title: Microtubule plus-end loading of p150(Glued) is mediated by EB1 and CLIP-170 but is not required for intracellular membrane traffic in mammalian cells.
abstract: Microtubule dynamics and function are regulated, at least in part, by a family of proteins that localize to microtubule plus-ends, and include EB1, CLIP-170 and the dynactin component p150(Glued). Plus-end pools of these proteins, notably dynactin, have been invoked in a number of ;search-and-capture' mechanisms, including the attachment of microtubules to kinetochores during mitosis and to endomembranes prior to the initiation of intracellular transport. Here we show that, in mammalian cells, EB1 is required for the plus-end localization of CLIP-170, and that this is in turn required to localize p150(Glued) to plus-ends. Specific depletion of CLIP-170 results in defects in microtubule dynamics, cell polarization in response to scratch wounding and a loss of p150(Glued) from plus ends. By contrast, removal of p150(Glued) from plus-ends by depletion of either EB1 or CLIP-170 caused no defects in the localization of intracellular organelles, the dynamics of ER-to-Golgi transport, the efficiency of transferrin uptake or the motility of early endosomes or lysosomes. In addition to labelling microtubule plus-ends, we show that GFP-p150(Glued) becomes incorporated into the dynactin complex and labels small, highly dynamic, punctate structures that move along microtubules. A subset of these structures colocalizes with ER-Golgi transport intermediates. Together, these data show that the function of CLIP-170 and p150(Glued) in membrane trafficking is not associated with their plus-end localization.
authors.affiliation: Department of Biochemistry, University of Bristol, School of Medical Sciences, University Walk, Bristol, BS8 1TD, UK.
authors.forename: Peter
authors.initials: P
authors.lastname: Watson
authors.forename: David J
authors.initials: DJ
authors.lastname: Stephens
mesh_terms: D001692:Biological Transport / Q000502:physiology; D002478:Cells, Cultured; D000072159:Dynactin Complex; D049452:Green Fluorescent Proteins / Q000378:metabolism; D006367:HeLa Cells; D006801:Humans; D008869:Microtubule-Associated Proteins / Q000378:metabolism* / Q000502:physiology; D008870:Microtubules / Q000378:metabolism*; D009363:Neoplasm Proteins / Q000378:metabolism* / Q000502:physiology; D015388:Organelles / Q000378:metabolism; D034741:RNA, Small Interfering / Q000378:metabolism; D011994:Recombinant Proteins / Q000378:metabolism; D014018:Tissue Distribution; D014162:Transfection; D022161:Transport Vesicles / Q000378:metabolism*
chemical_list: C000606360:DCTN1 protein, human; D000072159:Dynactin Complex; C405804:EB1 microtubule binding proteins; C485191:GFP10 Protein; D008869:Microtubule-Associated Proteins; D009363:Neoplasm Proteins; D034741:RNA, Small Interfering; D011994:Recombinant Proteins; D049452:Green Fluorescent Proteins; C076744:cytoplasmic linker protein 170