Quantitative distribution and colocalization of non-muscle myosin light chain kinase isoforms and cortactin in human lung endothelium.
Vascular barrier regulation is intimately linked to alterations in the distribution and configuration of the endothelial cell (EC) cytoskeleton in response to angiogenic and edemagenic agonists. Critical actin cytoskeletal rearrangement includes spatially directed increases in myosin light chain (MLC) phosphorylation, catalyzed by Ca(2+)/calmodulin-dependent non-muscle myosin light chain kinase variants (nmMLCK1- and -2), as well as association of nmMLCK with the actin-binding protein, cortactin. As these associations have proven difficult to quantify in a spatially specific manner, we now describe the utility of intensity correlation image analysis and the intensity correlation quotient (ICQ) to quantify colocalization in fixed and live cell imaging assays in human pulmonary artery EC. From baseline ICQ values averaging 0.216 reflecting colocalization of cortactin-DsRed with EGFP-nmMLCK fusion proteins in resting EC, thrombin-induced EC contraction significantly reduced cortactin-DsRed-EGFP-nmMLCK colocalization (nmMLCK1: ICQ=0.118; nmMLCK2: ICQ=0.091) whereas the potent EC barrier-protective agonist, sphingosine 1-phosphate (S1P), significantly increased nmMLCK-cortactin colocalization within lamellipodia (nmMLCK1: ICQ=0.275; nmMLCK2: ICQ=0.334). Over-expression of a cortactin-DsRed mutant fusion protein lacking the SH3 domain, known to be essential for cortactin-nmMLCK association, reduced baseline and S1P-mediated live cell colocalization with each nmMLCK variant (nmMLCK1: ICQ=0.160; nmMLCK2: ICQ=0.157). Similarly, expression of a truncated EGFP-nmMLCK2 mutant lacking cortactin- and actin-binding domains, markedly reduced basal localization in lamellipodia and abolished colocalization with cortactin-DsRed in lamellipodia after S1P (ICQ=-0.148). These data provide insights into the molecular basis for vascular barrier-regulatory cytoskeletal responses and support the utility of sophisticated imaging analyses and methodological assessment to quantify the critical nmMLCK and cortactin interaction during vascular barrier regulation.
Brown
Mary
M
Department of Medicine, Pritzker School of Medicine, University of Chicago, Chicago, IL 60637, USA.
Adyshev
Djanybek
D
Bindokas
Vytautus
V
Moitra
Jaideep
J
Garcia
Joe G N
JG
Dudek
Steven M
SM
D008841:Actin Cytoskeleton / Q000378:metabolism; D000199:Actins / Q000235:genetics / Q000378:metabolism; D000818:Animals; D002478:Cells, Cultured; D051356:Cortactin / Q000235:genetics / Q000378:metabolism*; D042783:Endothelial Cells / Q000166:cytology / Q000187:drug effects / Q000378:metabolism*; D006801:Humans; D007527:Isoenzymes / Q000235:genetics / Q000378:metabolism*; D008164:Luminescent Proteins / Q000235:genetics; D008168:Lung / Q000166:cytology*; D008246:Lysophospholipids / Q000494:pharmacology; D009219:Myosin-Light-Chain Kinase / Q000235:genetics / Q000378:metabolism*; D011485:Protein Binding / Q000502:physiology; D054730:Protein Interaction Domains and Motifs / Q000502:physiology; D021381:Protein Transport / Q000187:drug effects / Q000235:genetics; D011554:Pseudopodia / Q000378:metabolism; D051381:Rats; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D017384:Sequence Deletion / Q000235:genetics; D013110:Sphingosine / Q000031:analogs & derivatives / Q000494:pharmacology; D013917:Thrombin / Q000494:pharmacology; D014162:Transfection; D018909:src Homology Domains / Q000235:genetics
D000199:Actins; C495868:CTTN protein, human; D051356:Cortactin; C495870:Cttn protein, rat; D007527:Isoenzymes; D008164:Luminescent Proteins; D008246:Lysophospholipids; D011993:Recombinant Fusion Proteins; C060506:sphingosine 1-phosphate; D009219:Myosin-Light-Chain Kinase; D013917:Thrombin; D013110:Sphingosine