Regulation of the Smooth Muscle Cytoskeleton

肌动蛋白细胞骨架在分化的血管平滑肌细胞（dVSMCs）中起着重要作用， 它们与收缩器的活动一致地维持和改变细胞形状。大 许多肌动蛋白结合蛋白（ABP）参与这些过程，其中许多含有多个 结合模块，并进行磷酸化。项目2的基本假设是， 作为细胞信号传导的结果，磷酸化改变了ABPs的肌动蛋白结合特性，从而 允许肌动蛋白细胞骨架的重塑。这一假设将被测试的机械基础， 将研究dVSMC中的重塑及其调节。通过先前演示的方法， 磷酸化依赖的构象变化将使用五种选定的细胞骨架来确定 蛋白质（钙调蛋白、钙调蛋白、coronin、VASP和zyxin）作为模型病例。因此，结构信息 将获得的结果与项目3和项目4的结果进行比较。第二个假设是细胞骨架 蛋白质作为伙伴一起工作，协同维持肌动蛋白细胞骨架结构， 测试.为了支持这一观点，最近开发了一种小鼠模型，其中平滑肌钙调素 基因被破坏，表明许多细胞骨架蛋白表现出降低的水平， 表达沿着平滑肌钙调素的消除。之间的潜在相互作用 caldesmon和这些蛋白质将被检查。新型生物物理工具，如表面等离子体 将使用共振和等温滴定量热法。此外，互动的地点将是 将制备对应于结合的确定的和合成的肽或重组片段 序列的然后将这些肽/片段用作诱饵以测试肽/片段的功能意义。 在原代SMC和分离的dVSMC中的相互作用和磷酸化。细胞骨架的定位 蛋白质以及细胞骨架结构的形态将通过免疫荧光进行检查 和免疫电子显微镜，以及项目1的发现，以深入了解影响 对平滑肌收缩力的影响由于血管平滑肌的收缩控制着血液 压力在我们的身体，血管平滑肌功能障碍导致高血压和其他心血管疾病 疾病从这项研究中获得的信息将有助于开发这些疾病的治疗试剂。

Actin cytoskeleton plays an important role in differentiated vascular smooth muscle cells (dVSMCs) because they maintain and change the cell shape in concert with the activities of the contractile apparatus. A large number of actin-binding proteins (ABPs) participate in these processes, many of which contain multiple binding modules and are subject to phosphorylation. The underlying hypothesis of Project 2 is that phosphorylation alters the actin-binding properties of ABPs as a consequence of cell signaling, and thereby allows for remodeling of the actin cytoskeleton. This hypothesis will be tested the mechanistic basis for such remodeling and its regulation in dVSMCs will be investigated. With previously demonstrated approaches, the phosphorylation-dependent conformational changes will be determined using five selected cytoskeleton proteins (caldesmon, calponin, cortactin, VASP and zyxin) as model cases. The structural information thus obtained will be compared to the results from Projects 3 and 4. A second hypothesis that cytoskeleton proteins work together as partners to synergistically maintain the actin cytoskeleton structures will also be tested. In support of this idea a recently developed mouse model in which the smooth muscle caldesmon gene is disrupted, demonstrated that a number of cytoskeleton proteins exhibited decreased levels of expression along with the elimination of smooth muscle caldesmon. The potential interactions between caldesmon and these proteins will be examined. Novel biophysical tools such as surface plasmon resonance and isothermal titration calorimetry will be used. In addition, the sites of interaction will be determined and synthetic peptides or recombinant fragments will be prepared corresponding to the binding sequences. These peptides/fragments will then be used as decoys to test the functional significance of the interactions and phosphorylation in primary SMCs and in isolated dVSMCs. The localization of cytoskeleton proteins as well as the morphology of the cytoskeleton structure will be examined by immuno-fluorescence and immuno-electron microscopy, together with the findings from Project 1, to gain insights into the effects on smooth muscle contractility. Since the contraction of vascular smooth muscles controls the blood pressure in our body, mulfunction of vascular smooth muscle leads to hypertension and other cardiovascular diseases. Information obtained from this study will help develop therapeutic reagents for these diseases.