The membrane-tubulating potential of amphiphysin 2/BIN1 is dependent on the microtubule-binding cytoplasmic linker protein 170 (CLIP-170)
The membrane-tubulating potential of amphiphysin 2/BIN1 is dependent on the microtubule-binding cytoplasmic linker protein 170 (CLIP-170)
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DOI：
10.1016/j.ejcb.2008.08.006
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发表时间：
2009-02-01
影响因子：
6.6
通讯作者：
Leprince, Corinne
中科院分区：
文献类型：
--
作者：
Meunier, Brigitte;Quaranta, Muriel;Leprince, Corinne
双phiphysins是含有BIN-双phiphysin-RVS（BAR）结构域的蛋白质，其影响诸如肌细胞中的T-小管、神经元以及非神经元细胞中的内吞凹以及可能的细胞质内体的位点中的膜曲率。这种对脂质膜的影响是由不同的两性霉素2/BIN 1亚型，产生的选择性剪接，并显示出独特的结构和功能特性。在这项研究中，我们的目标是通过表征新的分子伴侣来表征无处不在表达的两性生理蛋白2/BIN 1的功能作用。我们进行了一个双杂交筛选与在HeLa细胞中表达的两性霉素2/BIN 1亚型。我们确定CLIP-170作为一个两栖physin 2/BIN 1相互作用的分子。CLIP-170是一种正末端跟踪蛋白，参与微管（MT）稳定性和dynactin的募集。两性生理素2/BIN 1和CLIP-170之间的结合依赖于两性生理素2的N-末端部分（主要是BAR结构域）和CLIP-170的内部卷曲螺旋区域。这种伙伴关系得到证实，GST下拉测定和共免疫沉淀在HeLa细胞表达内源性两性霉素2（主要亚型6，9和10）。当在HeLa细胞中过表达时，两性霉素2/BIN 1导致细胞内小管的形成，其可以与MT紧密对齐。MT解聚后，诺考达唑，两栖蛋白2染色的小管消失，并重新出现后诺考达唑洗脱。此外，通过RNAi消耗CLIP-170诱导具有两性生理蛋白2染色的小管的细胞比例降低，而不具有小管的细胞比例增加。这一结果表明两种类型的小管之间存在机械联系，这可能涉及+TIP蛋白CLIP-170。两性啡肽2/BIN 1可能是CLIP-170在膜上的锚定点，因此也是MT的锚定点。因此，MT聚合的推动力可以帮助两性生理蛋白2/BIN 1发挥其促分化潜力。我们建议，两栖physin 2/BIN 1参与的交通中间体和细胞内细胞器的微管，第一次通过其内在的微管电位，第二次通过其结合CLIP-170和MT的能力。(c)2009年Elsevier GmbH。All rights reserved.
Amphiphysins are BIN-amphiphysin-RVS (BAR) domain-containing proteins that influence membrane curvature in sites such as T-tubules in muscular cells, endocytic pits in neuronal as well as non-neuronal cells, and possibly cytoplasmic endosomes. This effect on lipid membranes is fulfilled by diverse amphiphysin 2/BIN1 isoforms, generated by alternative splicing and showing distinct structural and functional properties. In this study, our goal was to characterize the functional role of a ubiquitously expressed amphiphysin 2/BIN1 by the characterization of new molecular partners. We performed a two-hybrid screen with an isoform of amphiphysin 2/BIN1 expressed in HeLa cells. We identified CLIP-170 as an amphiphysin 2/BIN1-interacting molecule. CLIP-170 is a plus-end tracking protein involved in microtubule (MT) stability and recruitment of dynactin. The binding between amphiphysin 2/BIN1 and CLIP-170 is dependent on the N-terminal part of amphiphysin 2 (mostly the BAR domain) and an internal coiled-coil region of CLIP-170. This partnership was confirmed by GST pull-down assay and by co-immunoprecipitation in HeLa cells that express endogenous amphiphysin 2 (mostly isoforms 6, 9 and 10). When overexpressed in HeLa cells, amphiphysin 2/BIN1 leads to the formation of intracellular tubules which can closely align with MTs. After MT depolymerization by nocodazole, amphiphysin 2-stained tubules disappear, and reappear after nocodazole washout. Furthermore, depletion of CLIP-170 by RNAi induced a decrease in the proportion of cells with amphiphysin 2-stained tubules and an increase in the proportion of cells with no tubules. This result suggests the existence of a mechanistic link between the two types of tubules, which is likely to involve the +TIP protein, CLIP-170. Amphiphysin 2/BIN1 may be an anchoring point on membranes for CLIP-170, and consequently for MT. Then, the pushing force of polymerizing MT could help amphiphysin 2/BIN1 in its tubulation potential. We propose that amphiphysin 2/BIN1 participates in the tubulation of traffic intermediates and intracellular organelles first via its intrinsic tubulating potential and second via its ability to bind CLIP-170 and MT. (c) 2009 Elsevier GmbH. All rights reserved.