REGULATION OF ACTOMYOSIN DEPENDENT FUCTIONS IN CELLS

细胞内肌动球蛋白依赖性功能的调节

• 批准号: 2702294
• 负责人: KRISTINE E KAMM
• 金额: $ 32.23万
• 依托单位: UT SOUTHWESTERN MEDICAL CENTER
• 依托单位国家: 美国
• 财政年份: 1996
• 资助国家: 美国
• 起止时间: 1996-05-01 至 2000-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2702294
• 关键词: 3T3 cells; Adenoviridae; SDS polyacrylamide gel electrophoresis; actins; antisense nucleic acid; calponin; cell cycle; chemotaxis; collagen; complementary DNA; fibroblasts; laboratory rabbit; muscle contraction; myosin light chain kinase; myosins; phosphoproteins; respiratory muscles; smooth muscle; vascular smooth muscle; western blottings

The goals of experiments described in this proposal are to define mechanisms by which actomyosin-dependent contractile and motile functions are regulated in smooth and non-muscle cells. The proposed work will test hypotheses with regard to thick filament (myosin regulatory light chain (RLC) phosphorylation-dependent and independent activation) and thin filament (calponin) regulation. In smooth muscle tissues, RLC phosphorylation by Ca2+/CaM-dependent myosin light chain kinase (MLCK) is sufficient for force development or shortening; it has also been implicated in fibroblast contraction. Steady-state isometric force is linearly dependent upon the extent of RLC phosphorylation; however, the slope of this relation may change with different modes of stimulation. Regulation collateral to RLC phosphorylation may arise by activation of non-phosphorylated cross bridges or the action of the thin filament protein, calponin, which inhibits actin-activated myosin MgATPase in vitro in a Ca2+/CaM- and phosphorylation-reversible manner. FIRST, we will establish the dependence of isometric force on RLC phosphorylation in cultured aortic and tracheal smooth muscle cells and fibroblasts reconstituted in collagen gels. Conditions under which collateral regulation is maximal will be defined in these models. These experiments will indicate the relative extent of collateral regulation in smooth muscle as compared to fibroblast cells and will define optimal conditions for activation of non-phosphorylated cross bridges. SECOND, we will test the hypothesis that RLC phosphorylation is essential for contraction in smooth muscle cells and/or fibroblasts. Cultured smooth muscle cells and fibroblasts will be infected with adenovirus carrying cDNA sequences coding for 1) catalytically inactive MLCK (dominant negative), 2) antisense RNA to MLCK (enzyme depletion), 3) a non-phosphorylatable RLC (T18A/S19A), or 4) a combination of these. In addition, MLCK null mutant 3T3 fibroblasts will be generated by gene targeting through homologous recombination. After reconstituting treated cells into collagen gels, force, shortening and RLC phosphorylation will be measured in response to agents defined in Aim 1 as eliciting collateral regulation. These experiments will indicate whether non-phosphorylated cross bridges are capable of cycling. Comparison of results with overexpression of inactive enzyme to those with enzyme depletion will allow us to test the hypothesis that MLCK plays a structural role, in addition to its catalytic role. THIRD, MLCK-deficient cells generated as described in Specific Aim 2 will be evaluated for rates of cell migration (chemotaxis), rates of cellular proliferation (cytokinesis), and rates of cell attachment. These experiments will elucidate presently less well-defined functions for RLC phosphorylation in smooth muscle as well as non-muscle cells. FOURTH, the responses to calponin overexpression in smooth muscle cells and fibroblasts will be investigated in terms of force, cell migration, attachment, and proliferation. We will test the hypothesis that calponin depresses RLC-dependent motile responses.

本提案中描述的实验目标是定义 肌动蛋白依赖性收缩和运动功能的机制 在光滑和非肌肉细胞中调节。 拟议的工作将测试 关于厚丝的假设（肌球蛋白调节轻链 （RLC）磷酸化依赖性和独立激活）和薄 细丝（Calponin）调节。 在平滑肌组织中，RLC Ca2+/CAM依赖性肌球蛋白轻链激酶（MLCK）的磷酸化为 足以发育或缩短；也一直 与成纤维细胞收缩有关。 稳态等距为 线性依赖于RLC磷酸化的程度；但是， 这种关系的斜率可能会随着不同的刺激方式而变化。 可能通过激活而产生与RLC磷酸化的负责。 非磷酸化跨桥或细丝的作用 蛋白质，钙蛋白酶，在体外抑制肌动蛋白激活的肌球蛋白MGATPase 以CA2+/CAM和磷酸化 - 可逆的方式。 首先，我们会的 建立等距力对RLC磷酸化的依赖性 培养的主动脉和气管平滑肌细胞和成纤维细胞 在胶原蛋白凝胶中重构。 抵押品的条件 在这些模型中将定义最大调节。 这些实验 将指示平滑中侧外调节的相对程度 与成纤维细胞相比，肌肉将定义最佳条件 用于激活非磷酸化的跨桥。 第二，我们将测试 RLC磷酸化对于收缩至关重要的假说 平滑肌细胞和/或成纤维细胞。 培养的平滑肌细胞和 成纤维细胞将被携带cDNA序列的腺病毒感染 编码1）催化无效的MLCK（主要负），2） 反义RNA至MLCK（酶耗竭），3）不可磷酸的RLC （T18A/S19A）或4）这些组合。 另外，MLCK无效突变体 3T3成纤维细胞将通过基因靶向通过同源物产生 重组。 将处理的细胞重构为胶原蛋白凝胶后， 将测量力，缩短和RLC磷酸化。 AIM 1中定义为引发侧支调节的代理。 这些 实验将指示非磷酸化的跨桥是否是 能够骑自行车。 比较结果与非活性的过表达 酶耗竭的酶将使我们能够检验该假设 除了其催化作用外，MLCK还起着结构性的作用。 第三，如特定目标2中所述生成的MLCK缺陷细胞将 评估细胞迁移率（趋化性），细胞速率 增殖（细胞因子）和细胞附着速率。 这些 实验将阐明当前RLC定义明确的功能 平滑肌和非肌肉细胞中的磷酸化。 第四， 平滑肌细胞中对钙蛋白过表达的反应 成纤维细胞将以力，细胞迁移为角度进行研究 依恋和扩散。 我们将检验钙蛋白酶的假设 抑制依赖RLC的运动响应。