Intracellular calcium spikes trigger cytoskeletal reorganization, adhesion and migration

细胞内钙峰值触发细胞骨架重组、粘附和迁移多种细胞反应由胞质钙（[Ca2+]i）浓度的变化触发和/或调节。我们研究计划的长期愿景是更好地了解细胞如何创造和使用各种形式的Ca2+信号来交流细胞功能。我们之前一直在研究气道和血管平滑肌如何产生反复出现的Ca2+波，其频率调节收缩功能。三年前，我们开始研究成纤维细胞中的Ca2+波，发现它们可以调节基因表达。最近，我们实验室的一名学生开始观察嗜酸性粒细胞中的Ca2+峰值，发现它们具有完全不同的波形，并调节细胞粘附和转运。显然，Ca2+信号的产生及其转导成细胞反应具有复杂的多样性。我们还不了解嗜酸性粒细胞利用Ca2+瞬态来加强与血管内皮壁的粘附并诱导导致细胞变平和渗出的深刻的细胞骨架变化的机制。这个项目的直接目的是通过以下目标来探索这些问题：目标#1：嗜酸性粒细胞中由流动/压力引起的Ca2+尖峰的性质是什么？一名博士生将在监测[Ca2+]i（共聚焦荧光法）或离子电流（膜片钳电生理学）的同时，将粘附在灌注仪底部的嗜酸性粒细胞置于流量/压力的突然变化中。我们对这两种技术在气道/血管平滑肌、肺成纤维细胞、肾系膜细胞和DRG神经元上都有丰富的经验，现在已经将它们应用于嗜酸性粒细胞（图1-3）。同时，视频显微测量软件跟踪视野内细胞的运动和形状变化。药理学和遗传探针将用于识别产生和转导这些反应的效应物。目标#2:Ca2+尖峰如何加强细胞粘附并引起细胞骨架变化？另一名博士生将使用分子生物学和免疫组织化学技术研究压力/流量刺激引起的Ca2+峰值产生的细胞骨架变化（粘附、扁平和迁移），我们之前在气道平滑肌中使用过这种技术。学生将专注于整合素的作用和通过它们传导结构变化的效应物（特别是RhoA激酶和肌动蛋白聚合）。目的#3：流量/压力诱导的反应是否由eotaxin调节？Eotaxin是将嗜酸性粒细胞从循环中招募出来并使其迁移的主要刺激物。因此，两位学生将研究它如何调节Ca2+尖峰和细胞骨架的变化。我们将使用猪嗜酸性粒细胞，因为它们在当地屠宰场大量免费供应。提出的研究将产生新的见解解决Ca2+信号事件产生的细胞环境的流变特性，以及它们如何协调细胞内大规模的结构重排。这将补充我们正在进行的Ca2+信号在其他细胞类型响应配体介导的输入，并导致基因表达和主动收缩等多种功能的研究。

INTRACELLULAR CALCIUM SPIKES TRIGGER CYTOSKELETAL REORGANIZATION, ADHESION AND MIGRATION A diverse array of cellular responses are triggered and/or regulated by changes in the cytosolic concentration of calcium ([Ca2+]i). The long-term vision of our research program is to better understand how cells create and use various forms of Ca2+-signals to communicate cell function. We had previously been studying how airway and vascular smooth muscle generate recurring Ca2+-waves, the frequency of which regulate contractile function. Three years ago, we commenced studies of Ca2+-waves in fibroblasts, finding them to modulate gene expression. Most recently, a student in our laboratory began to look at Ca2+-spikes in eosinophils, finding those to have an entirely different wave-form and to modulate cell adherence and transmigration. Clearly there is a complex diversity in the generation of Ca2+-signals and their transduction into a cellular response. We do not yet understand the mechanisms by which eosinophils use Ca2+-transients to strengthen adhesion to the vascular endothelial wall and to induce the profound cytoskeletal changes which result in cell flattening and diapedesis. The immediate aim of this project is to explore those questions through the following Objectives: Objective #1: what is the nature of the Ca2+-spike which is evoked by flow/pressure in eosinophils? One PhD student will subject eosinophils adhered to the bottom of a perfusion apparatus to sudden changes in flow/pressure while monitoring [Ca2+]i (confocal fluorimetry) or ionic currents (patch-clamp electrophysiology). We have extensive experience with both techniques in airway/vascular smooth muscle, pulmonary fibroblasts, renal mesangial cells, and DRG neurons, and have now adapted them for eosinophils (Figs. 1-3). At the same time, videomicrometry software tracks the movement and shape changes of the cells within the field-of-view. Pharmacological and genetic probes will be used to identify the effectors which produce and transduce those responses. Objective #2: how does the Ca2+-spike strengthen cell adhesion and cause the cytoskeletal changes? A second PhD student will study cytoskeletal changes (adhesion, flattening and migration) produced by the Ca2+-spikes evoked by pressure/flow stimuli, using molecular biological and immunohistochemical techniques that we previously used in airway smooth muscle. The student will focus on the roles of integrins and the effectors through which they transduce the structural changes (esp. RhoA kinase and actin polymerization). Objective #3: is the flow/pressure-induced response modulated by eotaxin? Eotaxin is the primary stimulus which recruits the eosinophils out of the circulation and causes them to migrate. Both students will therefore examine how it modulates the Ca2+-spike and cytoskeletal changes. We will use porcine eosinophils, as they are freely available in large quantity at a local abattoir. The proposed research will yield novel insights addressing Ca2+-signalling events produced by the rheological properties of the cellular environment, and how they co-ordinate wide-scale structural rearrangements within the cells. This will complement our on-going studies of Ca2+-signaling in other cell types responding to ligand-mediated input and resulting in functions as diverse as gene expression and active contraction.