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Full Abstracts – CELL BIOLOGY AND SIGNAL TRASDUCTION I
157
101
Wingless is secreted on exosome-like vesicles in
Drosophila
S2 cells.
Karen Beckett
1
, Solange Monier
2
, Hannah Green
1
, Roland LeBorgne
2
, Jean-Paul
Vincent
1
. 1) Developmental Biology, National Institute for Medical Research, London, United Kingdom; 2) CNRS UMR 6061, Université de Rennes 1,
35043 Rennes Cedex, France.
Cell communication is essential for embryonic development and normal physiology of multicellular organisms. Wingless (Wg), the main
Drosophila
Wnt,
controls several developmental processes including growth, differentiation and survival. Wg is lipid-modified and thus tightly associated with cellular
membranes. However, Wg can be released to act at a distance from its source of production. Therefore an outstanding question is how Wg is packaged for
release from producing cells. To answer this question we have revisited the argosome model of Wg secretion and tested whether Wg is secreted on exosome-
like vesicles. Exosomes are 40-100nm microvesicles that are produced in multivesicular bodies (MVBs) and released by fusion of MVBs with the plasma
membrane. We have found that Wg is secreted on vesicles resembling exosomes by
Drosophila
S2 cells. These vesicles have the density, size and
morphology of exosomes and can activate downstream signalling. Proteomic analysis of Wg-containing vesicles has identified known mammalian exosome
proteins such as those involved in membrane trafficking, signalling and metabolism. We found that membrane trafficking proteins such as Hrs, Vps28 and
Rab35 are present in Wg-containing exosomes. However, so far they appear dispensable for exosome production. Wg secretion requires a multipass
transmembrane protein called Evi that is thought to transport Wg from the Golgi to the plasma membrane. Retromer-mediated recycling of Evi is
subsequently required to replenish Evi levels in the Golgi. We found that Evi is secreted on exosome-like vesicles in S2 cells independently of Wg. We
propose a model whereby Evi is continually trafficked to MVBs and released on exosomes. We suggest that, in Wg producing cells, following transport from
the golgi to the plasma membrane, Evi and Wg would be co-endocytosed, taken to MVBs and packaged into exosomes for release. We are currently
developing tools to test this model in vivo.
102
Polarized biosynthesis and secretion of Collagen IV during organ morphogenesis.
Sally Horne-Badovinac, David Lerner, Darcy McCoy, Gary Gerlach
II. Department of Molecular Genetics and Cell Biology, The University of Chicago, Chicago, IL.
The elongation of tissues and organs along a particular body axis is a common theme in the development of multicellular organisms. Though initially
spherical, Drosophila egg chambers lengthen along their anterior-posterior (A-P) axes to create the elongated shape of the mature egg. Egg chambers are
composed of an internal germ cell cluster surrounded by an epithelial layer of follicle cells. At the onset of egg chamber elongation, the entire organ
undergoes an unusual rotation perpendicular to its A-P axis. It has been proposed that this rotation leads to the remodeling of the Col IV matrix on the
outside of the egg chamber, which in turn promotes elongation morphogenesis. However, the molecular and cellular mechanisms that underlie Col IV matrix
remodeling remain obscure. Starting from a mutation that specifically blocks the secretion of Col IV, we have found that Col IV biosynthesis occurs in a
specialized sub-region of the endoplasmic reticulum (ER) near the basal follicle cell surface. Interestingly, these basal ER cisternae are also polarized within
the tissue plane, such that they are enriched at the back of each migrating follicle cell during rotation. We have further identified a specialized golgi
population associated with the basal ER, and have shown that disrupting proteins associated with the basal golgi leads to the mistrafficking of Col IV to the
apical surface. Together these data demonstrate a striking compartmentalization of the secretory machinery that controls polarized secretion of Col IV to the
basal surface. Moreover, the polarization of this machinery within the tissue plane may lend insight into the mechanisms controlling Col IV matrix
remodeling during egg chamber elongation.
103
A Novel Role for UDP-GlcNAC in Dpp Signal Antagonism.
Gregory B. Humphreys, Kate Monroe, Molly Jud, Anthea Letsou. Human Gen, Univ Utah,
Salt Lake City, UT.
mummy
(
mmy
), a member of the
raw
group of signaling antagonists, encodes the single Drosophila UDP-N-acetylglucosamine pyrophosphorylase. Mmy’s
effects on signal antagonism are most evident in the context of embryonic dorsal closure. In this developmental context, the JNK/AP-1 signaling cascade
transcriptionally activates Dpp signaling in leading edge (LE) epidermal cells. Whereas
dpp
is confined to LE cells in wild-type embryos, it expands
ectopically into the dorsolateral epidermis in
mmy
mutant embryos, establishing Mmy as a
dpp
antagonist. Considering that Jun is required for LE
dpp
transcription, and as Jun activity and localization are unaffected in
mmy
mutants, we utilized a
mmy Jra
double mutant to specifically test if Jun-initiated LE
dpp expression is required to ectopically express
dpp
in a
mmy
mutant background.
mmy Jra
mutant embryos lack both LE and ectopic
dpp
expression,
indicating a requirement for an initiating round of LE Dpp to enact ectopic expression. These data suggest that Mmy antagonizes paracrine Dpp in the
epidermis, consistent with earlier evidence identifying Dpp-dependent
dpp
transcription in the LE epidermis. To test this model, we assayed embryonic Dpp
signaling activity by probing Mad phosphorylation. P-Mad is found broadly in early embryonic epidermis, but undergoes a Mmy-dependent restriction to LE
cells prior to dorsal closure. P-Mad remains broad in the dorsolateral epidermis in
mmy
mutant embryos, suggesting that Dpp undergoes a Mmy-dependent
transition from paracrine to autocrine signaling in the embryonic epidermis. To identify downstream effectors of Dpp signal restriction, we screened the 25
Drosophila glycosyltransferases potentially utilizing UDP-GlcNAc downstream of Mmy. In embryos depleted of one of these transferases,
dpp
expanded
ectopically beyond the LE epidermis, identifying this transferase as a
dpp
antagonist and providing new insights into regulation of Dpp signaling via
glycosylation.