Project 4: A Binary Switch in Adhesion Maturation

概述：整合素启动的基质粘连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。这些粘连的多样性导致了基于形态的模糊分类，从而阻碍了对它们的组成、结构、信号特性以及与突起形态动力学等生物学功能的关系的研究。粘合会动态演变，从而通常会出现中间产物的连续体。我们对基质粘连的中心组织者整合素和Talin的分子连接的研究导致了一种假设，即两个相互排斥的二元蛋白质-蛋白质相互作用是基于整合素的粘连形成和成熟的中心开关。在这里，我们将测试这样一种想法，即这些蛋白质-蛋白质相互作用可以指定基于基质的粘连的两种不同的表型，这两种粘连类型在组成、精细结构、信号特性以及对突起形态动力学和力感觉的影响方面不同。 整合素启动的黏附在机械转导中的生物学意义。基于整合素的细胞黏附在后生动物生物学中发挥着核心作用(L)，控制细胞与细胞外基质的黏附、迁移、生长、分化和凋亡，尤其是这些黏附控制细胞外基质的组装和刚性以及细胞对刚性的感知能力(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猛烈的能力(5-7)。在以间充质模式迁移的细胞中，激活的整合素与前沿的聚合肌动蛋白结合，并在这样做时，在质膜内移动，可能寻找细胞外基质结合部位(8)。新生粘连形成于细胞前部，通常小于光镜下的分辨率(9)。一些新生的粘连在几分钟内解体(9)，其余的成长和成熟为焦点复合体(-0.5 PM)，然后是FA(1-5 PM)；这种演变是由力(5，10-13)驱动的，因此是细胞力觉的中心特征。人们一直有兴趣确定这些粘合剂的分子组成、信号特性以及最终的分子结构(14-18)。进展的一个关键障碍是这些粘连的异质性，因为在任何细胞中，都存在着一系列不断演变的形式，在组成、结构和功能上具有显著的多样性，整合素激活：粘连组装的第一步。大多数整合素在低亲和力状态下表达，需要细胞激活才能以足够的亲和力结合ECM形成黏附。在wfro(19)和活体(20，21)(22)中，talins与p亚单位胞浆尾部的结合是整合素激活的最后共同步骤。在纯化的系统中，talin结合足以激活整合素(23)，关于talin诱导的激活的细节已经了解很多(24)；在细胞中也需要kindlins(25-30)；然而kindlins的作用机制仍不清楚。新生到成熟的ECM粘连的谱系包括Talin(31)和Talin是粘连酵母菌所必需的(32-34)。最近的研究已经开始破译Talin-整合素相互作用是如何调节以控制整合素激活的？ RAS GTP酶是激活过程中的关键信号模块(35)，RAS亚家族成员RAP1 a和Raplb模拟整合素激活(36，37)(38-40)。一些RAPL效应器参与整合素的激活(41-43)，RAMM(RAPL-GTP相互作用的适配器分子)是一种RAPL效应器，是MRL(Mig-10/RIAM/LPD)适配器蛋白家族的成员(42)。RIAM包含RAS关联(RA)、Pleckstrin同源(PH)结构域和富含Pro的区域，这些都是MRL蛋白家族的定义特征。RIAM过表达诱导PI和32整合素介导的细胞黏附，RIAM基因敲除抑制RAP1依赖的白细胞黏附(42)，表明RIAM是RAP1依赖信号的下游调节因子。虽然RIAM在造血细胞中非常丰富，但LPD(LPD)是一种类似物，也存在于成纤维细胞和乙醚体细胞(44)。我们利用激动剂无法激活在CHO细胞中表达的重组allbB3这一事实来开发整合素激活途径的合成重建，并将其与正向和反向遗传学相结合来剖析整合素激活途径(45)。我们发现，Rap1诱导形成包含Rap1效应器、RiAM和talin的整合素激活复合体，导致talin募集到质膜和整合素allbB3。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的受体PARI来激活allbB3，并使用双分子荧光互补来表明RIAM的过度表达刺激和RIAM击倒阻止活细胞中talin的招募到allbB3(46)。此外，作图研究在RIAM和LPD中发现了与talin结合的短两亲性螺旋；将这些螺旋肽与Rap1的膜靶向序列连接导致Rap-riam模块最小化，足以将talin招募到整合素并激活整合素(47)。因此，RIAM作为连接Ras GTP酶中膜靶向序列和talin的支架，从而将talin招募到质膜上并激活整合素。 综上所述，这些结果使我们能够构建最初整合素激活的骨架“路线图”(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).