Isoform and Splice-Variant Specific Functions of Dynamin-2 Revealed by Analysis of Conditional Knock-Out Cells
Isoform and Splice-Variant Specific Functions of Dynamin-2 Revealed by Analysis of Conditional Knock-Out Cells
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DOI：
10.1091/mbc.e08-08-0890
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发表时间：
2008-12-01
影响因子：
3.3
通讯作者：
Schmid, Sandra L.
中科院分区：
文献类型：
--
作者：
Liu, Ya-Wen;Surka, Mark C.;Schmid, Sandra L.
发动蛋白（Dynamin，Dyn）是一种多功能的GT3，参与细胞内吞、胞内运输、细胞信号传导和胞质分裂等多种细胞活动。哺乳动物基因组编码三种亚型，Dyn 1，Dyn 2和Dyn 3，以及每种亚型的几种剪接变体，这表明不同的亚型和/或不同的剪接变体可能介导不同的细胞功能。我们产生了条件Dyn 2 KO细胞系，并进行敲除和重建实验，以探索广泛表达的Dyn 2的同种型和剪接变体特异性细胞功能。我们发现，Dyn 2所需的网格蛋白介导的内吞作用（CME），p75从高尔基体出口，和PDGF刺激的巨胞饮和胞质分裂，但不为其他内吞途径。令人惊讶的是，CME和p75胞吐被有效地救出Dyn 2的重新引入，但不是Dyn 1，这表明这两个亚型功能差异，在非神经元细胞的囊泡贩运。这两种亚型拯救巨胞饮和胞质分裂，这表明发动蛋白在这些过程中的功能可能是机械不同的作用，在CME。虽然所有四种Dyn 2剪接变体都可以同样地恢复CME，但Dyn 2ba和-bb在恢复p75胞吐方面更有效。这种剪接变体特异性与它们对高尔基体的差异靶向相关。这些研究揭示了Dyn 2的同种型和剪接变体的特异性功能。
Dynamin (Dyn) is a multifunctional GTPase implicated in several cellular events, including endocytosis, intracellular trafficking, cell signaling, and cytokinesis. The mammalian genome encodes three isoforms, Dyn1, Dyn2, and Dyn3, and several splice variants of each, leading to the suggestion that distinct isoforms and/or distinct splice variants might mediate distinct cellular functions. We generated a conditional Dyn2 KO cell line and performed knockout and reconstitution experiments to explore the isoform- and splice variant specific cellular functions of ubiquitously expressed Dyn2. We find that Dyn2 is required for clathrin-mediated endocytosis (CME), p75 export from the Golgi, and PDGF-stimulated macropinocytosis and cytokinesis, but not for other endocytic pathways. Surprisingly, CME and p75 exocytosis were efficiently rescued by reintroduction of Dyn2, but not Dyn1, suggesting that these two isoforms function differentially in vesicular trafficking in nonneuronal cells. Both isoforms rescued macropinocytosis and cytokinesis, suggesting that dynamin function in these processes might be mechanistically distinct from its role in CME. Although all four Dyn2 splice variants could equally restore CME, Dyn2ba and -bb were more effective at restoring p75 exocytosis. This splice variant specificity correlated with their differential targeting to the Golgi. These studies reveal isoform and splice-variant specific functions for Dyn2.