The proper regulation of actin dynamics is essential for many biological processes such as wound healing, immune response and morphogenesis, and plays a significant role in pathological processes such as cancer metastasis and cardiovascular disease, the two leading causes of death in the developed world. Despite its widespread involvement in normal physiology and disease, efforts to target actin dynamics for therapeutic purposes are at an early stage and clearly require a deeper understanding of the processes involved. The Arp2/3 complex is a critical player in actin dynamics that generates branched actin arrays which are thought to be important for many cellular processes including cell migration, phagocytosis and cell adhesion. Using cells derived from a conditional Arp2/3 knockout mouse (Arpc2 gene), we propose to address several important questions for the field of actin dynamics: 1) How are Arp2/3-branched actin networks disassembled and dynamically turned over in cells? We have developed a new optogenetic method to control Arp2/3 function in cells with light that will allow us to dissect the de-branching pathway. 2) Is Arp2/3 required for actin-dependent processes such as directed migration, phagocytosis and cell-cell junction establishment? This will be addressed using a clean, genetic deletion approach in primary cells both ex vivo using live-cell imaging approaches and in vivo using multiphoton intravital imaging. 3) How do cells coordinate Arp2/3 and non-Arp2/3 actin pathways to produce optimal actin dynamics? Using our Arp2/3- deficient cells, we will interrogate the Arp2/3-independent pathways that partially compensate for its loss and study how Arp2/3-dependent and ?independent pathway act in a coordinated pathway to produce optimal actin dynamics in cells.
Cell migration is essential for many physiological processes such as wound healing, immune response and morphogenesis, and plays a significant role in pathological processes such as cancer metastasis and cardiovascular disease, the two leading causes of death in the developed world. Despite its clear importance in normal human health and disease states, no therapeutic treatments directly target this process. This is mainly due to deficiencies in our knowledge about the mechanisms of cell migration. We propose to address some of these deficiencies using a multi-disciplinary approach involving molecular perturbations, microfluidics, cutting-edge microscopy and image analysis. These studies will directly contribute to understanding of disease states such as tumor metastasis, fibrosis and cardiovascular disease, and to our understanding of normal physiological processes such as wound repair.
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