Rondaij Mariska G, Bierings Ruben, Kragt Astrid, Gijzen Karina A, Sellink Erica, van Mourik Jan A, Fernandez-Borja Mar, Voorberg Jan
Department of Plasma Proteins, Sanquin Research, University of Amsterdam, The Netherlands.
Arterioscler Thromb Vasc Biol. 2006 Jan;26(1):49-55. doi: 10.1161/01.ATV.0000191639.08082.04. Epub 2005 Oct 20.
Perinuclear clustering is observed for several different organelles and illustrates dynamic regulation of the secretory pathway and organelle distribution. Previously, we observed that a subset of Weibel-Palade bodies (WPBs), endothelial cell-specific storage organelles, undergo centralization when endothelial cells are stimulated with cAMP-raising agonists of von Willebrand factor (vWF) secretion. In this study, we investigated this phenomenon of WPB clustering in more detail.
Our results demonstrate that the clustered WPBs are localized at the microtubule organizing center and that cluster formation depends on an intact microtubule network. Disruption of the microtubules by nocodazole completely abolished clustering, whereas treatment with the actin depolymerizing compound cytochalasin B had no effect on WPB clustering. Interfering with the dynein-dynactin interaction by overexpression of the p50 dynamitin subunit or the CC1 domain of the p150glued subunit of the dynactin complex completely inhibited perinuclear clustering of WPBs, suggesting that dynein activity mediates this process. Furthermore, we found that inhibition of dephosphorylation resulted in an increase in clustering, whereas inhibition of protein kinase A (PKA) markedly reduced WPB clustering.
These results suggest that perinuclear clustering of WPBs involves PKA-dependent regulation of the dynein-dynactin complex. Endothelial cell stimulation with epinephrine results in retrograde movement of a subset of WPBs to the microtubule organizing center. This minus-end directed transport requires an intact microtubular network and is mediated by the motor protein dynein. Together, our results suggest that epinephrine-induced clustering of WPBs involves PKA-dependent regulation of the dynein-dynactin complex.
观察到几种不同细胞器存在核周聚集现象，这说明了分泌途径和细胞器分布的动态调节。此前，我们观察到，当用升高环磷酸腺苷（cAMP）的血管性血友病因子（vWF）分泌激动剂刺激内皮细胞时，内皮细胞特异性储存细胞器——魏尔-帕拉德小体（WPB）的一个亚群会发生集中化。在本研究中，我们更详细地研究了WPB聚集这一现象。
我们的结果表明，聚集的WPB定位于微管组织中心，且聚集的形成依赖于完整的微管网络。用诺考达唑破坏微管会完全消除聚集，而用肌动蛋白解聚化合物细胞松弛素B处理对WPB聚集没有影响。通过过表达动力蛋白激活蛋白复合物中p50动力蛋白亚基或p150黏着蛋白亚基的CC1结构域来干扰动力蛋白-动力蛋白激活蛋白相互作用，会完全抑制WPB的核周聚集，这表明动力蛋白活性介导了这一过程。此外，我们发现抑制去磷酸化会导致聚集增加，而抑制蛋白激酶A（PKA）则会显著减少WPB聚集。
这些结果表明，WPB的核周聚集涉及PKA对动力蛋白-动力蛋白激活蛋白复合物的依赖性调节。用肾上腺素刺激内皮细胞会导致一部分WPB逆向移动至微管组织中心。这种向负端的运输需要完整的微管网络，并由驱动蛋白动力蛋白介导。总之，我们的结果表明，肾上腺素诱导的WPB聚集涉及PKA对动力蛋白-动力蛋白激活蛋白复合物的依赖性调节。
