Quantitative distribution and colocalization of non-muscle myosin light chain kinase isoforms and cortactin in human lung endothelium

Mary Brown, Djanybek Adyshev, Vytautus Bindokas, Jaideep Moitra, Joe GN Garcia, Steven M. Dudek

Research output: Contribution to journalArticle

35 Citations (Scopus)

Abstract

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 Ca2+/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.

Original languageEnglish (US)
Pages (from-to)75-88
Number of pages14
JournalMicrovascular Research
Volume80
Issue number1
DOIs
StatePublished - Jul 2010
Externally publishedYes

Fingerprint

Cortactin
Myosin-Light-Chain Kinase
Endothelium
Protein Isoforms
Lung
Endothelial cells
Endothelial Cells
Pseudopodia
Blood Vessels
Actins
Fusion reactions
Association reactions
Imaging techniques
Microfilament Proteins
Myosin Light Chains
Phosphorylation
src Homology Domains
Mutant Proteins
Calmodulin
Cytoskeleton

Keywords

  • Cytoskeleton
  • F-actin
  • Immunofluorescence
  • Intensity correlation image analysis
  • Lamellipodia
  • Permeability
  • Sphingosine 1-phosphate

ASJC Scopus subject areas

  • Biochemistry
  • Cardiology and Cardiovascular Medicine
  • Cell Biology

Cite this

Quantitative distribution and colocalization of non-muscle myosin light chain kinase isoforms and cortactin in human lung endothelium. / Brown, Mary; Adyshev, Djanybek; Bindokas, Vytautus; Moitra, Jaideep; Garcia, Joe GN; Dudek, Steven M.

In: Microvascular Research, Vol. 80, No. 1, 07.2010, p. 75-88.

Research output: Contribution to journalArticle

Brown, Mary ; Adyshev, Djanybek ; Bindokas, Vytautus ; Moitra, Jaideep ; Garcia, Joe GN ; Dudek, Steven M. / Quantitative distribution and colocalization of non-muscle myosin light chain kinase isoforms and cortactin in human lung endothelium. In: Microvascular Research. 2010 ; Vol. 80, No. 1. pp. 75-88.
@article{0047370d4e3f40079520c826a2411cfd,
title = "Quantitative distribution and colocalization of non-muscle myosin light chain kinase isoforms and cortactin in human lung endothelium",
abstract = "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 Ca2+/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.",
keywords = "Cytoskeleton, F-actin, Immunofluorescence, Intensity correlation image analysis, Lamellipodia, Permeability, Sphingosine 1-phosphate",
author = "Mary Brown and Djanybek Adyshev and Vytautus Bindokas and Jaideep Moitra and Garcia, {Joe GN} and Dudek, {Steven M.}",
year = "2010",
month = "7",
doi = "10.1016/j.mvr.2009.12.010",
language = "English (US)",
volume = "80",
pages = "75--88",
journal = "Microvascular Research",
issn = "0026-2862",
publisher = "Academic Press Inc.",
number = "1",

}

TY - JOUR

T1 - Quantitative distribution and colocalization of non-muscle myosin light chain kinase isoforms and cortactin in human lung endothelium

AU - Brown, Mary

AU - Adyshev, Djanybek

AU - Bindokas, Vytautus

AU - Moitra, Jaideep

AU - Garcia, Joe GN

AU - Dudek, Steven M.

PY - 2010/7

Y1 - 2010/7

N2 - 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 Ca2+/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.

AB - 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 Ca2+/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.

KW - Cytoskeleton

KW - F-actin

KW - Immunofluorescence

KW - Intensity correlation image analysis

KW - Lamellipodia

KW - Permeability

KW - Sphingosine 1-phosphate

UR - http://www.scopus.com/inward/record.url?scp=77953539413&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=77953539413&partnerID=8YFLogxK

U2 - 10.1016/j.mvr.2009.12.010

DO - 10.1016/j.mvr.2009.12.010

M3 - Article

VL - 80

SP - 75

EP - 88

JO - Microvascular Research

JF - Microvascular Research

SN - 0026-2862

IS - 1

ER -