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Full Abstracts – CELL BIOLOGY AND SIGNAL TRANSDUCTION II
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The GEF Vav regulates collective cell migration downstream of guidance receptors by locally activating Rac at the leading edge.
Cecilia H.
Fernandez-Espartero
1
, Damien Ramel
2
, Marganit Farago
3
, Malartre Marianne
1,5
, Carlos M. Luque
1,4
, Shiran Limanovich
3
, Shulamit Katzav
3
, Gregory
Emery
2
, María D. Martín-Bermudo
1
. 1) Centro Andaluz de Biologia del Desarrollo (CABD), CSIC, Sevilla, Spain; 2) IRIC, Université de Montréal, Quebec,
Canada; 3) IMRID, Jerusalem, Israel; 4) EMBL, Heidelberg, Germany; 5) Université Paris Sud, France.
Cell migration is essential in the development and maintenance of multicellular organisms. Guidance by spatial cues is essential for the migrating cells to
reach the correct target tissue. Thus, during wound healing and immune responses cells are guided to site of injury and tumor cells may be guided to target
tissues. One of our aims is to understand how guidance signals control cell migration. For this purpose, we use the migration of the border cells (BCs) of the
Drosophila ovary as a model. BCs are a group of 6-8 anterior specialized follicle cells that at stage 9 of oogenesis delaminate and migrate between the
germline toward the oocyte. This directed cell migration is controlled by two receptor tyrosine kinases, PVR and EGFR, which are activated in BCs in
response to their ligands produced by the oocyte. Although there is some evidence suggesting that Rac and its activator Mbc can act as mediators of these
guidance signals, the molecular mechanisms by which the activity of these two receptors control Rac are still unclear. Recently, we have identified Vav, a
guanine nucleotide exchange factor (GEF) for Rac, as putative downstream target of Pvr activation. Loss of Vav function impairs BC migration. Two hybrid
and co-IP experiments show that Vav interacts physically with Pvr. Furthermore, we find that stimulation of Pvr in S2 cells induces Vav activation. Finally,
we show by FRET analysis that Rac activity is reduced in the absence of Vav and that ectopic Vav activation leads to a non-polarized distribution of Rac
activity. Taken altogether, we propose a model in which Vav acts as a signal transducer that couples signalling downstream of guidance receptors to Rac
activation during directed cell migration.
120
Signal regulation by Rab5 GEFs in Drosophila melanogaster.
Katja L Vogt, Martin P Zeidler, Elizabeth Smythe. Biomedical Sciences, University of
Sheffield, Sheffield, United Kingdom.
A fundamental concept of the organisation and regulation of cellular communication is the control of signalling by endocytosis. The process of endocytic
membrane trafficking is intimately and bidirectionally linked to cell signalling. Crucially, spatial compartmentalisation enables the cell to shuttle distinct
signalling complexes into specific intracellular locations. In order to elicit specific responses not only the separation of signals in early and late endosomes,
but also their distinct localisation in micro-/ sub- domains (signalosomes). These signalosomes can influence distinct receptor signalling via a variety of
exclusive downstream signalling pathways. The small GTPase Rab5 protein not only controls early endosomal dynamics but is a central hub for unique
regulatory signalling platforms. Guanine exchange factors (GEFs) that exchange GDP for GTP act as Rab5 activators and appear to be initial important
regulators of Rab5.
We are investigating the interplay of endocytosis and signalling in Drosophila with a particular focus on the JAK/STAT pathway and the role of the four
Drosophila rab5GEFs (rabex5, alsin, sprint and dRME-6). Our initial results indicate that modulation of these GEFs differentially affects JAK/STAT
signalling outputs. We propose that each GEF establishes and activates distinct signalosomes, allowing discriminated signalling. Current studies centre on
structure / function analysis of the GEFs to determine the mechanism underpinning the role of endocytosis in JAK/STAT signalling.
121
Endocytic regulation of collective cell migration.
Gregory Emery
1
, Damien Ramel
1
, Xiaobo Wang
2
, Denise Montell
2
. 1) IRIC, University of Montreal,
Montreal, QC, Canada; 2) Department of Biological Chemistry,Center for Cell Dynamics, Johns Hopkins School of Medicine, Baltimore, MD.
Cell migration is a fundamental process to organize tissues but also in development of diseases such as cancer. Cells can migrate individually but growing
evidences suggest that many cells types, including tumorigenic cells migrate collectively. Collective migration implies that individual cells coordinate their
signaling to migrate as a coherent structure in response to attractive cues. Polarized activity of the small GTPase Rac has been demonstrated to control
collective guidance in vivo, however, nothing is known about the mechanisms that restrict Rac activation and localization during collective migration. Here,
we show that Rac activity and polarization is regulated by a trafficking cycle involving the recycling endosome compartment and its regulating small
GTPase Rab11. Real time analysis of Rac activity by Fluorescence Resonance Energy Transfert (FRET) in drosophila border cells reveals that expression of
a dominant negative version of Rab11 (Rab11SN) induces a complete depolarization of Rac activity. Morevover, Rab11 loss of function induces a
redistribution of actin protrusions during migration. We hypothesize that loss of polarity of Rac in Rab11SN background induces incoherent generation of
forces thereby blocking migration. To test this model, we used a photoactivable version Rac. We demonstrated that local activation of Rac fails to rescue
migration in a recycling deficient group indicating that Rab11 is required for generation of polarized forces that drive migration. Morevover, these results
implicate Rab11 in the proper sensing of Rac activation level in neighboring cells. Our work demonstrates that endocytosis and recycling are critical to
achieve spatial restriction of Rac activation in collective cell migration. These data implies Rab11 as a master regulator of these processes and provide new
insights in the role of endocytosis in the organization of individual cells in a coherent multicellular motile structure.