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

• 批准号: RGPIN-2014-05064
• 负责人: Janssen, Luke
• 金额: $ 3.42万
• 依托单位: McMaster University
• 依托单位国家: 加拿大
• 项目类别: Discovery Grants Program - Individual
• 财政年份: 2016
• 资助国家: 加拿大
• 起止时间: 2016-01-01 至 2017-12-31
• 项目状态: 已结题
• 来源: https://www.nserc-crsng.gc.ca/ase-oro/Details-Detailles_eng.asp?id=611416
• 关键词: Intracellular; calcium; spikes; trigger; cytoskeletal

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.

细胞内钙峰值触发细胞骨架重组、粘附和迁移 细胞质中钙 ([Ca2+]i) 浓度的变化会触发和/或调节多种细胞反应。我们研究计划的长期愿景是更好地了解细胞如何产生和使用各种形式的 Ca2+ 信号来传达细胞功能。我们之前一直在研究气道和血管平滑肌如何产生重复的 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+-尖峰，发现它们具有完全不同的波形并调节细胞粘附和迁移。显然，Ca2+ 信号的产生及其转导为细胞反应存在复杂的多样性。我们还不了解嗜酸性粒细胞利用 Ca2+ 瞬变来增强与血管内皮壁的粘附并诱导细胞骨架发生深刻变化（从而导致细胞变平和血细胞渗出）的机制。该项目的直接目标是通过以下目标探讨这些问题： 目标#1：嗜酸性粒细胞中的流量/压力引起的 Ca2+-尖峰的性质是什么？一名博士生将使粘附在灌注装置底部的嗜酸性粒细胞受到流量/压力的突然变化，同时监测 [Ca2+]i（共焦荧光测定法）或离子电流（膜片钳电生理学）。我们在气道/血管平滑肌、肺成纤维细胞、肾系膜细胞和 DRG 神经元方面拥有丰富的经验，现在已将其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器。 目标#2：Ca2+-尖峰如何增强细胞粘附并引起细胞骨架变化？第二名博士生将使用我们之前在气道平滑肌中使用的分子生物学和免疫组织化学技术，研究由压力/流量刺激引起的 Ca2+-尖峰产生的细胞骨架变化（粘附、压平和迁移）。学生将重点关注整合素的作用以及它们转导结构变化的效应器（特别是 RhoA 激酶和肌动蛋白聚合）。 目标#3：流量/压力引起的反应是否受嗜酸细胞趋化因子调节？嗜酸性粒细胞趋化因子是将嗜酸性粒细胞募集出循环并导致其迁移的主要刺激物。因此，两名学生都将研究它如何调节 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可以大量免费获得。拟议的研究将产生新的见解，解决细胞环境流变特性产生的 Ca2+ 信号传导事件，以及它们如何协调细胞内的大规模结构重排。这将补充我们正在进行的其他细胞类型中 Ca2+ 信号传导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。