Actin-related protein 2/3 complex is required for actin ring formation
Actin-related protein 2/3 complex is required for actin ring formation
复制标题
DOI：
10.1359/jbmr.0301238
复制
发表时间：
2004-03-01
影响因子：
6.2
通讯作者：
Holliday, LS
中科院分区：
文献类型：
--
作者：
Hurst, IR;Zuo, J;Holliday, LS
肌动蛋白环在骨细胞骨吸收过程中起重要作用，肌动蛋白相关蛋白2/3复合物是肌动蛋白聚合的关键调节因子。肌动蛋白相关蛋白2/3复合物存在于肌动蛋白环的足体中。一个短的干扰RNA敲低肌动蛋白相关蛋白2在破骨细胞和破坏肌动蛋白环的表达，这表明该复合物是至关重要的肌动蛋白环formation.Introduction：再吸收骨，破骨细胞形成一个细胞外的酸性隔室隔离的密封区。这取决于肌动蛋白环，该肌动蛋白环由丝状肌动蛋白组成，并被组织成称为podosomes的动态结构。肌动蛋白相关蛋白2/3（Arp 2/3）复合物是肌动蛋白聚合的重要调节剂。我们测试了Arp 2/3复合物是否是肌动蛋白环的一个组成部分，是重要的肌动蛋白环formation.Materials和方法：蛋白质印迹分析被用来确定Arp 2和Arp 3，Arp 2/3复合物在破骨细胞样cells的两个组件的水平。使用抗体进行免疫细胞化学的共聚焦显微镜研究证明了Arp 2/3复合物在破骨细胞中的定位。针对Arp 2制备短干扰RNA寡核苷酸（siRNA）并用于敲低其表达。结果：在RANKL诱导RAW 264.7细胞分化为破骨细胞样细胞的过程中，检测到Arp 2和Arp 3增加3倍。Arp 2/3复合物主要集中在肌动蛋白环中，并在封闭区附近富集。Arp 2/3复合物与corcocin共定位，corcocin是podosomes的一个组成部分，但不与包围podosomes的黏着斑蛋白共定位。与用无效siRNA转染的细胞相比，用RANKL刺激后5天转染到RAW 264.7细胞中的针对Arp 2的siRNA使Arp 2蛋白水平降低70%。RAW 264.7破骨细胞样细胞和骨髓破骨细胞中Arp 2被敲低的细胞化学特征显示较少的podosomes和没有肌动蛋白环，虽然许多细胞仍然很好地spreaded.Conclusions：这些数据表明，Arp 2/3复合物是肌动蛋白环的一个组成部分，Arp 2/3复合物的存在是至关重要的肌动蛋白环的形成。此外，结果显示siRNA用于RAW 264.7破骨细胞样细胞的研究。
Actin rings are vital for osteoclastic bone resorption, and actin-related protein 2/3 complex is a pivotal regulator of actin polymerization. Actin-related protein 2/3 complex was found in the podosomes of actin rings. A short interfering RNA knocked down expression of actin-related protein 2 in osteoclasts and disrupted actin rings, suggesting that the complex is crucial to actin ring formation.Introduction: To resorb bone, osteoclasts form an extracellular acidic compartment segregated by a sealing zone. This is dependent on an actin ring that is composed of filamentous actin organized into dynamic structures called podosomes. The actin-related protein 2/3 (Arp2/3) complex is a vital regulator of actin polymerization. We tested whether the Arp2/3 complex is a component of actin rings and is important for actin ring formation.Materials and Methods: Western blot analysis was used to determine levels of Arp2 and Arp3, two components of the Arp2/3 complex in osteoclast-like cells. Confocal microscopy studies using antibodies for immunocytochemistry demonstrated localization of Arp2/3 complex in osteoclasts. Short interfering RNA oligonucleotides (siRNAs) were made against Arp2 and used to knock down its expression. Results: A 3-fold increase in Arp2 and Arp3 was detected during RANKL-induced differentiation of RAW 264.7 cells into osteoclast-like cells. Arp2/3 complex was concentrated in actin rings and enriched near the sealing zone. Arp2/3 complex co-localized with cortactin, a component of podosomes, but not vinculin, which surrounds podosomes. siRNA against Arp2, transfected into RAW 264.7 cells 5 days after stimulation with RANKL, reduced Arp2 protein levels 70% compared with cells transfected with ineffective siRNAs. Cytochemical characterization of RAW 264.7 osteoclast-like cells and marrow osteoclasts in which Arp2 was knocked down revealed fewer podosomes and no actin rings, although many cells remained well spread.Conclusions: These data show that Arp2/3 complex is a component of actin rings and that the presence of Arp2/3 complex is vital to the formation of actin rings. In addition, the results show the use of siRNAs for the study of RAW 264.7 osteoclast-like cells.