Structure, dynamics and mechanism of Arpin, a novel inhibitor of the Arp2/3 complex that controls cell migration – Dynarpin

Notre consortium réunit l’expertise en biologie structurale (cristallographie, RMN, SAXS, microscopie) et en biologie et imagerie cellulaire. Les partenaires, en mettant ces expertises en commun, ont pour objectif d’établir une description intégrée de la structure, des mouvements, des interactions et de la fonction cellulaire de cette nouvelle protéine.

Le projet a démarré à la date prévue en janvier 2012. Les expériences sont actuellement en cours.

A terme, un nouveau regard sur la migration cellulaire avec des applications potentielles en recherche contre le cancer.

En cours

Cell migration is essential to many biological processes, such as the development of organs, immunological responses or healing of wound tissues. Likewise, defective or abnormal cell migration contributes to a variety of diseases. Notably, abnormal migration results in the invasion of cancer cells into adjacent tissues, leading to metastatic progression, the most dramatic clinical aspect of cancer. Cell movement is powered by the polarized and dynamic assembly of actin structures. At the leading edge of the cell, actin filaments assemble and branch in a sheet-like meshwork, which supports membrane protrusions called lamellipodia. Research efforts during the last decade have brought us a long way towards unravelling the molecular events that take place within the lamellipodium. The « minimal » machinery that is required to initiate and maintain the actin meshwork at the protrusive regions includes Rac, a small GTPase of the Rho family, the nucleation promoting factor WAVE, and the actin nucleator Arp2/3. Structural and biochemical studies combined to cell imaging have contributed major breakthrough into understanding the hierarchy of regulatory events in this pathway, leading to the current consensus model in which : 1) Rac is activated by GDP/GTP exchange; 2) Rac-GTP binds to inactive WAVE, thus exposing its WCA motif; 3) the WCA motif of activated WAVE binds to inactive Arp2/3, thus converting Arp2/3 into its active conformation; 4) activated Arp2/3 binds to an actin filament and nucleates a novel branched filament.

Remarkably, migrating lamellipodia maintain an essentially constant breadth, indicating that the balance between assembly and disassembly is exquisitely controlled once the lamellipodium has established. In contrast to the well-studied mechanisms of actin branches formation, the molecular mechanisms of their regulated disassembly are still poorly understood. Of the regulatory proteins that control actin disassembly, coronin is to date the only one that directly targets the Rac/WAVE/Arp2/3 machinery in the lamellipodium. Many modulators of lamellipodium turnover in this pathway thus clearly remain to be discovered and characterized. In this project, we will investigate the structure and regulation of Arpin, a newly discovered protein that has the hallmarks of a novel negative modulator of lamellipodia. Arpin was recently discovered by the Gautreau group (partner 2 in this consortium), and has no discernable homology to any known protein. Our preliminary experiments (partner 2, unpublished) found that Arpin inhibits the Rac/WAVE/Arp2/3 pathway through a Rac-GTP-dependent interaction with Arp2/3, and that its suppression in cells leads to exaggerated lamellipodial actity.

To address the molecular basis of Arpin regulation and interactions within Rac/WAVE/Arp2/3 pathway that lead to lamellipodia control, our consortium brings together experts in protein X-ray crystallography and biophysics (Cherfils group, coordinator), NMR (Zinn-Justin group, partner 3), structural electron microcopy (Jonic group, partner 4) and cell biology (Gautreau group, partner 2). Based on a consistent ensemble of preliminary results, the consortium will set out to (1) solve the structure of wild-type Arpin (partners 1 and 3); (2) realize a structure-based analysis of its regulation by conformational changes and phosphorylation in vitro and in cells (partners 1,2, and 3); (3) investigate its interactions with Rac, with Arp2/3 and with membranes by combining electron microscopy, crystallography, NMR and biophysical methods (partners 1, 3 and 4) and (4) undertake exploratory in-cell structural studies (partners 2 and 3). This should allow us to deliver an integrated structural and functional model of the negative modulation of cell migration by Arpin within the Rac/WAVE/Arp2/3 pathway, of potential application in cancer research.