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如何关注 调节血小板刺激。 静止的血小板包含280个妈妈 正如我们最近发现的那样，单体肌动蛋白主要是绑定的 到 胸腺素β4。 在初步研究中，我们发现身份不明 上清液中的钙不敏感的封盖和切割活动 透化血小板。 我们将纯化并表征蛋白质 负责这些活动。 我们将衡量总数 卡珀（S）和KD（S）确定是否在 静止的血小板被封顶。 完全封闭的细丝表明 稳态无g-肌动蛋白的浓度在刺的末端临界， 因此，仅通过自由而引起聚合可以引起聚合 带刺的末端。 这些可能是通过解开而产生的：我们将研究 切割蛋白是否会产生自由的刺末端。 我们已经获得了 血小板膜上成核位点的初步证据和 将进一步表征这些站点。 肌动蛋白聚合非常 快速：我们将确定profilin是否有作用 以及高亲和力profilin-actin复合物，在增加 聚合的速度。 我们的第二个目标是研究是否转移很大一部分 聚合肌动蛋白进入单体池会改变肌动蛋白的合成（即 或下调肌动蛋白合成）和细胞中总肌动蛋白。 初步证据表明，用含有质粒的转染 TBETA4的全长cDNA之后，应力减少了 纤维。 我们将研究此类转染的持续影响。 为了这 目的我们将使用使用TBETA4级别的细胞系生成细胞系 用含有电阻标记的质粒转染和诱导 发起人。