Dynamics of Alpha5 Integrin, Paxillin, and Alpha-Actinin During Formation and Disassembly of Adhesions in Migratory Cells
Laukaitis, Christina Marie
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Permalink
https://hdl.handle.net/2142/86632
Description
Title
Dynamics of Alpha5 Integrin, Paxillin, and Alpha-Actinin During Formation and Disassembly of Adhesions in Migratory Cells
Author(s)
Laukaitis, Christina Marie
Issue Date
2001
Doctoral Committee Chair(s)
Horwitz, Alan F.
Department of Study
Microbiology
Discipline
Microbiology
Degree Granting Institution
University of Illinois at Urbana-Champaign
Degree Name
Ph.D.
Degree Level
Dissertation
Keyword(s)
Biology, Cell
Language
eng
Abstract
In this study, I have probed the mechanisms of eukaryotic cell migration by fusing the genes encoding proteins involved in cell migration to the gene for a green fluorescent protein, GFP. I ascertained that the novel fusion protein expressed by cells transfected with alpha5-GFP is correctly localized, is surface-expressed in a heterodimeric form, and restores the ability of a cell line void of alpha5 expression to adhere and to spread on a fibronectin substrate. By viewing and comparing images taken from individual live cells at numerous stages of the migration cycle, I have characterized the role of the alpha5 integrin, paxillin, and alpha-actinin in the migration process. The alpha5 integrin as well as the actin-binding protein alpha-actinin are membrane-localized in actively protruding regions of the cell. The alpha5 integrin departs from protrusive regions, as well as from retracting regions of the cell, in vesicular structures. These vesicles co-localize with markers for endosomal recycling pathway, including the dye FM 4-64 and transferrin receptor staining. Paxillin organizes into dynamic complexes in areas of membrane activity and paxillin from older complexes dissipates in close temporal coordination with the formation of new ones. alpha-actinin binding stabilizes these dynamic adhesive structures and, when the alpha5 integrin joins them, centripetal motion ceases. Adhesion breakdown occurs between integrins and the intracellular proteins. Integrins are seen on the substrate behind areas of cell retraction, while paxillin and alpha-actinin move along the cell edge in areas of cell retraction. Observing cell migration in a chicken embryo slice system has allowed comparison of cell migration within an organism to the process commonly studied in tissue culture cells. Cell migration in vivo is similar to the in vitro process, except that each step is exaggerated in a cell migrating in an in vivo environment. Likewise, GFP-fused adhesion proteins localize similarly in the chicken slice system and in tissue culture cells.
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