SHIFTS IN ACTIN-MICROTUBULE FORCES IN THE ENDOTHELIUM

描述：(逐字摘自申请者摘要)：炎症性水肿 由基于细胞骨架的机械力介导，诱导内皮细胞 撤回。增加细胞内cAMP的药物保护内皮 来自炎性介质。了解水肿病的发生机制 药物和cAMP激动剂重塑内皮细胞骨架，进而， 调节屏障功能，是精确开发治疗方法的关键。我们 此前有报道称，水肿剂可独立干扰屏障功能 肌动蛋白-肌球蛋白收缩的表达。肌动蛋白-肌球蛋白的表达 收缩通过影响血管的修复而增加炎性水肿 屏障功能。细胞内cAMP升高不能保护屏障 通过抑制肌动蛋白-肌球蛋白张力的发展发挥作用，但通过 使这种收缩负荷从破坏细胞黏附中解脱出来。少年派，一种新的 研究人员将在人类内皮细胞中测试这一假设 微管通过平衡力调节内皮细胞黏附 有肌动蛋白-肌球蛋白细丝。通过调节微管组装，表达 向心性肌动蛋白-肌球蛋白张力对细胞有不同的影响 粘附力。为了检验这一假设，我们采取了跨学科的方法。 它使用细胞生物学和工程学来解决这个根本问题。在……里面 目的1，我们将测试微管组装的变化是否重塑机械 通过微管和微管之间的承重力的转移而产生的力 肌动蛋白-肌球蛋白成分。在目标2中，我们将定位微管和 肌动蛋白-肌球蛋白力直接作用于细胞-细胞或细胞-基质部位。在《目标3》中， 然后，我们将测试微管组装的变化是否会改变细胞黏附 基于负荷力的变化，以及生理刺激是否 利用这一基本机制。在目标4中，我们将测试另一种假设 微管组装的变化通过串扰改变细胞黏附 通过整合素-配体相互作用进行通讯。我们已经组装了一个 由生物学家、内科医生、物理学家和工程师组成的跨学科小组。 我们将使用跨内皮细胞阻抗的数学模型来定位 细胞黏附的变化。我们将直接测量向心张力和细胞 僵硬。我们将测量肌球蛋白、肌动蛋白和 微管。我们将利用分子方法和定量动力学 在活细胞中的成像方法进一步验证了我们的假设。我们 相信这些研究的结果将极大地促进血管和 细胞生物学。

DESCRIPTION: (Verbatim from Applicant's abstract): Inflammatory edema is mediated by cytoskeletal-based mechanical forces, which induce endothelial-cell retraction. Agents that increase intracellular cAMP protect the endothelium from inflammatory mediators. Understanding the mechanisms by which edemagenic agents and cAMP agonists remodel the endothelial cytoskeleton, which, in turn, regulates barrier function, is critical to precisely develop treatments. We previously reported that edemagenic agents disrupt barrier function independent of expression of actin-myosin contraction. Expression of actin-myosin contraction increases inflammatory edema by impacting the restoration of barrier function. Increased intracellular cAMP does not protect barrier function through inhibition of actin-myosin tension development, but does so by uncoupling this contractile load from disrupting cell adhesion. The PI, a new investigator, will test the hypothesis in human endothelial cells that microtubules modulate endothelial-cell adhesion through a counterbalance force with actin-myosin filaments. By modulating microtubule assembly, the expression of centripetal actin-myosin tension can have differential effects on cell adhesion. To test this hypothesis we have taken an interdisciplinary approach that uses cell biology and engineering to address this fundamental question. In Aim 1, we will test whether changes in microtubule assembly remodel mechanical forces through shifts in load-bearing forces between microtubules and actin-myosin elements. In Aim 2, we will localize whether microtubules and actin-myosin forces are directed at cell-cell or cell-matrix sites. In Aim 3, we will then test whether changes in microtubule assembly alter cell adhesion based on shifts in load-bearing forces, and whether physiological stimuli utilize this basic mechanism. In Aim 4, we will test an alternative hypothesis that changes in microtubule assembly alter cell adhesion through crosstalk communication through integrin-ligand interactions. We have assembled an interdisciplinary group of biologists, a physician, a physicist and engineers. We will use a mathematical model of transendothelial impedance to localize changes in cell adhesion. We will directly measure centripetal tension and cell stiffness. We will measure biochemical changes in myosin, actin and microtubules. We will utilize molecular approaches and quantitative dynamic imaging approaches in living cells to further validate our hypothesis. We believe that the results of these studies will greatly advance vascular and cell biology.