REGULATION OF ACTIN FILAMENT FORMATION IN PHAGOCYTES

中性粒细胞(PMN)和巨噬细胞，也称为吞噬细胞， 必须调节肌动蛋白细丝的组装才能改变形状，摄取 颗粒，释放颗粒，然后爬行到感染部位。一个 运动细胞肌动蛋白组装调控中的关键控制点 是对肌动蛋白细丝的带刺或(+)端的封盖和去封盖。 两种带刺末端封端蛋白可能在控制松材线虫病中起关键作用 将研究吞噬细胞运动过程中的肌动蛋白组装： 1.巨噬细胞封顶蛋白(MCP_：这是一种38 kDa的钙敏感蛋白 覆盖肌动蛋白细丝的带刺末端，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 细胞质总蛋白。使用免疫荧光和共聚焦 显微镜以及免疫金电子显微镜，MCP在 将检查静息和刺激的巨噬细胞。在体内，钙离子- 敏感度将通过研究MCP定位前后进行检查 EGTA/AM可降低细胞内钙离子浓度。为了检查体内的 MCP的作用，人MCP基因将永久性地转染入 单核细胞连体，U937和J774，使用β-肌动蛋白启动子驱动 LK4444向量。转基因细胞的爬行、脱颗粒和 然后将对吞噬作用进行研究。基因组MCP DNA已从一株 人类胎盘基因组文库，目前正在进行测序。MCP 基因被定位在特定的染色体位置。序列分析 结果表明，MCP是明胶蛋白/绒毛蛋白的一员 蛋白质家族。基于一级结构的MCP基因突变研究 通过聚合酶链式反应将其与明胶蛋白和绒毛蛋白进行比较。变种人 MCP蛋白已在大肠杆菌中表达，具有新的功能，即 既能切断又能封盖的。这一成就是 在肌动蛋白结合蛋白领域史无前例，并将允许探索 关于相互关系的 在单体结合、封盖和使用荧光探针切断之间， 吡喃肌动蛋白。重组MCP的X-射线晶体结构分析 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部位的评估。 PMN肌动蛋白聚合抑制剂(Annexin VI)。这个65 kDa的蛋白质 结合膜脂，也覆盖肌动蛋白细丝的带刺末端。 Annexin VI占PMN总蛋白的3%-4%，很可能 在激动剂介导的膜-肌动蛋白相互作用中发挥重要作用。 这种蛋白质隔离肌动蛋白单体和切断肌动蛋白的能力 细丝将使用吡喃肌动蛋白进行研究。共聚焦免疫荧光 将用于在刺激前后将该蛋白定位在PMN中。 膜联蛋白VI的磷酸化将使用免疫沉淀和 P32标记。除了提供对吞噬细胞的更好的理解 运动性，这些研究有望为巨噬细胞提供新的见解 发展，转移癌细胞的行为，控制 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.