Tropomyosin and tyrosine kinases in mechanics of cancer

描述(申请人提供)：我们实验室的主要目标是了解细胞-基质粘连中涉及刚性和力感应的分子途径。在这项资助中，我们将探索这些通路是如何在与原肌球蛋白耗尽和酪氨酸激酶修饰相关的癌症中改变的。癌细胞通常表现出生长的锚定独立性，细胞骨架蛋白原肌球蛋白(TM)在许多癌症中都是耗尽的。此外，恢复正常的Tm1表达会逆转转化的表型。最近，我们的实验室表明，细胞刚性感知依赖于局部收缩单位，局部收缩单位使基质位移50-70 nm，如果迅速超过阈值力，则会形成刚性粘连。局部收缩单位在大小、功能和组成(肌动蛋白、肌球蛋白II、α-肌动蛋白、原调节蛋白和原肌球蛋白)上与肌肉肌节相似(~2�)。在Tm1被敲除后，局部收缩发生了戏剧性的变化，细胞不再感觉到纤维连接蛋白包裹的基质的刚性。同样，酪氨酸激酶的敲除改变了刚性感知和收缩模式，这些模式是通过直径500 nm的PDMS柱子的位移来测量的。我们现在建议跟踪力依赖的时间进程以及黏附和收缩蛋白在柱子上的聚集和消散。这将告诉我们 结合并提供有关收缩和释放循环中所涉及的分子步骤的线索。然后，我们将解决参与刚性感知的酪氨酸激酶(Ax1、ROR2和EGF)如何与早期黏附复合体相互作用的问题，作为复合体上的力的函数。由于原肌球蛋白抑制转化和肿瘤生长，我们将确定原肌球蛋白的耗竭如何改变软表面上的黏附成熟模式，从而使转化成为可能。因此，我们将能够更好地理解转化的机械力化学基础。

DESCRIPTION (provided by applicant): The major goal of our lab is to understand the molecular pathways involved in rigidity and force sensing at cell-matrix adhesions. In this grant we will explore how these pathways are altered in cancers related to depletion of tropomyosin and modifications of tyrosine kinases. Cancer cells typically show anchorage independence of growth and the cytoskeletal protein, tropomyosin (Tm), is depleted in many cancers. Further, restoration of normal Tm1 expression reverses the transformed phenotype. Recently our lab has shown that cell rigidity sensing depends upon local contraction units that displace matrix by 50-70nm and if a threshold force is exceeded rapidly, then rigid adhesions form. Local contraction units resemble muscle sarcomeres in size (~2 �, function, and composition (actin, myosin II, alpha-actinin, tropomodulin and tropomyosin). After knockdown of Tm1, the local contractions are dramatically altered and the cells no longer sense the rigidity of fibronectin-coated substrates. Similarly, the knockdown of tyrosine kinases alters both rigidity sensing and the pattern of contractions that are measured from displacements of 500nm diameter PDMS pillars. We propose now to follow the time course of force dependence and the concentration and dissipation of adhesion and contractile proteins at the pillars. This will tell us the order of binding and provide clues about the molecular steps involved in the cycles of contraction and release. We will then address the question of how the tyrosine kinases involved in rigidity sensing (AXL, ROR2 and EGF) interplay with the early adhesion complexes as a function of the force on the complexes. Since tropomyosin inhibits transformation and tumor growth, we will determine how tropomyosin depletion alters the pattern of adhesion maturation on soft surfaces that enables transformation. Thus, we will be able to better understand the mechanochemical basis of transformation.