Protrusion of the leading edge is the first step in directed cell migration, a critical process in both cancer metastasis and normal development. It requires polymerization of actin filaments (F-actin) at the leading edge plasma membrane in exquisite balance with both network adhesion and contraction, all under the control of regulatory signals. The long term goal of my research program is to create systems-level models of the integration of these mechanical and chemical pathways as a non-steady state and spatially distributed process. The project proposed here is based on our discovery of two dynamically, molecularly, and functionally distinct but spatially overlapping F-actin networks, named lamella (La) and lamellipodium (Lp) (Ponti et al, Science 2004). The central hypothesis of this proposal suggests that cell protrusion is driven by elongation of actin filaments in the contractile La network which engages with adhesions and spans the region between the cell body and the leading edge. The Lp is a narrow, fast treadmilling F-actin network that assembles off of La-filaments via Arp2/3 and cofilin activity. However, according to our hypothesis the Lp only makes minor contributions to the generation of propulsive forces itself, but rather modulates the dynamics of La filament assembly. Thus, our model defines the Lp as a regulatory organelle of cell protrusion. To test this prediction we will integrate quantitative high-resolution live cell microcopy and image data driven modeling approaches to achieve three Specific Aims: 1.) Test the hypothesis that Lp F-actin assembles off elongating La filaments and transiently dissociates under the competing activities of tropomyosin and cofilin; 2.) Test the hypothesis that La F-actin assembly is coupled to La network contraction via RhoA signals. 3.) Test the hypothesis that Lp dynamics modulate La-mediated cell protrusion in both EGF-stimulated and random cell migration. The cell biological deliverable of this project is the deconvolution of several mechanochemical pathways by systematic in situ analysis of their modes of action and timing during cell protrusion. The technological deliverables will be a unique and robust methodology to probe the dynamic interaction of two protein assemblies and their relationship to intracellular forces in live cell analyses. Second, the non-steady state measurements provided by these methods will initiate novel numerical modelling to recapitulate the non-linear dynamics of cell protrusion. ? ? ?
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