Caldesmon effects on the actin cytoskeleton and cell adhesion in cultured HTM cells
Caldesmon effects on the actin cytoskeleton and cell adhesion in cultured HTM cells
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DOI：
10.1016/j.exer.2006.01.006
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发表时间：
2006-06-01
影响因子：
3.4
通讯作者：
Bershadsky, Alexander D.
中科院分区：
文献类型：
--
作者：
Grosheva, Inna;Vittitow, Jason L.;Bershadsky, Alexander D.
Caldesmon是一种多功能的肌动蛋白细胞骨架调节剂，它可以影响肌动蛋白的收缩和肌动蛋白的聚合。以前的研究表明，钙调蛋白在培养的成纤维细胞中过度表达，产生的效果类似于细胞收缩的化学抑制剂。由于这些抑制剂(H-7、Y-27632等)已被证明具有降低眼压和增加眼前房流出能力的作用，我们建议将钙离子蛋白用于青光眼的基因治疗。在本研究中，我们研究了腺病毒携带的大鼠非肌钙蛋白与绿色荧光蛋白融合蛋白(AdCaldGFP)的表达对培养的人眼小梁细胞(HTM)肌动蛋白细胞骨架和基质黏附的影响。此外，我们还评估了钙调蛋白对肾上皮MDCK细胞细胞-细胞连接稳定性的影响。培养的HTM细胞显示出发达的肌动蛋白细胞骨架，主要由平行的肌球蛋白束(应力纤维)组成。还可观察到含有致密肌动蛋白网络的片状脂膜突起。细胞基质粘连主要由与应力纤维末端相关的局灶性粘连(FA)、片状脂膜中的局灶性复合体和铺展细胞中央的纤维粘连所主导。在感染后24-48小时内，AdCaldGFP处理HTM细胞可引起剂量依赖性的形态变化。中度表达钙调蛋白的细胞显示含有肌动蛋白和肌球蛋白11的直束，比对照细胞短得多。在钙调蛋白过度表达的细胞中，短丝状束形成了由三角形肌动蛋白结构组成的阵列，在它们的顶点上有小的纽蛋白阳性FA。此外，显示大片状脂血的细胞比例增加。约40-50%的钙调蛋白表达细胞高水平表达GFP-钙调蛋白，肌动蛋白细胞骨架发生严重改变，表现为应力纤维消失，形成弯曲的肌动蛋白和肌球蛋白纤维束。这些束一起形成了一个动态网络，由充满整个细胞质的脉动环组成。加入thapsigarin可增加细胞内钙离子浓度，使曲线束伸直。另一种由caldesmon过表达诱导的新型肌动蛋白结构是高度动态的环形波，它以大约10微米/分钟的速度在受影响的细胞中传播。在应力纤维断裂的细胞中，含有纽蛋白的FAs和富含张力蛋白的纤维粘连也基本上消失了。然而，磷酸酪氨酸阳性的焦点复合体在这些细胞的板脂中仍然很明显。在MDCK细胞中过表达钙调蛋白以剂量依赖的方式减少了细胞-细胞贴壁连接处的β-连环素含量，在某些情况下导致了贴壁连接的物理破坏。因此，钙调蛋白的过表达诱导了受影响细胞中肌动蛋白细胞骨架的独特重组，伴随着局灶性和纤维性细胞-基质黏附的破坏，以及细胞-细胞黏附连接的不稳定。在体内诱导青光眼HTM细胞的收缩能力和肌动蛋白细胞骨架的这种变化可以产生治疗上有用的流出能力的增加。(C)2006爱思唯尔有限公司。保留所有权利。
Caldesmon is a multifunctional ubiquitous regulator of the actin cytoskeleton, which can affect both actomyosin contractility and actin polymerization. Previous studies showed that caldesmon over-expression in cultured fibroblasts produces effects that resemble those of chemical inhibitors of cellular contractility. Since these inhibitors (H-7, Y-27632, etc.) have been shown to lower intraocular pressure and increase outflow facility from the anterior chamber of the eye, we proposed that caldesmon might be used for gene therapy of glaucoma. In the present study we examined the effects of expression of adenovirus-delivered rat non-muscle caldesmon fused with green fluorescent protein (AdCaldGFP) on the actin cytoskeleton and matrix adhesions in cultured human trabecular meshwork (HTM) cells. In addition, we assessed the effect of caldesmon on the stability of cell-cell junctions in kidney epithelial MDCK cells. Cultured HTM cells demonstrate a well-developed actin cytoskeleton, comprising mainly arrays of parallel actomyosin bundles (stress fibers). Lamellipodial protrusions containing dense actin networks are also observed. Cellmatrix adhesions are dominated by focal adhesions (FAs) associated with the ends of the stress fibers, focal complexes in lamellipodia, and fibrillar adhesions in the central part of the spread cells. Treatment of HTM cells with AdCaldGFP resulted in dose-dependent morphological changes within 24-48 hr post-infection. Cells expressing moderate levels of caldesmon exhibited straight bundles containing actin and myosin 11, which were considerably shorter than those in control cells. Short filament bundles in caldesmon over-expressing cells formed arrays consisting of triangular actin structures with small vinculin-positive FAs at their vertices. In addition, the fraction of cells displaying large lamellipodia increased. About 40-50% of the population of caldesmon-expressing cells demonstrated high levels of GFP-caldesmon expression and severe changes in the actin cytoskeleton, manifested by the disappearance of stress fibers and the formation of curved actin- and myosin-containing bundles. These bundles formed together a dynamic network consisting of pulsating loops filling the entire cytoplasm. Addition of thapsigargin, which increases intracellular Ca++ concentration, resulted in a straightening of the curved bundles. Another type of novel actin structures induced by caldesmon over-expression were highly dynamic circular waves that propagated over the affected cells with a velocity about 10 mu m min. In cells with disrupted stress fibers, vinculin-containing FAs and tensin-rich fibrillar adhesions had also essentially vanished. However, phosphotyrosine-positive focal complexes were still prominent throughout the lamellipodia of these cells. Over-expression of caldesmon in MDCK cells reduced, in a dose dependent manner, the beta-catenin content at cell-cell adherens junctions and in some cases led to physical disruption of adherens junctions. Thus, caldesmon overexpression induces unique reorganization of the actin cytoskeleton in affected cells, accompanied by disruption of focal and fibrillar cell-matrix adhesions, and destabilization of cell-cell adherens junctions. Inducing such changes in the contractility and actin cytoskeleton of HTM cells in glaucomatous eyes in vivo could produce a therapeutically useful increase in outflow facility. (c) 2006 Elsevier Ltd. All rights reserved.