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

四聚 JAM - 四跨膜蛋白 - αvβ5 整合素复合物对肿瘤细胞中运动和集体细胞迁移的接触抑制的调节

• 批准号: 398471960
• 负责人: Professor Dr. Klaus Thomas Ebnet
• 金额: --
• 依托单位: Institut für Medizinische Biochemie
• 依托单位国家: 德国
• 项目类别: Research Grants
• 财政年份: 2018
• 资助国家: 德国
• 起止时间: 2017-12-31 至 2021-12-31
• 项目状态: 已结题
• 来源: https://gepris.dfg.de/gepris/projekt/398471960?language=en
• 关键词: Regulation; Contact; Inhibition; Locomotion; Collective

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.

细胞迁移需要在细胞的前缘极化形成膜突起，特别是板状伪足。片状伪足的形成是一个高度动态的过程，主要受Arp 2/3复合物的调节，Arp 2/3复合物触发在先前存在的肌动蛋白丝的侧面形成新的肌动蛋白丝，从而产生肌动蛋白丝的分支网络。当单独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的发育期间，必须停止板状伪足形成，该过程称为运动的接触抑制（CIL）。当细胞作为群集迁移时，称为集体细胞迁移（CCM），板状伪足活性被高度极化，被限制在群集内细胞的前缘（隐蔽板状伪足），但在细胞的侧缘不存在。这两个过程，CIL和CCM，指出存在的细胞-细胞接触为基础的机制，抑制板状伪足形成的界面结合的细胞。我们的工作重点是连接粘附分子（JAMs）。我们已经鉴定了一种四聚体蛋白复合物，其中JAM-A通过两个四跨膜蛋白CD 9和CD 81与αvβ5整合素连接（JAM-Tspn-αvβ5复合物）。通过同时消耗CD 9和CD 81或通过消耗JAM-A来破坏复合物，增加c-Src，Erk 1/2和Abi 1的活性，它们是调节Arp 2/3复合物的信号级联的一部分。因此，我们的研究结果强烈表明，JAM-Tspn-αvβ5复合物负调节Arp 2/3复合物和板脂醛活性。根据这一点，CD 9和CD 81的同时消耗增加了细胞运动性，并且JAM-A的消耗或不定位于细胞连接处的JAM-A突变体的表达导致细胞的多层生长，这让人联想到受损的CIL。本研究的目的是详细描述JAM-Tspn-αvβ5复合物在CIL和CCM中的作用。我们将对JAM-Tspn-αvβ5复合物进行生物化学表征，并确定复合物调节Arp 2/3复合物的下游靶点。我们将研究它在单细胞的片状伪足动力学过程中的作用，依赖于Arp 2/3复合物，包括细胞扩散，细胞迁移在2D和3D中，并在机械力的产生。我们将分析其贡献的板状脂质体活性的下调，在CIL细胞间接触的形成。我们将讨论它在抑制CCM期间成簇迁移的细胞侧边缘的板脂酶活性方面的假定作用。我们期望表征一种分子机制，该机制可以防止细胞-细胞接触时的片状脂质体活性，这对胚胎发育，伤口愈合和肿瘤传播等过程具有强烈的影响。