ACTOMYOSIN SYSTEMS AND CELL MOTILITY

肌动球蛋白系统和细胞运动性

• 批准号: 5213161
• 负责人: VIVIANNE T NACHMIAS
• 金额: --
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/5213161
• 关键词: 3T3 cells; acrosome; actin binding protein; actins; affinity chromatography; cell motility; clone cells; cytoskeleton; human subject; myosins; plasmids; platelet activation; platelets; polymerization; thymosin; transfection; tropomyosin

Our major goal is to understand how the polymerization of actin following platelet stimulation is regulated. Resting platelets contain 280 muM monomeric actin which, as we have recently discovered, is mostly bound to thymosin beta4. In preliminary studies we have found unidentified capping and cutting activities, both calcium insensitive, in supernatants of permeabilized platelets. We will purify and characterize the proteins responsible for these activities. We will measure the total amount of capper(s) and the Kd(s) to determine whether the actin filaments in resting platelets are capped. Fully capped filaments would indicate that the steady state free G-actin concentration is above barbed end critical, so that polymerization could be elicited solely by the generation of free barbed ends. These could be produced by uncapping: we will study whether the cutting protein generates free barbed ends. We have obtained preliminary evidence for nucleating sites at the platelet membrane and will characterize these sites further. Actin polymerization is very rapid: we will determine whether there is a role for profilin, in low as well as high affinity profilin-actin complexes, in increasing the speed of polymerization. Our second goal is to study whether shifting a significant fraction of polymerized actin into the monomer pool alters actin synthesis (i.e. up or down regulation of actin synthesis) and total actin in the cell. Preliminary evidence shows that transfection with a plasmid containing full length cDNA for Tbeta4 is followed by a striking reduction in stress fibers. We will study sustained effects of such transfections. For this purpose we will generate cell lines with increased levels of Tbeta4 using transfection with plasmid containing resistance markers and inducible promoters.

我们的主要目标是了解肌动蛋白的聚合是如何进行的 血小板刺激受到调节。 静息血小板含有 280 muM 正如我们最近发现的，单体肌动蛋白大部分是结合的 到 胸腺素β4。 在初步研究中我们发现了不明 上清液中的加盖和切割活性，对钙不敏感 透化血小板。 我们将纯化并表征蛋白质 负责这些活动。 我们将测量总金额 capper(s) 和 Kd(s) 以确定肌动蛋白丝是否存在 静息血小板被封盖。 完全覆盖的灯丝表明 稳态游离 G-肌动蛋白浓度高于倒刺末端临界值， 因此聚合反应只能通过产生游离的 末端有倒刺。 这些可以通过开盖来生产：我们将研究 切割蛋白是否产生游离的倒刺末端。 我们已经获得了 血小板膜成核位点的初步证据 将进一步描述这些站点的特征。 肌动蛋白聚合非常 快速：我们将确定 profilin 在低浓度下是否有作用 以及高亲和力的 profilin-actin 复合物，在增加 聚合速度。 我们的第二个目标是研究是否转移了很大一部分 聚合肌动蛋白进入单体池会改变肌动蛋白合成（即向上 或肌动蛋白合成的下调）和细胞中的总肌动蛋白。 初步证据表明，转染含有 Tbeta4 的全长 cDNA 后应激显着减少 纤维。 我们将研究此类转染的持续影响。 为了这 目的，我们将使用以下方法生成 Tbeta4 水平增加的细胞系 用含有抗性标记和诱导标记的质粒转染 发起人。