Distinct temporal-spatial roles for rho kinase and myosin light chain kinase in epithelial purse-string wound closure.
OBJECTIVE
Small epithelial wounds heal by purse-string contraction of an actomyosin ring that is regulated by myosin light chain (MLC) kinase (MLCK) and rho kinase (ROCK). These studies aimed to define the roles of these kinases in purse-string wound closure.


METHODS
Oligocellular and single-cell wounds were created in intestinal epithelial monolayers. Fluorescence imaging and electrophysiologic data were collected during wound closure. Human biopsies were studied immunohistochemically.


RESULTS
Live-cell imaging of enhanced green fluorescent protein-beta-actin defined rapid actin ring assembly within 2 minutes after wounding. This progressed to a circumferential ring within 8 minutes that subsequently contracted and closed the wound. We therefore divided this process into 2 phases: ring assembly and wound contraction. Activated rho and ROCK localized to the wound edge during ring assembly. Consistent with a primary role in the assembly phase, ROCK inhibition prevented actin ring assembly and wound closure. ROCK inhibition after ring assembly was complete had no effect. Recruitment and activation of MLCK occurred after ring assembly was complete and coincided with ring contraction. MLCK inhibition slowed and then stopped contraction but did not prevent ring assembly. MLCK inhibition also delayed barrier function recovery. Studies of human colonic biopsy specimens suggest that purse-string wound closure also occurs in vivo, because MLC phosphorylation was enhanced surrounding oligocellular wounds.


CONCLUSIONS
These results suggest complementary roles for these kinases in purse-string closure of experimental and in vivo oligocellular epithelial wounds; rho and ROCK are critical for actin ring assembly, while the activity of MLCK drives contraction.
Russo
John M
JM
Department of Pathology, University of Chicago, IL 60637, USA.
Florian
Peter
P
Shen
Le
L
Graham
W Vallen
WV
Tretiakova
Maria S
MS
Gitter
Alfred H
AH
Mrsny
Randall J
RJ
Turner
Jerrold R
JR
D008841:Actin Cytoskeleton; D000199:Actins / Q000378:metabolism; D018938:Caco-2 Cells; D003106:Colon / Q000378:metabolism / Q000473:pathology / Q000503:physiopathology; D003199:Computer Systems; D006801:Humans; D015212:Inflammatory Bowel Diseases / Q000378:metabolism / Q000473:pathology / Q000503:physiopathology; D007413:Intestinal Mucosa / Q000201:enzymology / Q000378:metabolism / Q000503:physiopathology*; D047908:Intracellular Signaling Peptides and Proteins; D018994:Myosin Light Chains / Q000378:metabolism; D009219:Myosin-Light-Chain Kinase / Q000378:metabolism*; D010539:Permeability; D010766:Phosphorylation; D017346:Protein Serine-Threonine Kinases / Q000378:metabolism*; D013997:Time Factors; D014018:Tissue Distribution; D014945:Wound Healing; D054460:rho-Associated Kinases
D000199:Actins; D047908:Intracellular Signaling Peptides and Proteins; D018994:Myosin Light Chains; D017346:Protein Serine-Threonine Kinases; D054460:rho-Associated Kinases; D009219:Myosin-Light-Chain Kinase