Intracellular calcium spikes trigger cytoskeletal reorganization, adhesion and migration

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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.