REGULATION OF ACTIN FILAMENT FORMATION IN PHAGOCYTES

多形核白细胞（PMNs）和巨噬细胞需要一个 用于形状变化和运动的收缩装置。 两 纯化的肌动蛋白调节蛋白和完整的吞噬细胞将被 研究这些细胞是如何调节 肌动蛋白丝浓度的变化与它们的 动作 I）从巨噬细胞纯化的肌动蛋白调节蛋白： A）41 K蛋白是Ca 2+敏感的单体螯合剂， 可以在细胞中磷酸化的加帽蛋白。 的 41 K蛋白对肌动蛋白单体和肌动蛋白丝的亲和力 使用荧光探针芘基-肌动蛋白测定末端。 钙离子浓度的变化对神经末梢阻滞和细胞凋亡的影响 将使用Ca/EGTA溶液检查单体螯合， 以及钙电极。 41 K蛋白对Ca 2+的亲和力 将通过Ca 45平衡透析测定。 体内 41 K的磷酸化将使用P32标记的 巨噬细胞和免疫沉淀以及免疫印迹的两个 三维电泳凝胶 在体外的影响 还将检查磷酸化对41 K功能的影响; B） Acumentin是一种非常不稳定的末端封闭蛋白， 使用芘基-肌动蛋白解聚和延伸测定， 确定该蛋白质是否阻断有倒钩的细丝末端; c）Profilin， 肌动蛋白单体螯合蛋白，其也可以直接 与丝相互作用，将与肌动蛋白单体结合， 研究profilin对尖 端部组件。 鲎顶体肌动蛋白束将用于 测量这种蛋白质如何有效地阻止肌动蛋白组装， 有倒钩的细丝末端。 Profilin也以高亲和力 与actin复合。 离子交换和凝胶过滤色谱法 将用于尝试纯化一种或多种辅因子， 调节profilin-actin转换从中度到高度 亲和络合物 II）肌动蛋白丝组装和轮廓蛋白 在完整PMN Triton可溶性提取物中的功能， 聚-L-脯氨酸和DNA酶I-缀合的葡聚糖凝胶珠以及 芘基肌动蛋白将被用来评估的相对重要性， 各种肌动蛋白调节蛋白在控制大肌动蛋白 PMN中发现的单体池。 高亲和力的浓度 前纤维蛋白-肌动蛋白复合物以及肌动蛋白螯合活性将 在趋化刺激之前和之后测量。 最近 有证据表明，profilin可能不能解释高肌动蛋白单体 PMNs中的内容。 我们将寻找其他蛋白质， 螯合肌动蛋白单体，并尝试使用HPLC离子色谱纯化它们 交换层析III）最后，严重的PMN运动 与肌动蛋白组装缺陷相关的疾病，称为 中性粒细胞肌动蛋白功能障碍，将重新研究。 的 这种疾病可能是由一个点突变引起的， β作用基因将通过克隆和测序进行研究 技术. 了解肌动蛋白在吞噬细胞中的功能， 导致更好地了解运动过程在其他 细胞，特别是血小板，内皮和侵袭性肿瘤 细胞 这些知识也可能导致新的发展。 改变白细胞运动并由此改善 增强宿主防御和控制炎症的能力。

Polymorphonuclear leukocytes (PMNs) and macrophages required a contractile apparatus for shape changes and motility. Both purified actin-regulatory proteins and intact phagocytes will be studied to understand of how these cells regulate the dynamic changes in actin filament concentration associated with their movements. I) Actin-regulatory proteins purified from macrophages: A) The 41K protein is a Ca2+-sensitive monomer sequestering and capping protein which may be phosphorylated in the cell. The affinities of 41K protein for actin monomers and actin filament ends will be determined using the fluorescent probe pyrenyl-actin. The effects of changes in ionized calcium on end-blocking and monomer sequestering will be examined using Ca/EGTA solutions as well as a calcium electrode. The 41K protein's affinity for Ca2+ will be determined by equilibrium dialysis with Ca45. In vivo phosphorylation of 41K will be examined using P32 labeled macrophages and immunoprecipitation as well as immunoblots of two dimensional electrophoretic gels. The effects of in vitro phosphorylation on 41K function will also be examined; b) Acumentin, a very labile end blocking protein, will be studied using pyrenyl-actin depolymerization and elongation assays to determine if this protein blocks barbed filament ends; c) Profilin, an actin monomer sequestering protein which may also directly interact with filament, will be combined with actin monomers and barbed-end capped filaments to study profilin's effects on pointed end assembly. Limulus acrosomal actin bundles will be used to measure how effectively this protein can block actin assembly at the barbed filament end. Profilin also exists as a high affinity complex with actin. Ion exchange and gel filtration chromatography will be used to attempt to purify a cofactor or cofactors which may regulate profilin-actin conversion from a moderate to a high affinity complex. II) Actin filament assembly and profilin function in intact PMN Triton-soluble extracts in combination with poly-L-proline and DNAase I-conjugated sephadex beads as well as pyrenyl actin will be used to assess the relative importance of the various actin-modulating protein in controlling the large actin monomer pool found in PMNs. The concentrations of high affinity profilin-actin complex as well as actin sequestering activity will be measured before and after chemotactic stimulation. Recent evidence suggest profilin may no account for the high actin monomer content in PMNs. We will look for other proteins which may also sequester actin monomers and attempt to purify them using HPLC ion exchange chromatography. III) Finally the severe PMN motility disorder associated with defective actin assembly, called neutrophil actin dysfunction, will be reinvestigated. The possibility that this disorder is caused by a point mutation in the beta acting gene will be studied by cloning and sequencing techniques. Understanding how actin functions in phagocytes may result in a better understanding of motile processes in other cells, in particular platelets, endothelial and invasive neoplastic cells. This knowledge may also lead to the development of new methods for altering leukocyte movement and thereby improvement ability to enhance host defense and control inflammation.