Role of clathrin-coated plaques in regulating cell migration

网格蛋白是一种独特的支架蛋白，它在质膜上形成多面体笼，组装成平面和弯曲的晶格。弯曲的网格蛋白包被的凹坑在形成内吞结构中的功能被很好地研究。相反，扁平六边形网格蛋白阵列（也称为斑块）的作用仍然不明确。几项研究表明，斑块参与细胞粘附，然而，导致这种扁平网格蛋白晶格形成的分子起源和机制尚不清楚。此外，斑块的生理相关性和功能仍有待确定。在准备这项建议时，我们确定了导致斑块形成的机制及其在细胞迁移过程中的功能。在迁移细胞中，前缘的局灶性粘连以协调的方式分解。使用活细胞显微镜，我们可以证明这些粘着斑被网格蛋白包被的斑块所取代，我们称之为“粘着斑转换”。我们可以证明，斑块募集后直接积极消化细胞外基质的局灶性粘连。使用人工3D微模式或基因操作的局部粘连的蛋白水解活性，我们可以表明，斑块招募，在整合素依赖的方式，局部粘连创建的3D地形线索。通过基因沉默，我们已经确定Eps 15/R作为一个重要的调节斑块形成过程中的局灶性粘附开关。通过对野生型细胞和耗尽Eps 15/R的细胞进行迁移测定，我们可以将细胞方向性和速度与斑块读取细胞外地形线索的能力相关联。因此，我们已经确定了斑块在细胞迁移过程中的起源和功能，我们提出网格蛋白包被的斑块可以作为非常规集体迁移的调节剂，以细胞与细胞接触独立的方式。该提案建立在这些初步研究结果的基础上，旨在阐明以下开放的问题：在粘着斑转换和细胞迁移过程中驱动网格蛋白募集的分子/生物物理信号是什么？使网格蛋白涂层稳定为网格蛋白涂层菌斑的分子组分是什么？斑块如何调节细胞迁移？具体目标如下：目的1：确定整合素和地形学线索在调节斑块形成和稳定性中的重要性目的2：Eps 15/R的功能，从介导FA转换到调节细胞迁移目的3：鉴定调节斑块形成和稳定性的分子决定因素

Clathrin is a unique scaffold protein, which forms polyhedral cages at the plasma membrane assembling into flat and curved lattices. The function of the curved clathrin-coated pits in forming endocytic structures is well studied. On the contrary, the role of the flat hexagonal clathrin arrays, also known as plaques remains ambiguous. Several studies have suggested that plaques are involved in cell adhesion however, the molecular origins and mechanisms leading to the formation of such flat clathrin lattices are unknown. Furthermore, the physiological relevance and functions of plaques remains to be determined. In preparation of this proposal, we identified the mechanisms leading to plaque formation and their functions during cell migration. In migrating cells, focal adhesions at the leading edge disassemble in a co-ordinated manner. Using live-cell microscopy, we could demonstrate that these focal adhesions get replaced by clathrin-coated plaques, a process we termed “focal-adhesion switch”. We could demonstrate that plaque recruitment directly follows after the active digestion of the extracellular matrix by focal adhesions. Using artificial 3D-micropatterns or genetic manipulation of the proteolytic activity of focal adhesions we could show that plaques are recruited, in an integrin-dependent manner, to 3D-topographical cues created by focal adhesions. Through genetic silencing, we have identified Eps15/R as an important regulator of plaque formation during focal-adhesion switch. By performing migration assays of wild type cells and cells depleted of Eps15/R, we could correlate cell directionality and velocity with the ability of plaques to read extracellular topographical cues. As such, we have identified the origin and function of plaques during cell migration and we propose that clathrin-coated plaques may act as regulator of unconventional collective migration in a cell-to-cell contact independent manner. This proposal builds on these preliminary findings and aims at shedding lights on the following opened questions: what are the molecular/biophysical signals that drive clathrin recruitment during focal-adhesion switch and cell migration? What are the molecular components that stabilize a clathrin coat into a clathrin-coated plaque? How do plaques regulate cell migration? The specific aims will be:Aim 1: Defining the importance of integrins and topographical cues in regulating plaque formation and stability Aim 2: Function of Eps15/R, from mediating FA switch to regulating cell migrationAim 3: Identification of the molecular determinants regulating plaque formation and stability