REGULATION OF ACTIN FILAMENT FORMATION IN PHAGOCYTES

多形核白细胞（PMN）和巨噬细胞，也称为吞噬细胞， 必须调节肌动蛋白丝的组装，以改变形状，摄取 颗粒，释放颗粒并爬行到感染部位。 一 运动细胞肌动蛋白组装调节的关键控制点 是肌动蛋白丝的倒刺或（+）端的帽化和去帽化。 两个倒钩末端加帽蛋白可能在控制 将研究吞噬细胞运动期间的肌动蛋白组装： 1. 巨噬细胞加帽蛋白（MCP_）：该38 kDa的Ca~（2+）敏感蛋白 覆盖肌动蛋白丝的倒刺末端，但不切断它们。 mcp是 巨噬细胞中最丰富的肌动蛋白结合蛋白，占巨噬细胞的1%， 总细胞质蛋白。 使用免疫荧光和共聚焦 显微镜以及免疫金电子显微镜，MCP的位置， 将检查"静息"和刺激的巨噬细胞。 体内Ca2 +- 通过研究MCP定位前后的敏感性将进行检查 EGTA/AM处理可降低细胞内Ca~（2+）。 为了检查体内 MCP的作用，人MCP cDNA将被永久转染到 单核细胞系u937和J774，使用β-肌动蛋白启动子驱动 IK4444载体。 转染的细胞的爬行、脱髓鞘和 然后研究吞噬作用。 基因组MCP DNA已经从一个 人胎盘基因组文库，目前正在测序。 主控制程序 基因被定位在特定的染色体位点。 序列分析 人MCP cDNA的克隆表明MCP是一个 凝溶胶蛋白/绒毛蛋白 蛋白质家族 基于一级结构的MCP cDNA突变 通过PCR引入了与凝溶胶蛋白和绒毛蛋白的比较。 的突变 MCP蛋白在E.它有一个新的功能， 能够切断和封盖。 这一成就是 这在肌动蛋白结合蛋白领域是前所未有的， 的相互关系 在使用荧光探针的单体结合、加帽和切断之间， 芘基肌动蛋白 重组MCP的X-射线晶体学分析和 切断突变体将补充我们的功能研究， 三级结构和肌动蛋白接触位点的评估。 二. PMN肌动蛋白聚合抑制剂（Annexin VI）。 该65kDa蛋白 结合膜脂并覆盖肌动蛋白丝的倒刺末端。 膜联蛋白VI占PMN中总蛋白的3 - 4%， 在激动剂介导的膜-肌动蛋白相互作用中起重要作用。 这种蛋白质螯合肌动蛋白单体和切断肌动蛋白的能力 将使用芘基肌动蛋白研究细丝。 共聚焦免疫荧光 将被用来定位这种蛋白在中性粒细胞刺激前后。 膜联蛋白VI磷酸化将使用免疫沉淀法进行研究， p32标记。 除了提供对吞噬细胞更好的理解外， 运动，这些研究有望提供新的见解巨噬细胞 发展，转移性癌细胞的行为， 炎症和宿主防御。

Polymorphonuclear leukocytes (PMN) and macrophages, also called phagocytes, must regulate actin filament assembly in order to change shape, ingest particles, release granules and crawl to the sites of infection. A critical control point in the regulation of actin assembly in motile cells is the capping and uncapping of the barbed or (+) ends of actin filaments. Two barbed end capping protein likely to play key roles in the control of actin assembly during phagocyte movement will be investigated: 1. Macrophage capping protein (MCP_: This 38 kDa, Ca2+-sensitive protein caps the barbed ends of actin filaments, but does not sever them. MCP is the most abundant actin-binding protein in macrophages, representing 1% of the total cytoplasmic protein. Using immunofluorescence and confocal microscopy as well as immunogold electron microscopy, MCP's location in "resting' and stimulated macrophages will be examined. In vivo Ca2+- sensitivity will be examined by studying MCP localization before and after intracellular Ca2+ is lowered by EGTA/AM treatment. To examine the in vivo effects of MCP, human MCP cDNA will be permanently transfected into monocyte cell liens, u937 and J774, using the beta-actin promoter driven lK4444 vector. The ability of transfected cells to crawl, degranulate and phagocytose will then be studied. Genomic MCP DNA has been cloned from a human placenta genomic library and is presently being sequenced. The MCP gene is being localized to a specific chromosomal site. Sequence analysis of human MCP cDNA reveals that MCP is a member of the gelsolin/villin protein family. Mutations in MCP cDNA based on primary structural comparisons to gelsolin and villin are being introduced by PCR. A mutant MCP protein has been expressed in E. Coli which has a new function, being capable of severing as well as capping. This accomplishment is unprecedented in the actin-binding protein field and will allow exploration of the interrelationships between monomer binding, capping and severing using the fluorescent probe, pyrenyl actin. X-ray crystallographic analysis of recombinant MCP and the severing mutant will complement our functional studies by allowing assessment of tertiary structure and actin contact sites. II. PMN actin polymerization inhibitor (Annexin VI). This 65kDa protein binds membrane lipids and also caps the barbed ends of actin filaments. Annexin VI represents 3-4% of the total protein in PMN and is likely to play an important role in agonist mediated membrane-actin interactions. The ability of this protein to sequester actin monomers and to sever actin filaments will be studied using pyrenyl actin. Confocal immunofluorescence will be used to localize this protein in PMN before and after stimulation. Annexin VI phosphorylation will be studied using immunoprecipitation and p32 labeling. In addition to providing a better understanding of phagocyte motility, these studies promise to provide new insights into macrophage development, the behavior of metastatic cancer cells, control of inflammation nd host defense.