Rab GTPases and the collective migration of MDCK cells

集体细胞迁移在多细胞生物的发育过程中起着关键作用。大多数集体细胞迁移需要维持细胞-细胞粘附，使得细胞群作为单个实体迁移。此外，在集体细胞运动过程中，需要严格调节迁移决定因素的定位和激活位点。是什么控制这些决定因素和结构的时空调节知之甚少。RAB家族的小GTP酶是囊泡运输的调节剂。因此，它们在各种蛋白质的亚细胞定位中发挥重要作用，包括细胞迁移决定因子。RAB蛋白因此是作为集体细胞迁移的关键调节剂的优秀候选者。Mitsunori Fukuda团队最近使用CRISPR/CAS 9为我们将利用的所有哺乳动物Rab蛋白（Homma，JCB 2019）生成了一系列MDCK II KO细胞系。事实上，MDCK II细胞集体迁移，并在迁移前沿的领导细胞和跟随细胞之间显示出强烈的层次结构。 我们已经获得了KO细胞的集合，并筛选了它们的迁移能力。我们确定了几个RAB蛋白所需的正常，集体迁移的MDCK II细胞。在这个节目中，我们将分析我们的顶级候选人的作用。首先，我们将记录这些细胞迁移的时间推移，并测量不同的参数。有了这些，我们将确定他们的迁移能力（速度，方向性），建立领导者和非领导者细胞之间的层次结构，并协调他们的运动与邻居细胞。接下来，我们将标记不同的迁移决定因素，包括肌动蛋白细胞骨架和粘着斑，细胞通过其对底物施加力，Rho GTP酶（包括使用FRET生物传感器的活性Rho GTP酶库）和细胞-细胞连接。将生成亲本MDCK细胞和KO细胞之间的嵌合体，以确定观察到的表型是细胞自主的还是非细胞自主的。最后，我们将通过研究RAB蛋白的亚细胞区室，他们调节，他们运输的货物和他们的监管机构进一步表征的作用。如果需要，我们将通过使用邻近生物素化测定来确定它们的相互作用组。 虽然在这个计划的过程中，我们将限制自己的几个RAB蛋白，从长远来看，我们将扩大我们的候选人名单使用相同的方法，我们将描述RAB调节器的功能，特别是鸟嘌呤交换因子（GEF）和GTdR激活蛋白（GAP）。在未来，我们还计划通过观察苍蝇中的边界细胞迁移和斑马鱼侧线器官的迁移等过程来验证我们的体内发现。因此，我们的计划将导致不同的Rab蛋白在各种集体迁移中的作用的全面表征。

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.