ABSTRACT
To investigate the mechanisms by which adhesions form and disperse in migrating cells, we expressed alpha 5 integrin, alpha-actinin, and paxillin as green fluorescent protein (GFP) fusions. All localized with their endogenous counterparts and did not perturb migration when expressed at moderate levels. alpha 5-GFP also rescued the adhesive defects in CHO B2 cells, which are alpha 5 integrin deficient. In ruffling cells, alpha 5-GFP and alpha-actinin--GFP localized prominently at the leading edge in membrane protrusions. Of the three GFP fusion proteins that we examined, paxillin was the first component to appear visibly organized in protrusive regions of the cell. When a new protrusion formed, the paxillin appeared to remodel from older to newer adhesions at the leading edge. alpha-Actinin subsequently entered adhesions, which translocated toward the cell center, and inhibited paxillin turnover. The new adhesions formed from small foci of alpha-actinin--GFP and paxillin-GFP, which grew in size. Subsequently, alpha 5 integrin entered the adhesions to form visible complexes, which served to stabilize the adhesions. alpha 5-GFP also resided in endocytic vesicles that emanated from the leading edge of protrusions. Integrin vesicles at the cell rear moved toward the cell body. As cells migrated, alpha 5 vesicles also moved from a perinuclear region to the base of the lamellipodium. The alpha 5 vesicles colocalized with transferrin receptor and FM 4-64 dye. After adhesions broke down in the rear, alpha 5-GFP was found in fibrous structures behind the cell, whereas alpha-actinin--GFP and paxillin-GFP moved up the lateral edge of retracting cells as organized structures and then dissipated.
Subject(s)
Actinin/metabolism , Antigens, CD/metabolism , Cell Movement/physiology , Cytoskeletal Proteins/metabolism , Phosphoproteins/metabolism , Actinin/genetics , Animals , Antigens, CD/genetics , Antigens, CD/pharmacology , CHO Cells , Cell Adhesion/drug effects , Cell Adhesion/physiology , Cell Line , Cell Membrane Structures/metabolism , Cricetinae , Cytoskeletal Proteins/genetics , Cytoskeleton/metabolism , Fluorescent Dyes , Green Fluorescent Proteins , Integrin alpha5 , Intracellular Fluid/metabolism , Luminescent Proteins/genetics , Macromolecular Substances , Microscopy, Video , Paxillin , Phosphoproteins/genetics , Protein Transport/physiology , Pseudopodia/metabolism , Pseudopodia/ultrastructure , Receptors, Transferrin/metabolism , Recombinant Fusion Proteins/genetics , Recombinant Fusion Proteins/metabolism , Transport Vesicles/metabolism , Transport Vesicles/ultrastructureABSTRACT
We studied the effects of a single genetic change on a complex mammalian behavior using animals congenic for two variants of Abpa, the gene for the alpha subunit of mouse salivary androgen-binding protein (ABP), in two-way preference tests. Females exhibited a preference for investigating salivas of males of their own genetic type of ABP but not for urines of either type of male. This preference behavior is consistent for samples of mice from geographically diverse populations of Mus musculus domesticus and M. m. musculus. These findings provide an explanation for the observation that this gene is evolving under strong selection.
