Actin remodeling in motile cells

Thesis (Ph. D.)--Harvard-MIT Division of Health Sciences and Technology, 2004.Includes bibliographical references.Non-muscle cell shape change and motility depend primarily on the dynamics and distributions of cytoplasmic actin. In cells, actin cycles between monomeric and polymeric phases tightly regulated by actin binding proteins that control cellular architecture and movement. Here, we characterize actin remodeling in shear stress stimulated endothelial cells and in actin networks reconstituted with purified proteins. Fluid shear stress stimulation induces endothelial cells to elongate and align in the direction of applied flow. Alignment requires 24 h of exposure to flow, but the cells respond within minutes to flow by diminishing their movements by 50%. Although movement slows, actin filament turnover times and the amount of polymerized actin in cells decreases, increasing actin filament remodeling in individual cells composing a confluent endothelial monolayer to levels used by disperse, non-confluent cells for rapid movement. Hours later, motility returns to pre-shear stress levels, but actin remodeling remains highly dynamic in many cells. We conclude that shear stress initiates a cytoplasmic actin remodeling response that is used to modify endothelial cell shape instead of bulk cell translocation. We determine the steady state dynamics of purified actin filament networks in the entangled state and after orthogonal cross-linking with filamins using a novel, non-perturbing fluorescence system. Human filamin A or Dictyosteliun discoidium filamin slow actin filament turnover by [approximately] 50% and recruit much of a significant population of actin oligomers that we measure are present in polymerized purified actin solutions into the immobile filament fraction. Surprisingly, these observations occur at very low stoichiometry to actin, approximately requiring only one(cont.) filamin molecule bound per actin filament, similar to the amount required for actin filament gelation in vitro. Networks formed with filamin truncates localize this activity to the actin binding domain and reveal that dimerization and orthogonal cross-linking are not required for dynamic stabilization. Re-expression of filamin A with or without the actin binding domain in human melanoma cells that naturally lack this protein support the findings in purified actin networks. These results indicate that filamin cross-linking stabilizes filament dynamics by, slowing filament subunit cycling rates and by either decreasing spontaneous filament fragmentation or promoting filament annealing.by Eric A. Osborn.Ph.D

The dynamics and regulation of actin filaments in vascular endothelial cells and in a reconstituted purified protein system

Thesis (S.M.)--Massachusetts Institute of Technology, Dept. of Mechanical Engineering, 2001.Includes bibliographical references.This electronic version was submitted by the student author. The certified thesis is available in the Institute Archives and Special Collections.Cell motility and shape change are complex processes that depend primarily on the cytoplasmic dynamics and distribution of actin monomer and polymer. Proteins that regulate actin cycling control cellular architecture and movement. One method to measure parameters that characterize actin dynamics is photo activation of fluorescence (PAF), which can simultaneously estimate the fraction of total actin polymerized (PF) and the lifetime of actin filaments (t). By deciphering the relationships between actin dynamics and regulatory proteins, the complicated motions of cells and biological consequences of these movements can be better understood. In purified actin solutions at steady-state, actin filament dynamics can be analyzed with PAF at long times following photoactivation. By increasing the width of the photoactivated band, actin filament turnover (t ~ 8 hours) can be distinguished from actin filament diffusion. Proteins believed to stabilize actin filaments against depolymerization markedly slow actin filament turnover in wide photoactivated bands (t ~ 65 hours). Decreasing the band width causes photoactivated fluorescence to decay more rapidly (t ~ 3 hours) due to a combination of actin filament diffusion and turnover. Addition of actin binding protein forms crosslinked actin gels that hinder filament diffusion and slow filament turnover (t ~ 12 hours) in narrow photoactivated bands. Endothelial cells decrease t and PF in order to accelerate their migration speed, consistent with mechanisms attributed to ADF/cofilin in vitro. Removal of gelsolin in fibroblasts produces a similar correlation between motility, t, and PF. Consistent with increased actin filament severing, fast-moving endothelial cells have an increased number of short actin filaments and more uncapped barbed ends, but paradoxically bind less cofilin. A mechanism of increasing endothelial cell motility is proposed that relies on actin filament severing to create uncapped pointed ends for ADF/cofilin-mediated depolymerization.by Eric A. Osborn.S.M