Project 4: A Binary Switch in Adhesion Maturation

概览：整合素引发的基质粘附在发育和许多病理过程中起着核心作用，因为它们对于形成和塑造细胞外基质以及决定细胞对基质组成、硬度和施加在基质上的力的变化的反应是必不可少的。这些粘连的可变性导致了模糊的基于形态学的分类，从而阻碍了研究其组成、结构、信号传导特性以及与生物学功能（如突起形态动力学）的关系的努力。粘附动态地演变，使得通常存在连续的中间体。我们的研究整合素和talins的分子连接，基质粘连的中央组织者，导致的假设，两个相互排斥的二元蛋白质-蛋白质相互作用的基础上的中央开关的形成和成熟的整合素为基础的粘连。在这里，我们将测试的想法，这些蛋白质-蛋白质相互作用可以指定两种不同的表型的基质为基础的粘连，这两种粘连类型不同的组成，精细结构，信号传导特性和后果突起形态动力学和力传感。 整合素启动的粘附在机械力传导中的生物学意义。基于整合素的细胞粘附在后生细胞的生物学中起中心作用（1）控制细胞粘附到细胞外基质（ECM）、迁移、生长、分化和凋亡。特别地，这些粘附控制细胞外基质的组装和刚性以及细胞感知刚性的能力（2-4）。同样，来自这些结构的信号有助于细胞发挥和感知外部激烈的能力（5-7）。在以间充质模式迁移的细胞中，活化的整合素与前沿的聚合肌动蛋白相关联，并且在这样做的过程中，在质膜内移动，可能寻找ECM结合位点（8）。新生粘连形成于细胞前部，通常小于光显微镜的分辨率（9）。一些新生的粘连在几分钟内分解（9），其余的生长并成熟为焦点复合物（约0.5 μ m），然后是FA（1-5 μ m）;这种演变是由力驱动的（5，10-13），因此是细胞力感知的核心特征。人们对定义这些粘附剂的分子组成、信号性质以及最终的分子结构感兴趣（14-18）。进展的一个关键障碍是这些粘附的异质性，因为在任何细胞中，存在连续的进化形式，在组成、结构和功能上具有显著的可变性，整合素活化：粘附组装的第一步。大多数整联蛋白以低亲和力状态表达，并且需要细胞活化以足够的亲和力结合ECM以形成粘附。talins与p亚基胞质尾区的结合是wfro（19）和体内（20，21）（22）中整合素活化的最后共同步骤。在纯化的系统中，talin结合足以激活整联蛋白（23），并且已经了解了很多关于talin诱导的激活的细节（24）;在细胞中也需要kindlin（25-30）;然而，kindlin的作用机制仍然未知。新生至成熟ECM粘连的范围包含talin（31），并且talin是粘连形成所需的（32-34）。最近的研究已经开始破译talin整合素的相互作用是如何调节控制整合素激活？ Ras GTP酶是活化过程中的关键信号传导模块（35）。Ras亚家族成员Rapla和Raplb模拟整联蛋白活化（36，37）（38-40）。几种Rapl效应物已经涉及整联蛋白活化（41-43），RIAM（Rapl-GTP相互作用衔接子分子）是Rapl效应物，其是衔接子蛋白的MRL（Mig-10/RIAM/Lpd）家族的成员（42）。RIAM包含Ras结合（RA）和普列克底物蛋白同源（PH）结构域以及富含脯氨酸的区域，这些是MRL蛋白家族的定义特征。RIAM过表达诱导Pl和β 2整合素介导的细胞粘附，RIAM敲低抑制Rapl依赖性白细胞粘附（42），表明RIAM是Rapl依赖性信号传导的下游调节剂。RIAM在造血细胞中大量富集，而Lpd（Lpd）是一种也存在于成纤维细胞和醚体细胞中的paramount（44）。我们利用激动剂不能激活CHO细胞中表达的重组allbB 3的事实，开发了整合素激活途径的合成重建，并将其与正向和反向遗传学结合使用，以剖析整合素激活途径（45）。我们发现，Rap 1诱导形成的整合素激活复合物含有Rap 1效应，RIAM，和塔林，导致塔林招聘质膜和整合素allbB 3。最近，我们进一步重建了这一途径，以证明通过刺激生理相关受体PARI激活allbB 3，并使用双分子荧光互补显示RIAM过表达刺激和RIAM敲低阻断talin在活细胞中向allbB 3募集（46）。此外，图谱研究确定了RIAM和Lpd中结合talin的短两亲性螺旋;将这些螺旋肽与Rap 1的膜靶向序列连接导致最小化的Rap-RIAM模块，该模块足以将talin招募到整联蛋白并激活整联蛋白（47）。因此，RIAM作为一个支架，将Ras GTP酶中的膜靶向序列连接到talin，从而将talin募集到质膜并激活整联蛋白。 总之，这些结果使我们能够构建一个骨架“路线图”，以初始整合素激活（图1）。

Overview: Integrin-initiated matrix adhesions play a central role in development and numerous pathological processes because they are essential for forming and shaping the extracellular matrix and for dictating the responses of cells to variations in matrix composition, stiffness, and to forces imposed on the matrix. The variability of these adhesions has led to ambiguous morphology-based classifications and thus hindered efforts to study their composition, structure, signaling properties, and relationship to biological functions such as protrusion morphodynamics. Adhesions dynamically evolve such that a continuum of intermediates is generally present. Our studies of the molecular connections of integrins and talins, the central organizers of matrix adhesions, have lead to the hypothesis that two mutually-exclusive binary protein-protein interactions underlie a central switch in the formation and maturation of integrin-based adhesions. Here we will test the idea that these protein-protein interactions can specify two distinct phenotypes of matrix-based adhesions, that these two adhesion types differ in composition, fine structure, signaling properties and consequences for protrusion morphodynamics and force sensing. Biological Significance of integrin-initiated Adhesions in Mechanotransduction. Integrin-based cell adhesions play central roles in the biology of metazeans(l) controlling cell adhesion to the extracellular matrix (ECM), migration, growth, differentiation and apoptosis In particular, these adhesions control both the assembly and rigidity of the extracellular matrix and the cell's capacity to sense rigidity(2-4). Similarly, signaling from these structures contributes to the ability of cells to exert and sense external fierce (5-7). In cells migrating in a mesenchymal mode, activated integrins associate with polymerizing actin at the leading edge and, in doing so, move within the plasma membrane, possibly seeking ECM binding sites(8). Nascent adhesions form at the cell anterior, and are generally smaller than the resolution of the light micrescepe(9). Some of the nascent adhesions disassemble within minutes(9), and the remainder grow and mature into focal complexes (-0.5 pm) and then FAs (1-5 pm); this evolution is driven by force(5, 10-13) and is thus a central feature of force-sensing by cells. There has been interest in defining the molecular composition, signaling properties, and ultimately the molecular structure of these adhesiens(14-18). A critical barrier to progress is the heterogeneity of these adhesions since, in any cell, a continuum of evolving forms is present, with marked variability in compositions, structures, and functions, Integrin Activation: A First Step in Adhesion Assembly. Most integrins are expressed in a low affinity state and require cellular activation to bind ECM with sufficient affinity to form adhesions. The binding of talins to the p subunit cytoplasmic tail is a final common step in integrin activation in wfro(19) and in vivo{20, 21) (22). In purified systems, talin binding is sufficient to activate integrins(23) and much has been learned about the details of talin-induced activation(24); in cells there is also a requirement for kindlins(25-30); however, the mechanism of kindlins' actions remains unknown. The spectrum of nascent to mature ECM adhesions contain talin(31) and talin is required for adhesion fermatien(32-34). Recent studies have begun to decipher how the talin-integrin interaction is regulated to control integrin activation? Ras GTPases are critical signaling modules in the activation process (35), The Ras subfamily members, Rapl a and Raplb, simulate integrin activation(36, 37) (38-40). Several Rapl effectors have been implicated in integrin activation(41-43), RIAM (Rapl-GTP-interacting adapter molecule) is a Rapl effector that is a member of the MRL (Mig-10/RIAM/Lpd) family of adaptor proteins(42). RIAM contains Ras association (RA) and pleckstrin homology (PH) domains and proline-rich regions, which are defining features ofthe MRL protein family. RIAM over expression induces pi and 32 integrin-mediated cell adhesion, and RIAM knockdown inhibits Rap1-dependent leukocyte adhesien(42), indicating RIAM is a downstream regulator of Rap1 dependent signaling. Whereas RIAM is greatly enriched in hematopoietic cells, Lpd (Lpd) is a paralogue also present in fibroblasts and ether somatic cells(44). We exploited the fact that agonists fail to activate recombinant allbB3 expressed in CHO cells to develop a synthetic reconstruction of an integrin activation pathway and used it in combination with forward and reverse genetics to dissect a pathway to integrin activation(45). We found that Rap1-induced formation of an integrin activation complex containing the Rap1 effector, RIAM, and talin that lead to talin recruitment to the plasma membrane and to integrin allbB3. More recently, we further reconstructed this pathway to demonstrate activation of allbB3 by stimulation of a physiologically relevant receptor, PARI, and used bimolecular fluorescence complementation to show that RIAM over expression stimulates and RIAM knockdown blocks talin recruitment to allbB3 in living cells(46). Furthermore, mapping studies identified short amphipathic helices in RIAM and Lpd that bind talin; joining those helical peptides to the membrane targeting sequences of Rap1 led to a minimized Rap-RIAM module that was sufficient to recruit talin to integrins and to activate the integrins(47). Thus, RIAM functions as a scaffold that connects the membrane-targeting sequences in Ras GTPases to talin, thereby recruiting talin to the plasma membrane and activating integrins. Together, these results enable us to construct a skeletal "Roadmap" to initial integrin activation (Fig.1).