Neurons are the most polarized cells known. Regulated assembly and disassembly of actin filament and microtubule cytoskeletal proteins is fundamental to generation of neuronal polarity and regulation of neuronal growth. Recent developments in imaging technology including fluorescent speckle microscopy (FSM) enable direct visualization of cytoskeletal polymer assembly, disassembly and translocation of in living cells. New algorithms for automated fluorescent feature tracking allow quantitative analysis of protein movements on an image-wide basis, allowing characterization of cytoskeletal system responses. These computational advances also pave the way for mathematical modeling of neuronal motility events. We have been using these techniques to characterize the actin filament and microtubule protein dynamics involved in neuronal growth. Our focus has been on the highly motile structure located at the distal end of developing axonal and dendritic processes called the growth cone. Growth cones regulate the rate and direction of neurite advance by responding to diffusible and/or bound molecular cues in their environment. Cue recognition feeds into signal transduction pathways that ultimately impinge on the final cytoskeletal protein effectors. Retrograde actin flow is a ubiquitous aspect of growth cone motility. It results from constant actin network assembly at the leading edge plus rearward network """"""""pulling"""""""" due to myosin II contractility in a more central region of the growth cone called the T zone. Much recent work has focused on proteins that coordinate the actin filament assembly at the leading edge necessary to sustain retrograde flow. However, actin filament turnover is also necessary to maintain the steady state polymer flux characteristic of retrograde flow. This project is aimed at addressing the problem of actin turnover and network recycling in neuronal growth cones since this important function is not understood. We are motivated by our recent discovery that localized myosin II contractility plays an unexpected role in the steady turnover of polarized actin bundles that comprise filopodia. These actin bundles are assembled at the leading edge, and then transported by retrograde flow into the T zone where they undergo minus end severing and turnover. Our studies suggest that localized myosin II contractility potentiates the activity of a separate actin filament severing factor. We propose to investigate cofilin as an actin bundle recycling candidate and characterize cofilin function elsewhere as well. Although cofilin has been implicated in growth cone function, its mechanism of action in cells is not well understood;indeed, recent biochemical evidence suggests cofilin actions are complex and concentration dependent. To address this outstanding problem, novel molecular imaging assays will be used to correlate actin filament turnover rates directly with actin filament-cofilin interactions in living growth cones. We will also look at cofilin function in the context of acute growth cone advance stimulated by application of permissive cell adhesion molecule substrates.
Neuronal growth cone motility is necessary for axon growth and guidance during development and for regeneration after nerve injury. Growth cone motility depends on the steady assembly and disassembly of actin filament networks. How and where the relevant actin networks assemble is relatively well understood;however, the network disassembly process is a mystery. This project addresses this outstanding problem.
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