Dynamics of microtubules bundled by microtubule associated protein 2C (MAP2C).
MAP2C is a microtubule-associated protein abundant in immature nerve cells. We isolated a cDNA clone encoding whole mouse MAP2C of 467 amino acid residues. In fibroblasts transiently transfected with cDNA of MAP2C, interphase microtubule networks were reorganized into microtubule bundles. To reveal the dynamic properties of microtubule bundles, we analyzed the incorporation sites of exogenously introduced tubulin by microinjection of biotin-labeled tubulin and the turnover rate of microtubule bundles by photoactivation of caged fluorescein-labeled tubulin. The injected biotin-labeled tubulin was rapidly incorporated into distal ends of preexisting microtubule bundles, suggesting a concentration of the available ends of microtubules at this region. Although homogenous staining of microtubule bundles with antibiotin antibody was observed 2 h after injection, the photoactivation study indicated that turnover of microtubule bundles was extremely suppressed and < 10% of tubulin molecules would be exchanged within 1 h. Multiple photoactivation experiments provided evidence that neither catastrophic disassembly at the distal ends of bundles nor concerted disassembly due to treadmilling at the proximal ends could explain the observed rapid incorporation of exogenously introduced tubulin molecules. We conclude that microtubules bundled by MAP2C molecules are very stable while the abrupt increase of free tubulin molecules by microinjection results in rapid assembly from the distal ends within the bundles as well as free nucleation of small microtubules which are progressively associated laterally with preexisting microtubule bundles. This is the first detailed study of the function of MAPs on the dynamics of microtubules in vivo.
Umeyama
T
T
Department of Anatomy and Cell Biology, School of Medicine, University of Tokyo, Japan.
Okabe
S
S
Kanai
Y
Y
Hirokawa
N
N
D000818:Animals; D000831:Animals, Newborn; D001483:Base Sequence; D001921:Brain / Q000502:physiology*; D003001:Cloning, Molecular; D004247:DNA / Q000235:genetics; D004591:Electrophoresis, Polyacrylamide Gel; D005347:Fibroblasts / Q000166:cytology / Q000378:metabolism / Q000648:ultrastructure; D005455:Fluorescent Antibody Technique; D007399:Interphase; D051379:Mice; D008869:Microtubule-Associated Proteins / Q000032:analysis / Q000235:genetics / Q000378:metabolism*; D008870:Microtubules / Q000378:metabolism / Q000648:ultrastructure*; D008969:Molecular Sequence Data; D009838:Oligodeoxyribonucleotides; D011994:Recombinant Proteins / Q000032:analysis / Q000378:metabolism; D015183:Restriction Mapping; D017384:Sequence Deletion; D014162:Transfection
D008869:Microtubule-Associated Proteins; C079240:Mtap2 protein, mouse; D009838:Oligodeoxyribonucleotides; D011994:Recombinant Proteins; D004247:DNA