Meunier Brigitte, Quaranta Muriel, Daviet Laurent, Hatzoglou Anastassia, Leprince Corinne
Analysis of Signal Transduction Group, INSERM U830, Institut Curie, Paris, France.
Eur J Cell Biol. 2009 Feb;88(2):91-102. doi: 10.1016/j.ejcb.2008.08.006. Epub 2008 Nov 12.
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.
发动蛋白是含有BIN-发动蛋白-RVS（BAR）结构域的蛋白质，可影响肌肉细胞中T小管、神经元及非神经元细胞中的内吞小窝以及可能的细胞质内体等部位的膜曲率。对脂质膜的这种作用由多种通过可变剪接产生且具有不同结构和功能特性的发动蛋白2/BIN1亚型实现。在本研究中，我们的目标是通过鉴定新的分子伴侣来表征普遍表达的发动蛋白2/BIN1的功能作用。我们用在HeLa细胞中表达的发动蛋白2/BIN1的一种亚型进行了双杂交筛选。我们鉴定出CLIP-170是一种与发动蛋白2/BIN1相互作用的分子。CLIP-170是一种正端追踪蛋白，参与微管（MT）的稳定性维持和动力蛋白激活蛋白的募集。发动蛋白2/BIN1与CLIP-170之间的结合依赖于发动蛋白2的N端部分（主要是BAR结构域）和CLIP-170的一个内部卷曲螺旋区域。这种伙伴关系通过GST下拉实验以及在表达内源性发动蛋白2（主要是亚型6、9和10）的HeLa细胞中的共免疫沉淀得以证实。当在HeLa细胞中过表达时，发动蛋白2/BIN1会导致形成可与微管紧密排列的细胞内小管。在用诺考达唑使微管解聚后，被发动蛋白2染色的小管消失，并在诺考达唑洗脱后重新出现。此外，通过RNA干扰耗尽CLIP-170会导致被发动蛋白2染色的小管的细胞比例下降以及无小管的细胞比例增加。这一结果表明这两种类型的小管之间存在一种机制联系，这可能涉及正端追踪蛋白CLIP-170。发动蛋白2/BIN1可能是CLIP-170在膜上的一个锚定点，因此也是微管的锚定点。然后，聚合微管的推力可能有助于发动蛋白2/BIN1发挥其形成小管的潜力。我们提出，发动蛋白2/BIN1首先通过其内在的形成小管的潜力，其次通过其结合CLIP-170和微管的能力，参与运输中间体和细胞内细胞器的小管形成。
