title: Visualization of microtubule growth in cultured neurons via the use of EB3-GFP (end-binding protein 3-green fluorescent protein).
abstract: Several microtubule binding proteins, including CLIP-170 (cytoplasmic linker protein-170), CLIP-115, and EB1 (end-binding protein 1), have been shown to associate specifically with the ends of growing microtubules in non-neuronal cells, thereby regulating microtubule dynamics and the binding of microtubules to protein complexes, organelles, and membranes. When fused to GFP (green fluorescent protein), these proteins, which collectively are called +TIPs (plus end tracking proteins), also serve as powerful markers for visualizing microtubule growth events. Here we demonstrate that endogenous +TIPs are present at distal ends of microtubules in fixed neurons. Using EB3-GFP as a marker of microtubule growth in live cells, we subsequently analyze microtubule dynamics in neurons. Our results indicate that microtubules grow slower in neurons than in glia and COS-1 cells. The average speed and length of EB3-GFP movements are comparable in cell bodies, dendrites, axons, and growth cones. In the proximal region of differentiated dendrites approximately 65% of EB3-GFP movements are directed toward the distal end, whereas 35% are directed toward the cell body. In more distal dendritic regions and in axons most EB3-GFP dots move toward the growth cone. This difference in directionality of EB3-GFP movements in dendrites and axons reflects the highly specific microtubule organization in neurons. Together, these results suggest that local microtubule polymerization contributes to the formation of the microtubule network in all neuronal compartments. We propose that similar mechanisms underlie the specific association of CLIPs and EB1-related proteins with the ends of growing microtubules in non-neuronal and neuronal cells.
authors.affiliation: Medical Genetics Center Department of Cell Biology and Genetics, Erasmus University, 3000 DR Rotterdam, The Netherlands.
authors.forename: Tatiana
authors.initials: T
authors.lastname: Stepanova
authors.forename: Jenny
authors.initials: J
authors.lastname: Slemmer
authors.forename: Casper C
authors.initials: CC
authors.lastname: Hoogenraad
authors.forename: Gideon
authors.initials: G
authors.lastname: Lansbergen
authors.forename: Bjorn
authors.initials: B
authors.lastname: Dortland
authors.forename: Chris I
authors.initials: CI
authors.lastname: De Zeeuw
authors.forename: Frank
authors.initials: F
authors.lastname: Grosveld
authors.forename: Gert
authors.initials: G
authors.lastname: van Cappellen
authors.forename: Anna
authors.initials: A
authors.lastname: Akhmanova
authors.forename: Niels
authors.initials: N
authors.lastname: Galjart
mesh_terms: D000818:Animals; D015415:Biomarkers / Q000032:analysis; D019556:COS Cells; D002454:Cell Differentiation; D002478:Cells, Cultured; D003598:Cytoskeletal Proteins / Q000032:analysis* / Q000235:genetics / Q000378:metabolism; D003712:Dendrites / Q000737:chemistry / Q000648:ultrastructure; D049452:Green Fluorescent Proteins; D006624:Hippocampus / Q000166:cytology; D008164:Luminescent Proteins / Q000032:analysis* / Q000235:genetics; D051379:Mice; D018613:Microscopy, Confocal; D008869:Microtubule-Associated Proteins; D008870:Microtubules / Q000648:ultrastructure*; D009068:Movement; D009474:Neurons / Q000737:chemistry / Q000378:metabolism / Q000648:ultrastructure*; D011689:Purkinje Cells / Q000737:chemistry / Q000648:ultrastructure; D011993:Recombinant Fusion Proteins / Q000032:analysis; D014162:Transfection
chemical_list: D015415:Biomarkers; D003598:Cytoskeletal Proteins; D008164:Luminescent Proteins; C403664:MAPRE3 protein, human; D008869:Microtubule-Associated Proteins; D011993:Recombinant Fusion Proteins; D049452:Green Fluorescent Proteins