Urokinase anchors uPAR to the actin cytoskeleton.
OBJECTIVE
To investigate the expression and localization of urokinase plasminogen activator (uPA) and its receptor (uPAR) and their interaction with the actin cytoskeleton in human corneal fibroblasts.


METHODS
Primary cultured human corneal fibroblasts were exposed to exogenous uPA to investigate its effect on the distribution of uPAR under resting conditions and in a scrape-wound model. Fluorescence microscopy, immunolocalization, immunoprecipitation, and the actin depolymerizing drug cytochalasin D were used to evaluate uPAR's interaction with the actin cytoskeleton.


RESULTS
uPA/uPAR was immunodetected in large (200 microm2) aggregates devoid of detectable F-actin. However, when uPA was added to corneal fibroblasts before fixation, a dynamic association between uPAR and the actin cytoskeleton was revealed: the uPA/uPAR complex was immunodetected throughout the surface of the plasma membrane in the form of dispersed small aggregates (0.05 microm2). Association of uPAR with actin stress fibers was visualized when FITC-labeled uPA was added to the cells. This codistribution of uPA/uPAR and actin was not detected when the cells were pretreated with the actin-depolymerizing drug, cytochalasin D. uPAR was found also in focal adhesions, the termination points of F-actin, where it colocalized with the integrin alphavbeta3 in cells migrating into a scrape wound. Coimmunoprecipitation experiments confirmed the physical association of uPAR with alphavbeta3 in fibroblasts.


CONCLUSIONS
The authors propose that uPA/uPAR ligation anchors the complex to the actin cytoskeleton and is a part of the mechanism responsible for uPA-induced cell migration in fibroblasts.
Department of Ophthalmology, Monut Sinai School of Medicine, New York, New York 10029-6574, USA. audrey.bernstein@mssm.edu
Audrey M
AM
Bernstein
Roseanne S
RS
Greenberg
Lavinia
L
Taliana
Sandra K
SK
Masur
D000199:Actins / Q000378:metabolism*; D000328:Adult; D002462:Cell Membrane / Q000378:metabolism; D002465:Cell Movement; D002478:Cells, Cultured; D003315:Cornea / Q000378:metabolism*; D015638:Cytochalasin D / Q000494:pharmacology; D003599:Cytoskeleton / Q000378:metabolism*; D005347:Fibroblasts / Q000187:drug effects / Q000378:metabolism* / Q000502:physiology; D022001:Focal Adhesions / Q000378:metabolism; D006801:Humans; D007150:Immunohistochemistry / Q000379:methods; D039302:Integrin alphaVbeta3 / Q000378:metabolism; D008875:Middle Aged; D008954:Models, Biological; D011956:Receptors, Cell Surface / Q000378:metabolism*; D055293:Receptors, Urokinase Plasminogen Activator; D013194:Staining and Labeling; D014018:Tissue Distribution; D014568:Urokinase-Type Plasminogen Activator / Q000378:metabolism* / Q000494:pharmacology
D000199:Actins; D039302:Integrin alphaVbeta3; C524898:PLAUR protein, human; D011956:Receptors, Cell Surface; D055293:Receptors, Urokinase Plasminogen Activator; D015638:Cytochalasin D; D014568:Urokinase-Type Plasminogen Activator