Rab GTPases and the collective migration of MDCK cells

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Collective cell migrations play critical roles during the development of pluricellular organisms. Most collective cell migrations require the maintenance of cell-cell adhesions so that the group of cell migrates as a single entity. Furthermore, the localization and the site of activation of determinants of migration needs to be tightly regulated during collective cell movements. What control the spatiotemporal regulation of these determinants and structures is poorly understood. Small GTPases of the RAB family are regulators of vesicular trafficking. As such, they play fundamental roles in the subcellular localization of various proteins, including cell migration determinants. RAB proteins are thus excellent candidates to be key regulators of collective cell migration. The group of Mitsunori Fukuda has recently used CRISPR/CAS9 to generate a collection of MDCK II KO cell lines for all the mammalian Rab proteins (Homma, JCB 2019) that we will exploit. Indeed, MDCK  II cells migrate collectively and display a strong hierarchy between leader cells at the migration front and follower cells. We have obtained the collection of KO cells and we have screened them for their ability to migrate. We identified several RAB proteins that are required for the normal, collective migration of MDCK II cells. In this program, we will analyze the role of our top candidates. First, we will record time lapse of these cells migrating, and we will measure different parameters. With these, we will determine their ability to migrate properly (speed, directionality), to establish a hierarchy between leader and non-leader cells and to coordinate their movements with neighbour cells. Next, we will label different migration determinants, including the actin cytoskeleton and focal adhesion by which cells exert force on the substrate, Rho GTPases (including the pool of active Rho GTPases by using FRET biosensor) and cell-cell junctions. Mosaic between parental MDCK cells and KO cells will be generated to determine if the phenotype observed are cell autonomous or non-cell autonomous. Finally, we will further characterize the role of the RAB proteins of interest by investigating the subcellular compartment they regulate, the cargoes they transport and their regulators. If required, we will determine their interactome by using a proximity biotinylation assay. While in the course of this program, we will limit ourselves to a few RAB proteins, in the long run, we will extend our list of candidates using the same approach and we will characterize the function of RAB regulators, in particular Guanine Exchange Factors (GEFs) and GTPase Activating Proteins (GAPs). In the future, we also plan to verify our findings in vivo by looking at processes such as border cell migration in flies and migration of the lateral line organ in Zebrafish. Our program will thus lead to a comprehensive characterization of the role of different Rab proteins in various collective migrations.