Deng Maoxian, Mohanan Sunish, Polyak Erzsebet, Chacko Samuel
Division of Urology, Department of Surgery, University of Pennsylvania, Glenolden, Pennsylvania 19036, USA.
Cell Motil Cytoskeleton. 2007 Dec;64(12):951-65. doi: 10.1002/cm.20236.
Caldesmon (CaD), a component of microfilaments in all cells and thin filaments in smooth muscle cells, is known to bind to actin, tropomyosin, calmodulin, and myosin and to inhibit actin-activated ATP hydrolysis by smooth muscle myosin. Thus, it is believed to regulate smooth muscle contraction, cell motility and the cytoskeletal structure. Using bladder smooth muscle cell cultures and RNA interference (RNAi) technique, we show that the organization of actin into microfilaments in the cytoskeleton is diminished by siRNA-mediated CaD silencing. CaD silencing significantly decreased the amount of polymerized actin (F-actin), but the expression of actin was not altered. Additionally, we find that CaD is associated with 10 nm intermediate-sized filaments (IF) and in vitro binding assay reveals that it binds to vimentin and desmin proteins. Assembly of vimentin and desmin into IF is also affected by CaD silencing, although their expression is not significantly altered when CaD is silenced. Electronmicroscopic analyses of the siRNA-treated cells showed the presence of myosin filaments and a few surrounding actin filaments, but the distribution of microfilament bundles was sparse. Interestingly, the decrease in CaD expression had no effect on tubulin expression and distribution of microtubules in these cells. These results demonstrate that CaD is necessary for the maintenance of actin microfilaments and intermediate-sized filaments in the cytoskeletal structure. This finding raises the possibility that the cytoskeletal structure in smooth muscle is affected when CaD expression is altered, as in smooth muscle de-differentiation and hypertrophy seen in certain pathological conditions.
钙调蛋白（CaD）是所有细胞微丝和平滑肌细胞细肌丝的组成成分，已知它能与肌动蛋白、原肌球蛋白、钙调蛋白和肌球蛋白结合，并抑制平滑肌肌球蛋白的肌动蛋白激活的ATP水解。因此，人们认为它能调节平滑肌收缩、细胞运动和细胞骨架结构。利用膀胱平滑肌细胞培养和RNA干扰（RNAi）技术，我们发现，在细胞骨架中，肌动蛋白组织成微丝的过程会因siRNA介导的CaD沉默而减少。CaD沉默显著降低了聚合肌动蛋白（F-肌动蛋白）的含量，但肌动蛋白的表达没有改变。此外，我们发现CaD与10nm中等大小的中间丝（IF）相关，体外结合试验表明它能与波形蛋白和结蛋白结合。波形蛋白和结蛋白组装成IF也受到CaD沉默的影响，尽管当CaD沉默时它们的表达没有显著改变。对经siRNA处理的细胞进行电子显微镜分析，结果显示存在肌球蛋白丝和一些周围的肌动蛋白丝，但微丝束的分布稀疏。有趣的是，CaD表达的降低对这些细胞中微管蛋白的表达和微管的分布没有影响。这些结果表明，CaD对于维持细胞骨架结构中的肌动蛋白微丝和中等大小的中间丝是必需的。这一发现增加了这样一种可能性，即当CaD表达改变时，如在某些病理状况下出现的平滑肌去分化和肥大过程中，平滑肌的细胞骨架结构会受到影响。
