REGULATION OF ACTIN FILAMENT FORMATION IN PHAGOCYTES

中性粒细胞和巨噬细胞需要 用于形状变化和运动的收缩装置。两者都有 纯化的肌动蛋白调节蛋白和完整的吞噬细胞 研究了解这些细胞是如何调节动态的 肌动蛋白细丝浓度的变化与其相关 动静。I)从巨噬细胞中纯化的肌动蛋白调节蛋白： A)41K蛋白是一种钙离子敏感的单体隔离和 可能在细胞内被磷酸化的封端蛋白。这个 41K蛋白与肌动蛋白单体和肌动蛋白细丝的亲和力 末端将使用荧光探针Py renyl-Actin来确定。 钙离子变化对终末阻断和血管紧张素转换酶的影响 单体隔离将使用Ca/EGTA溶液进行检查 和钙电极一样。41K蛋白与钙离子的亲和力 用Ca45平衡透析法测定。活体内 41K的磷酸化将用P32标记进行检测 巨噬细胞和免疫沉淀以及两者的免疫印迹 立体电泳胶。体外效应的研究 41K功能的磷酸化也将被检测；b) Acumentin是一种非常不稳定的末端阻断蛋白，将被研究 使用吡喃-肌动蛋白解聚和伸长率测定 确定该蛋白质是否阻止了带刺的细丝末端；c)Profilin， 一种肌动蛋白单体隔离蛋白，也可以直接 与细丝相互作用，将与肌动蛋白单体结合 研究Profilin对尖端有刺细丝的影响 结束装配。海带顶体肌动蛋白束将用于 测量这种蛋白质在多大程度上阻止肌动蛋白组装 带刺的花丝末端。Profilin也作为一种高亲和力存在 肌动蛋白复合体。离子交换和凝胶过滤层析 将用于尝试提纯一个或多个辅因子，该辅因子可以 将Profilin-肌动蛋白的转化从中等水平调整为高水平 亲和力情结。二)肌动蛋白细丝组装和轮廓分析 在完整的PMN Triton可溶性提取物中的作用 多聚L-脯氨酸和脱氧核糖核酸酶I标记的葡聚糖凝胶小球 将使用吡喃肌动蛋白来评估 多种肌动蛋白调节蛋白对大肌动蛋白的调控 在PMN中发现单体池。高亲和力的浓度 Profilin-肌动蛋白复合体以及肌动蛋白隔离活性将 在趋化刺激前后进行测量。近期 有证据表明，Profilin可能不是肌动蛋白单体含量高的原因 PMN中的内容。我们将寻找其他蛋白质，也可能 分离肌动蛋白单体并尝试用高效液相色谱离子进行纯化 交换层析。三)最后是严重的中性粒细胞运动 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，将重新调查。这个 这种疾病可能是由基因突变引起的 将通过克隆和测序来研究β作用基因 技巧。了解肌动蛋白在吞噬细胞中的功能可能 结果更好地理解了其他 细胞，特别是血小板、内皮细胞和侵袭性肿瘤 细胞。这一认识也可能导致新的 改变白细胞运动并由此改进的方法 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.