EB1 identifies sites of microtubule polymerisation during neurite development.
EB1 is a microtubule associated protein which interacts with the APC tumour suppressor protein and components of the cytoplasmic dynein/dynactin complex. EB1 is also a specific marker of growing microtubule tips. Here we demonstrate that EB1 protein levels are increased during axon but not dendrite formation in differentiated N2A neuroblastoma cells, and that EB1 localises to microtubule tips throughout extending neurites in these cells. In N2A axons, analysis of the ratio of EB1/beta-tubulin fluorescence demonstrated that the distal tip region contained the highest proportion of polymerising microtubules. Time-lapse confocal imaging of an EB1-GFP fusion protein in transfected N2A cells directly revealed the dynamics of microtubule extension in neurites, and demonstrated the existence of unusual, discrete knots of microtubule polymerisation at the periphery of non-process bearing cells which may represent an early event in neurite outgrowth. We conclude that EB1 localisation can be used to identify and analyse sites of microtubule polymerisation at a high resolution during neurite development, a process to which it may contribute.
Morrison
E E
EE
Molecular Medicine Unit, Clinical Sciences Building, St. James's University Hospital, Beckett St., LS9 7TF, Leeds, UK. e.e.morrison@leeds.ac.uk
Moncur
P M
PM
Askham
J M
JM
D000818:Animals; D001369:Axons / Q000378:metabolism* / Q000648:ultrastructure; D001704:Biopolymers; D003712:Dendrites / Q000737:chemistry / Q000648:ultrastructure; D049452:Green Fluorescent Proteins; D008164:Luminescent Proteins / Q000032:analysis / Q000235:genetics; D046911:Macromolecular Substances; D051379:Mice; D018613:Microscopy, Confocal; D008856:Microscopy, Fluorescence; D008869:Microtubule-Associated Proteins / Q000032:analysis* / Q000235:genetics; D008870:Microtubules / Q000737:chemistry / Q000378:metabolism*; D009419:Nerve Tissue Proteins / Q000032:analysis* / Q000235:genetics; D009447:Neuroblastoma / Q000473:pathology; D025941:Protein Interaction Mapping; D011993:Recombinant Fusion Proteins / Q000032:analysis; D014404:Tubulin / Q000032:analysis; D014407:Tumor Cells, Cultured / Q000737:chemistry / Q000648:ultrastructure
D001704:Biopolymers; C405804:EB1 microtubule binding proteins; D008164:Luminescent Proteins; D046911:Macromolecular Substances; D008869:Microtubule-Associated Proteins; D009419:Nerve Tissue Proteins; D011993:Recombinant Fusion Proteins; D014404:Tubulin; D049452:Green Fluorescent Proteins