CYSTOLIC-FREE CALCIUM AND CELL MOTILITY

描述[来自申请的逐字记录]：本研究的总体目标是 了解白细胞如何响应外部刺激，并迁移到 感染和炎症。以前的努力一直集中在了解 细胞内游离钙[Ca 2 +i]的变化在调节 粘附相互作用和细胞骨架。结果发现， [Ca 2 +i]的增加是中性粒细胞从 玻连蛋白和纤连蛋白。[Ca 2 +i]-敏感结合这些基质 蛋白质分别通过α v β 3和α 5 β 1整联蛋白。 在正常条件下，发现这两种整联蛋白都是 分布在粘附膜上，梯度在 细胞的前面。这些整合素也存在于内吞囊泡中。当[Ca 2 +i] 瞬时被阻断，整合素被发现在细胞的后部， 粘附膜和内吞囊泡不含整联蛋白。基于 根据这些和其他数据，提出[Ca 2 +i]瞬变需要 从紧密结合中释放α v β 3和α 5 β 1整联蛋白， 释放后，它们被内化并回收到一个 迁移细胞本研究的目的之一是探讨定向 中性粒细胞的再循环数字荧光显微镜，共聚焦 显微镜和电子显微镜将用于检查内吞 中性粒细胞中的循环途径，以及整合素通过这些途径 将详细研究各种途径。基于微管的囊泡的作用 将通过中性粒细胞动力蛋白运动功能的破坏来检查运动 通过过表达和/或细胞质递送， p50-dynamitin的。肌球蛋白II是一种主要的细胞骨架蛋白， [2][3][4][5][6][7][8]肌球蛋白II在2D中性粒细胞迁移中的作用 基板和通过自然的3D矩阵将被检查。使用抗体 针对肌球蛋白II的亲和纯化抗体和针对肌球蛋白II的磷酸化形式的亲和纯化抗体。 肌球蛋白轻链、肌球蛋白II的分布及其在 通过免疫荧光测定各种条件。肌球蛋白II功能 将通过将抑制性肽递送至细胞质而被抑制， 迁移细胞最后，肌球蛋白和定向再循环的作用将是 在通过内皮细胞单层迁移的中性粒细胞中检查， 通过天然的生物基质，类似于 中性粒细胞功能

DESCRIPTION [Verbatim from application]: The overall goal of this study is to understand how leukocytes respond to external stimuli and migrate to sites of infection and inflammation. Previous efforts have been focused on understanding the role of changes in intracellular free calcium [Ca2+i], in regulating adhesive interactions and the cytoskeleton. It was found that transient increases in [Ca2+i] are required for neutrophils to dissociate from vitronectin and fibronectin. The [Ca2+i]-sensitive binding to these matrix proteins is via alpha v beta 3 and alpha 5 beta 1 integrins, respectively. Under normal conditions, it was found that both of these integrins are distributed on the adherant membrane with a gradient that is higher at the front of the cell. These integrins are also in endocytic vesicles. When [Ca2+i] transients are blocked, the integrins are found at the rear of the cells on the adherant membrane, and endocytic vesicles do not contain the integrins. Based on these and other data it was proposed that [Ca2+i] transients are required to release the alpha v beta 3 and alpha 5 beta 1 integrins from tight binding and that after release they are internalized and recycled toward the front of a migrating cell. One aim of the proposed research is to investigate the oriented recycling in migrating neutrophils. Digital fluorescence microscopy, confocal microscopy, and electron microscopy will be used to examine the endocytic recycling pathways in neutrophils, and the passage of integrins through these pathways will be examined in detail. The role of microtubule-based vesicle motors will be examined by disruption of dynein motor function in neutrophils and neutrophil-like HL-60 cells by overexpression and/or cytoplasmic delivery of p50-dynamitin. Myosin II is a major cytoskeletal protein that is activated by increases in [Ca2+i]. The role of myosin II in neutrophil migration on 2D substrates and through natural 3D matrices will be examined. Using antibodies to myosin II and affinity purified antibodies to the phosphorylated form of myosin light chain, the distribution of myosin II and its activation under various conditions will be determined by immunofluorescence. Myosin II function will be inhibited by delivery of inhibitory peptides to the cytoplasm of migrating cells. Finally, the role of myosin and of oriented recycling will be examined in neutrophils migrating through endothelial cell monolayers and through natural biological matrices, which resemble the physiological sites of neutrophil function.