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.
| Identifer | oai:union.ndltd.org:MIT/oai:dspace.mit.edu:1721.1/28600 |
| Date | January 2004 |
| Creators | Osborn, Eric A. (Eric Alan), 1975- |
| Contributors | C. Forbes Dewey., Harvard University--MIT Division of Health Sciences and Technology., Harvard University--MIT Division of Health Sciences and Technology. |
| Publisher | Massachusetts Institute of Technology |
| Source Sets | M.I.T. Theses and Dissertation |
| Language | en_US |
| Detected Language | English |
| Type | Thesis |
| Format | 142 leaves, 7801872 bytes, 7820724 bytes, application/pdf, application/pdf, application/pdf |
| Rights | M.I.T. theses are protected by copyright. They may be viewed from this source for any purpose, but reproduction or distribution in any format is prohibited without written permission. See provided URL for inquiries about permission., http://dspace.mit.edu/handle/1721.1/7582 |
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