and Adults.
Pedro Alvarez-Ortiz, Bryan Ballif, Shawna Guillemette, Rachel Humphrey, Jim
Vigoreaux. Biology, University of Vermont, Burlington, VT.
The basal lamina is a specialized extracellular matrix (ECM) that plays an essential role in tissue organization and integrity. One component of the
Drosophila basal lamina is glutactin, a highly acidic and sulfated glycoprotein with calcium binding activity and sequence similarity to serine esterases, but
lacking a critical catalytic serine residue. During embryogenesis, glutactin has been shown to be expressed in the basement membranes enclosing the gut,
brain, nerve cord, and sensory bodies. Here we show that glutactin is abundantly expressed in Drosophila larva and adults. The protein is highly resistant to
non-ionic detergent extraction and remains associated with larval body wall. Immunostaining of larval sections show glutactin localizes to regions
enveloping the body wall musculature and, to a lesser extent, visceral muscle. The protein is expressed in all regions of the adult fly, including the head,
thorax, legs, and abdomen. Immunostaining is found predominantly along the alimentary canal and digestive tract, including the proventriculus (a
specialization of the anterior alimentary canal), midgut, and the rectal ampulla. The presence of the protein in the pericellular matrices surrounding muscles
of the thorax and legs, but not of the large indirect flight muscles, suggest specialized differences in matrices associated with adult muscles. Differences are
also detected in adult female and male gonads, with staining being more prominent along the reproductive tract in females. The functional consequences of
up-regulating and down-regulating the expression of glutactin will be presented.
195C
Gon1 is a matrix metalloproteinase required for migrating cells to detach from the ECM.
Afshan Ismat, Alan Cheshire, Deborah Andrew. Dept Cell
Biol, Johns Hopkins Sch Med, Baltimore, MD.
Proper migration of cells through the three-dimensional extracellular matrix (ECM) requires clearing a path at the cell front or leading edge, and
detachment from the ECM at the rear or trailing edge. Among the extracellular enzymes that could modify the ECM to allow for proper cell migration is the
ADAMTS family of matrix metalloproteinases. The Drosophila genome encodes three ADAMTS genes, including
gon1 (CG14869)
, which is expressed in
many migratory tissues. Loss of
gon1
causes migration defects in tissues that express
gon1
, the caudal visceral mesoderm and tracheal visceral branches, and
in tissues that do not, the germ cells. Tissue-specific rescue experiments show that
gon1
functions both cell autonomously and non-cell autonomously to
rescue migration defects. The salivary gland (SG) migrates as an intact organ, and comprises adherent polarized epithelial cells surrounding an inner, matrix-
filled lumen. The basal surfaces of SG cells face outward, acting as the leading edge of this migrating collective, and the apical surfaces face inward toward
the lumen, potentially acting as the trailing edge. In the absence of
gon1
, the SG exhibits severe apical membrane retractions resulting from an apparent
failure of the apical membrane to detach from the apical ECM of the lumen. The distal-most SG cells are more elongated than in wild type, and the lateral
membranes appear stretched and torn. Thus, without
gon1
, migratory forces are pulling the SG forward, but the trailing edge of individual SG cells cannot
detach from the apical ECM. This apparent failure in trailing edge detachment in the SG brings up the issue of whether
gon1
functions the same in other cell
types. To address this, I am exploring where Gon1 localizes in individually migrating cells, the germ cells. I hope to learn if
gon1
functions at the leading
edge to clear a path for forward movement or at the trailing edge to allow cells to detach from the ECM. Through this work, I expect to learn how the
ADAMTS family of extracellular proteinases contributes to cell migration and tissue morphogenesis during development.
196A
Genetic interaction between POSH and Zasp52.
Ashley Lennox, Rebecca Garlena, Beth Stronach. Univ of Pittsburgh Sch Med, Pittsburgh, PA.
The POSH protein consists of a RING domain with ubiquitin E3-ligase activity and four SH3 domains. Evidence supports a proapoptotic role for POSH as
a scaffold protein linking Rac GTPase and the Jun Kinase pathway. Yet POSH has been tied to other processes including innate immunity, longevity,
apoptotic resistance, synaptic growth, and virus trafficking. Though Drosophila POSH mutants are viable, our previous studies have shown that
overexpression of POSH in the embryo leads to loss of amnioserosa integrity and caspase-dependent cell death. In these studies, we noted that the
phenotypic consequences of POSH overexpression were enhanced by inclusion of a GFP protein trap line, zcl423, which labels dorsal ectodermal cells. We
find that the genetic interaction between POSH overexpression and zcl423 is recapitulated in several tissues, including the larval eye and wing discs. While
zcl423 expression is predominant in muscle tissue, the phenotypes we observe are manifest in epithelial tissues, prompting us to explore the expression of
zcl423 more closely. Zcl423 is detectable at relatively reduced levels in many non-muscle tissues of the larva, including the imaginal discs, salivary glands,
hemocytes, central nervous system, and ring gland, though it is absent from fat body, for instance. Within epithelial cells, zcl423 localizes in discrete foci at
the basal membrane, alternating with patches of βPS integrin staining, and in relatively punctate fashion at cell junctions in association with E-cadherin. To
determine what protein is labeled with GFP in the zcl423 line, we performed plasmid rescue and sequenced the flanking DNA. Zcl423 is a GFP protein trap
in Zasp52, the Z-band alternatively-spliced PDZ protein at position 52 on chromosome 2R. It is likely that zcl423 is a viable hypomorph, because we can
recapitulate the genetic interaction with POSH expression using a Zasp52 RNAi line. To get at the mechanism underlying the genetic interaction, we are
pursuing studies to determine whether POSH is altering Zasp52 levels or localization to bring about epithelial tissue dysmorphology.