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Full Abstracts – CELL BIOLOGY AND SIGNAL TRANSDUCTION II
165
122
Intercellular protein movement in syncytial Drosophila follicle cells.
Peter McLean, Stephanie Airoldi, Lynn Cooley. Genetics, Yale School of Medicine,
333 Cedar St, New Haven, CT 06520.
Ring canals are stabilized cytoplasmic bridges formed by incomplete cytokinesis, and thus connect sibling cells. They have an essential role in mammalian
spermatogenesis and both male and female gametogenesis in Drosophila. Interestingly, ring canals are also present in Drosophila somatic tissues such as the
follicle cells, imaginal discs, and the larval brain. However, little is known about somatic ring canal structure, composition, or function. In contrast to the
Drosophila germline, we did not observe strict synchronization of follicle cell mitoses or specific orientation of the mitotic spindles. Quantitation of somatic
ring canals throughout ovarian development demonstrated that ~89% of main-body follicle cell divisions in stages 2-6 result in a stable ring canal, but ring
canals are not present between stalk cells or polar cells. We demonstrated intercellular exchange between main-body follicle cells by observing the spread of
fluorescent photoactivatable GFP (PAGFP) from a single activated cell. A computational analysis of the movement of PAGFP between cells suggests that
the observed intercellular movement occurred by simple diffusion. Interestingly, the observed frequency of somatic ring canal formation and the extent of
spread of activated PAGFP are consistent with the presence of syncytia averaging eight cells, much smaller than the complete mitotic lineage of ~500
follicle cells. We used Fluorescence Loss in Photobleaching (FLIP) of GFP-tagged proteins to monitor intercellular movement of Drosophila proteins. Our
data indicated that ring canals in follicle cells allow rapid equilibration of small, endogenous, cytoplasmic proteins between cells. In contrast, ribosomal and
mRNA-associated proteins do not show significant intercellular movement. Together, our results suggest a function for ring canals in equilibrating a subset
of cytoplasmic proteins in patches of cells around the egg chamber. This implies a broader significance for syncytial organization of cells outside the
germline, and provides insight into possible roles for ring canals in other Drosophila tissues.
123
Destabilization of Integrin-dependent adhesion leads to epidermal cell-cell fusion in
Drosophila
larvae.
Yan Wang, Michael Galko. Biochemistry and
Molecular Biology, University of Texas MD Anderson Cancer Center, Houston, TX.
The molecular basis of cell-cell fusion is not yet well understood despite its importance to development and physiology. In the
Drosophila
larval
epidermis, where cells are normally mononuclear, cell-cell fusion can be induced by wounding. Nuclear division followed by failed cytokinesis cannot
explain the appearance of the multinucleate cells. We thus performed a reporter-based tissue-specific
in vivo
screen for genes that regulate epidermal cell-
cell fusion. RNAi-mediated epidermal knockdown of the focal adhesion components βPSintegrin, integrin-linked kinase (ILK) and PINCH leads to
epidermal cell-cell fusion even in the unwounded tissue. This suggests that destabilization of cell adhesion, either by knockdown of focal adhesion
components or by physical wounding, triggers epidermal cell-cell fusion. Consistent with this model, ILK and PINCH are relocalized from the plasma
membrane to the nucleus and the cytoplasm, respectively, in wound-proximal cells where fusion occurs. RNAi-mediated knockdown of focal adhesion
components in patches of epidermal cells (genetically mimicking a wound) induces both autonomous (within the patch) and non-autonomous (involving
neighboring cells not targeted by the RNAi) syncytium formation. In the
UAS-PINCH
RNAi
-expressing larval epidermis we observed that the level of a Jun N-
terminal kinase (JNK) activity reporter is elevated preferentially near sites where fusion tends to occur. This resembles the JNK hyperactivation that occurs
normally in the proximal cells that are most likely to fuse following wounding. Indeed, we found that local JNK hyperactivation can also drive relocalization
of focal adhesion components and cell-cell fusion. This work provides mechanistic insight into the molecular basis of cell-cell fusion in epidermal tissues
and provides a platform for further identification of genes that both positively and negatively regulate fusion.
124
Wunen, a
Drosophila
lipid phosphate phosphatase, is required for septate junction mediated barrier function.
Andrew D. Renault
1
, Kristina E. Ile
1
,
Ratna Tripathy
1
, Valentina Goldfinger
1,2
. 1) Max Planck Institute for Developmental Biology, Tübingen, Germany; 2) Department of
Microbiology/Biotechnology, University of Tübingen, Tübingen, Germany.
Lipid phosphate phosphatases (LPPs) are integral membrane enzymes than can regulate the levels of bioactive lipids such as sphingosine 1-phosphate and
lysophosphatidic acid. The
Drosophila
LPPs
wunen
(
wun
) and
wunen2
(
wun2
) have a well-established role in regulating the survival and migration of germ
cells. We now show that Wun has an independent and essential cell autonomous role in development of the trachea. In particular Wun is required to maintain
septate junction (SJ) paracellular barrier function, loss of which causes failure to accumulate critical luminal components. We find the integrity of the blood
brain barrier is also lost in
wun
mutants indicating a general role for LPPs in SJ function. Furthermore by comparing the rescue ability of different LPP
homologs we show that Wun function in the trachea is distinct from its role in germ cell migration, although in both cases, catalytic activity is essential for
function.
125
Macroglobulin complement related is a secreted core septate junction protein whose localization is mediated through the transmembrane protein
Neuroglian.
Sonia Hall, Robert Ward. Molecular Biosciences, University of Kansas, Lawrence, KS.
Polarized epithelia play critical roles as barriers to the outside environment and enable the formation of specialized compartments for organs to carry out
essential functions. Barrier functions are mediated by cellular junctions, principally tight junctions in vertebrates and septate junctions (SJs) in invertebrates,
that line the lateral plasma membrane between cells. Over the last two decades, more than twenty genes have been identified to function in SJ biogenesis.
We recently identified mutations in
Macroglobulin complement related
(
Mcr
) that are embryonic lethal and show reduced epithelial cuticle, chitinous
deposits in the salivary glands, tracheal length and width control defects, and incomplete dorsal closure. These phenotypes suggest that
Mcr
has a role in SJ
formation or maintenance.
Mcr
encodes a protein with α-2-Marcroglobulin and LDL receptor A domains. Previous work has indicated a role for
Mcr
in the
phagocytosis of
Candida
albicans
, suggesting a role for
Mcr
in innate immunity. Here, we demonstrate the first essential developmental role for
Mcr
. We
found that
Mcr
localizes to SJs in ectodermally derived epithelial tissues, and that loss of
Mcr
results in structural and functional defects in SJs. Surprisingly,
RNAi of
Mcr
in embryos and imaginal discs suggests a cell autonomous role for
Mcr
in SJ structure and function. We hypothesized that
Mcr
localizes to the
SJ by binding to a core SJ transmembrane protein. To identify this protein, we expressed RNAi against twelve SJ transmembrane genes in the dorsal wing
compartment using
Ap-Gal4
, and observed a dramatic decrease in
Mcr
expression and localization in cells expressing
Neuroglian-RNA
i. Consistent with this
finding,
Mcr
expression is reduced and not membrane associated in
Neuroglian
mutant embryos.