Regulation of MDCK cell-substratum adhesion by RhoA and myosin light chain kinase after ATP depletion.
The attachment of epithelial cells to the extracellular matrix substratum is essential for their differentiation and polarization. Despite this, the precise adhesion mechanism and its regulation are poorly understood. In the kidney, an ischemic insult causes renal tubular epithelial cells to detach from the basement membrane, even though they remain viable. To understand this phenomenon, and to probe the regulation of epithelial cell attachment, we used a model system consisting of newly adherent Madin-Darby canine kidney (MDCK) cells subjected to ATP depletion to mimic ischemic injury. We found that MDCK cells detach from collagen I after 60 min of ATP depletion but reattach when resupplied with glucose. Detachment is not caused by degradation or endocytosis of beta(1)-integrins, which mediate attachment to collagen I. Basal actin filaments and paxillin-containing adhesion complexes are disrupted by ATP depletion and quickly reform on glucose repletion. However, partial preservation of basal actin by overexpression of constitutively active RhoA does not significantly affect cell detachment. Furthermore, Y-27632, an inhibitor of the RhoA effector Rho-kinase, does not prevent reattachment of cells on glucose addition, even though reformation of central stress fibers and large adhesion complexes is blocked. In contrast, reattachment of ATP-depleted cells and detachment of cells not previously subjected to ATP depletion are prevented by ML-7, an inhibitor of myosin light chain kinase (MLCK). We conclude that initial adherence of MDCK cells to a collagen I substratum is mediated by peripheral actin filaments and adhesion complexes regulated by MLCK but not by stress fibers and adhesion complexes controlled by RhoA.
Prahalad
Priya
P
Department of Biology, Massachusetts Institute of Technology, Cambridge 02142, USA.
Calvo
Ignacio
I
Waechter
Holly
H
Matthews
Jeffrey B
JB
Zuk
Anna
A
Matlin
Karl S
KS
D000255:Adenosine Triphosphate / Q000378:metabolism; D000818:Animals; D002460:Cell Line; D004847:Epithelial Cells / Q000166:cytology / Q000378:metabolism*; D022001:Focal Adhesions / Q000502:physiology*; D019012:Integrin beta1 / Q000378:metabolism; D007668:Kidney / Q000166:cytology; D018994:Myosin Light Chains / Q000378:metabolism*; D010455:Peptides / Q000378:metabolism; D010766:Phosphorylation; D022502:Stress Fibers / Q000378:metabolism; D020742:rhoA GTP-Binding Protein / Q000378:metabolism*
D019012:Integrin beta1; D018994:Myosin Light Chains; D010455:Peptides; C065738:MLCK peptide; D000255:Adenosine Triphosphate; D020742:rhoA GTP-Binding Protein