Quantitative distribution and colocalization of non-muscle myosin light chain kinase isoforms and cortactin in human lung endothelium.
Quantitative distribution and colocalization of non-muscle myosin light chain kinase isoforms and cortactin in human lung endothelium.
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DOI：
10.1016/j.mvr.2009.12.010
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发表时间：
2010-07
影响因子：
3.1
通讯作者：
Dudek SM
中科院分区：
文献类型：
--
作者：
Brown M;Adyshev D;Bindokas V;Moitra J;Garcia JG;Dudek SM
血管屏障调节与内皮细胞（EC）细胞骨架的分布和结构的改变密切相关，这是对血管生成和消肿激动剂的反应。关键的肌动蛋白细胞骨架重排包括肌球蛋白轻链（MLC）磷酸化的空间定向增加，这是由Ca2+/钙调素依赖的非肌肉肌球蛋白轻链激酶变体（nmMLCK1-和-2）催化的，以及nmMLCK与肌动蛋白结合蛋白的关联。由于这些关联已被证明难以以空间特异性的方式量化，我们现在描述了强度相关图像分析和强度相关商（ICQ）在人类肺动脉EC固定和活细胞成像分析中量化共定位的效用。从平均为0.216的基线ICQ值来看，反映了静息EC中接触蛋白- dsred与EGFP-nmMLCK融合蛋白的共定位，凝血酶诱导的EC收缩显著降低了接触蛋白- dsred -EGFP-nmMLCK的共定位（nmMLCK1: ICQ = 0.118; nmMLCK2: ICQ = 0.091），而有效的EC屏障保护激动剂鞘糖苷1-磷酸（S1P）显著增加了板足内nmmlck -接触蛋白的共定位（nmMLCK1: ICQ = 0.275; nmMLCK2: ICQ = 0.334）。缺乏SH3结构域的cortactin-DsRed突变融合蛋白（已知是cortactin-nmMLCK关联所必需的）的过表达降低了基线和s1p介导的活细胞与每个nmMLCK变体的共定位（nmMLCK1: ICQ = 0.160; nmMLCK2: ICQ = 0.157）。同样，缺乏接触蛋白和肌动蛋白结合结构域的EGFP-nmMLCK2突变体的表达在S1P后显著降低了板足的基础定位，并消除了与接触蛋白- dsred在板足的共定位（ICQ = - 0.148）。这些数据为血管屏障调节细胞骨架反应的分子基础提供了见解，并支持了复杂成像分析和方法学评估的应用，以量化血管屏障调节过程中关键的nmMLCK和接触相互作用。
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.
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影响因子：
3.3
作者：
Khanna, R.;Li, Q.;Stanley, E. F.
通讯作者：
Stanley, E. F.
DOI：
10.1152/ajplung.2001.280.6.l1168
发表时间：
2001-06-01
影响因子：
4.9
作者：
Petrache, I;Verin, AD;Garcia, JGN
通讯作者：
Garcia, JGN
影响因子：
5.3
作者：
Li, Q;Lau, A;Stanley, EF
通讯作者：
Stanley, EF
DOI：
10.1152/ajplung.00343.2005
发表时间：
2006-08-01
影响因子：
4.9
作者：
Jacobson, Jeffrey R.;Dudek, Steven M.;Garcia, Joe G. N.
通讯作者：
Garcia, Joe G. N.
影响因子：
4.8
作者：
Liu, F;Schaphorst, KL;Garcia, JGN
通讯作者：
Garcia, JGN