STUDIES ON AN ACTIN GELLING PROTEIN FROM HELA CELLS

海拉细胞肌动蛋白胶凝蛋白的研究

• 批准号: 3291654
• 负责人: ROBERT R WEIHING
• 金额: $ 12.65万
• 依托单位: CLARK UNIVERSITY (WORCESTER, MA)
• 依托单位国家: 美国
• 财政年份: 1985
• 资助国家: 美国
• 起止时间: 1985-12-01 至 1987-11-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3291654
• 关键词: HeLa cells; crosslink; crystallization; cytoplasm; cytoskeleton; electron microscopy; fluorescence microscopy; gel electrophoresis; histochemistry /cytochemistry; immunochemistry; ion exchange chromatography; physical model; smooth muscle

The applicant has purified a high molecular weight actin binding protein (designated HMWP) from HeLa cells. HMWP is homologous to the filamins and, therefore, may help establish cytoarchitecture in HeLa cells by cross-linking actin filaments in vivo. In situ localization of filamins using electron microscopy has not yet been reported and, therefore, we shall explore the intracellular localization of HMWP using immunocytochemistry at the electron microscopic level. This will allow us to determine if HMWP is actually concentrated in cortical cytoplasm (suggested by our immunofluorescence studies), to study the distribution of HMWP relative to the dense regions of the stress fibers, and to confirm that HMWP is present in stress fibers but absent from microvilli (suggested by our immunofluorescence studies). To study how intrinsic properties of the interaction of HMWP with actin might influence its in situ localization, we shall determine the polarity of the actin filaments in the bundles induced in vitro by HMWP. The observation or random polarity would suggest that HMWP should be present in situ only in structures with a similar organization, such as stress fibers; the observation of a single polarity would suggest that it should also be present in situ in structures containing actin filaments of 1 polarity, such as the microvillus and, therefore that the random polarity of the filaments in stress fibers might be caused by interdigitation of bundles of opposite intrinsic polarity. The applicant recently observed incorporation of HMWP into striated paracrystals in vitro. To determine if this is a general property of the filamins, we shall investigate smooth muscle filamin for the ability to form paracrystals. If it does not, then we shall investigate HeLa cells for the presence of factor(s) that induce incorporation of HMWP as well as filamin into paracrystals. We shall use electron microscopy to investigate how HMWP is incorporated into paracrystals. We shall examine HMWP and paracrystals after rotary shadowing, thin sections of paracrystals, and negatively stained paracrystals after decoration with antibody to HMWP tagged with colloidal gold or ferritin and after controlled proteolysis. These studies will test the hypothesis that HMWP is incorporated into paracrystals by end-to-end association of HMWP molecules and permit mapping of the domain(s) that cause incorporation into paracrystals relative to the actin binding and the dimerization domains that have previously been recognized on other filamins.

申请人已经纯化了一种高分子量肌动蛋白结合蛋白 (命名为HMWP)。HMWP与丝蛋白同源， 因此，可能有助于建立HeLa细胞的细胞结构，通过 体内的交联型肌动蛋白细丝。丝蛋白的原位定位 使用电子显微镜还没有报道，因此，我们 将探索HMWP的细胞内定位 电子显微镜水平的免疫细胞化学。这将使我们能够 要确定HMWP是否确实集中在皮质细胞质中 (由我们的免疫荧光研究建议)，以研究 HMWP相对于应力纤维的致密区域，并确认 HMWP存在于应力纤维中，但不存在于微绒毛中(提示 通过我们的免疫荧光研究)。要研究的内在属性如何 HMWP与肌动蛋白的相互作用可能影响其原位表达 定位，我们将确定肌动蛋白细丝的极性 HMWP体外诱导的丛生组织。观察值或随机极性将 建议HMWP应仅在具有 类似的组织，如应力纤维；观察到的单个 极性表明，它也应该在结构中原位存在 含有1个极性的肌动蛋白细丝，如微绒毛和， 因此，应力纤维中细丝的随机极性可能 是由相反的固有极性的束状物相互交错造成的。 申请人最近观察到HMWP在Striated中的合并 体外培养的准晶体。来确定这是否是 细丝蛋白，我们将研究平滑肌细丝蛋白的能力 形成准晶体。如果没有，那么我们将研究HeLa细胞 对于诱导HMWP掺入的因子(S)的存在以及 丝状物变成了准晶体。我们将使用电子显微镜来研究 HMWP是如何结合到准晶体中的。我们将研究HMWP和 旋转阴影后的准晶、准晶的薄片和 HMWP抗体修饰后阴性的准晶体 用胶体金或铁蛋白标记，经过受控的蛋白质分解。 这些研究将检验HMWP被合并到 通过HMWP分子的端到端缔合形成的准晶并允许作图 导致并入到准晶体中的结构域(S)相对于 肌动蛋白结合和先前已被 在其他细丝上被识别。