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网架，组装成平面和弯曲的晶格。研究了包覆网格蛋白的弯曲凹坑在形成内吞结构中的作用。相反，扁平六边形网格蛋白阵列（也称为空斑）的作用仍然不明确。一些研究表明，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。在准备本提案时，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，前缘的黏附以协调的方式解体。使用活细胞显微镜，我们可以证明这些黏附被网格蛋白包裹的斑块所取代，我们将这一过程称为“黏附切换”。我们可以证明，斑块招募直接发生在局灶黏附对细胞外基质的积极消化之后。利用人造3d微模式或对局灶粘连蛋白水解活性的基因操作，我们可以证明斑块以整合素依赖的方式被招募到局灶粘连产生的3d地形线索上。通过基因沉默，我们发现Eps15/R在病灶-粘附转换过程中是斑块形成的重要调节因子。通过对野生型细胞和Eps15/R缺失的细胞进行迁移实验，我们可以将细胞的方向性和速度与斑块读取细胞外地形信号的能力联系起来。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能以细胞间接触独立的方式作为非常规集体迁移的调节剂。该建议建立在这些初步发现的基础上，旨在阐明以下开放性问题：在聚焦-粘附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？是什么分子成分使网格蛋白外壳稳定成网格蛋白包裹的空斑？斑块如何调节细胞迁移？具体目标将是：目标1：定义整合素和地形线索在调节斑块形成和稳定性中的重要性；目标2:Eps15/R的功能，从介导FA开关到调节细胞迁移；目标3：确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