Regulation of Contact Inhibition of Locomotion and Collective Cell Migration in tumor cells by a tetrameric JAM - Tetraspanin - αvβ5 integrin complex

细胞迁移需要在细胞的前缘极化形成膜突起，特别是片状脂膜。片脂蛋白的形成是一个高度动态的过程，主要受Arp2/3复合体的调控，Arp2/3复合体触发在原有肌动蛋白细丝的外侧形成新的肌动蛋白细丝，从而产生肌动蛋白细丝的分支网络。当个别迁移的细胞形成新的细胞-细胞接触时，例如在伤口愈合期间，必须停止片状脂血症的形成，这一过程被称为接触运动抑制(CIL)。当细胞以簇的形式迁移时，称为集体细胞迁移(CCM)，板状脂的活动被高度极化，仅限于簇内细胞的前沿(隐层状脂)，而在细胞的侧缘则不存在。这两个过程，CIL和CCM，都指出存在基于细胞-细胞接触的机制，这种机制抑制了结合细胞界面上片状脂膜的形成。我们的工作重点是连接黏附分子(JAMs)。我们已经确定了一个四聚体蛋白复合体，其中JAM-A通过两个四聚体蛋白CD9和CD81与αvβ5整合素相连(JAM-TSPN-αvβ5复合体)。通过同时耗尽CD9和CD81或耗尽JAM-A来破坏该复合体，可以增加c-Src、ERK1/2和ABI1的活性，这些都是调节Arp2/3复合体的信号级联反应的一部分。因此，我们的发现有力地表明，JAM-TSPN-αvβ5复合体对Arp2/3复合体和膜脂活性具有负性调节作用。与此相应，CD9和CD81的同时缺失增加了细胞的运动性，而JAM-A的缺失或表达不定位于细胞连接处的JAM-A突变体导致了细胞的多层生长，使人想起受损的CIL。本研究的目的是详细研究JAM-TSPN-αvβ5复合体在慢性粒细胞白血病和慢性粒细胞白血病中的作用。我们将对JAM-TSPN-αvβ5复合体进行生化表征，并确定该复合体调节Arp2/3复合体的下游靶点。我们将研究它在依赖Arp2/3复合体的过程中在单细胞板脂运动中的作用，包括细胞在2D和3D中的扩散、细胞迁移以及在机械力产生过程中的作用。我们将分析它在CIL过程中对细胞-细胞接触形成反应中层脂膜活性下调的贡献。我们将讨论它在CCM过程中抑制簇内迁移细胞侧缘的片脂活性的可能作用。我们希望描述一种阻止细胞-细胞接触的片脂活动的分子机制，这对胚胎发育、伤口愈合和肿瘤扩散等过程具有重要意义。

Cell migration requires the polarized formation of membrane protrusions, in particular lamellipodia, at the leading edge of the cell. The formation of lamellipodia is a highly dynamic process and is regulated predominantly by the Arp2/3 complex, which triggers the formation of new actin filaments at the lateral sides of preexisting ones thus generating a branched network of actin filaments. When individually migrating cells develop new cell-cell contacts, for example during development of during wound healing, lamellipodia formation must be halted, a process referred to as contact inhibtion of locomotion (CIL). When cells migrate as clusters, called collective cell migration (CCM), lamellipodia activity is highly polarized being restricted to the leading edges of the cells within the cluster (cryptic lamellipodia) but absent at the lateral edges of the cells. Both processes, CIL and CCM, point to the existence of cell-cell contact-based mechanisms that inhibit of lamellipodia formation at the interface of bounded cells. The focus of our work are Junctional Adhesion Molecules (JAMs). We have identified a tetrameric protein complex, in which JAM-A is linked to αvβ5 integrin through the two tetraspanins CD9 and CD81 (JAM-Tspn-αvβ5 complex). Disrupting the complex, either by simultaneous depletion of CD9 and CD81 or by depletion of JAM-A, increases the activities of c-Src, Erk1/2 and Abi1, which are part of a signalling cascade that regulates the Arp2/3 complex. Our findings therefore strongly suggest that the JAM-Tspn-αvβ5 complex negatively regulates the Arp2/3 complex and lamellipodial activity. In accordance with this, simultaneous depletion of CD9 and CD81 increased cell motility, and depletion of JAM-A or expression of a JAM-A mutant that does not localize at cell junctions resulted in multilayered growth of cells reminiscent of impaired CIL. The aim of this study is to characterize the role of the JAM-Tspn-αvβ5 complex during CIL and CCM in detail. We will biochemically characterize the JAM-Tspn-αvβ5 complex and identify the downstream targets through which the complex regulates the Arp2/3 complex. We will study its role in lamellipodia dynamics of single cells during processes that depend on the Arp2/3 complex, including cell spreading, cell migration in 2D and in 3D, and during mechanical force generation. We will analyze its contribution to the downregulation of lamellipodial activity in response to cell-cell contact formation during CIL. And we will address its putative role in inhibiting lamellipodial activity at the lateral borders of cells migrating in a cluster during CCM. We expect to characterize a molecular mechanism that prevents lamellipodial activity at cell-cell contacts, which has strong implications for processes like embryonic development, wound healing and tumor dissemination.