title: The membrane-tubulating potential of amphiphysin 2/BIN1 is dependent on the microtubule-binding cytoplasmic linker protein 170 (CLIP-170).
abstract: 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.
authors.affiliation: Analysis of Signal Transduction Group, INSERM U830, Institut Curie, Paris, France.
authors.forename: Brigitte
authors.initials: B
authors.lastname: Meunier
authors.forename: Muriel
authors.initials: M
authors.lastname: Quaranta
authors.forename: Laurent
authors.initials: L
authors.lastname: Daviet
authors.forename: Anastassia
authors.initials: A
authors.lastname: Hatzoglou
authors.forename: Corinne
authors.initials: C
authors.lastname: Leprince
mesh_terms: D002462:Cell Membrane / Q000187:drug effects / Q000378:metabolism*; D022081:Cell Surface Extensions / Q000187:drug effects / Q000378:metabolism*; D005786:Gene Expression Regulation / Q000187:drug effects; D006367:HeLa Cells; D006801:Humans; D042541:Intracellular Space / Q000187:drug effects / Q000378:metabolism; D008869:Microtubule-Associated Proteins / Q000737:chemistry / Q000378:metabolism*; D008870:Microtubules / Q000187:drug effects / Q000378:metabolism*; D009363:Neoplasm Proteins / Q000737:chemistry / Q000378:metabolism*; D009419:Nerve Tissue Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D015739:Nocodazole / Q000494:pharmacology; D011485:Protein Binding / Q000187:drug effects; D020033:Protein Isoforms / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D017434:Protein Structure, Tertiary; D021381:Protein Transport / Q000187:drug effects; D014404:Tubulin / Q000378:metabolism; D020798:Two-Hybrid System Techniques
chemical_list: D008869:Microtubule-Associated Proteins; D009363:Neoplasm Proteins; D009419:Nerve Tissue Proteins; D020033:Protein Isoforms; D014404:Tubulin; C075637:amphiphysin; C076744:cytoplasmic linker protein 170; D015739:Nocodazole