Abstract
Microtubule-associated proteins (MAPs) that copurify with tubulin through multiple cycles of in vitro assembly have been implicated as regulatory factors and effectors in the in vivo activity of microtubules. As an approach to the analysis of the functions of these molecules, a collection of lymphocyte hybridoma monoclonal antibodies has been generated using MAPs from HeLa cell microtubule protein as antigen. Two of the hybridoma clones secrete IgGs that bind to distinct sites on what appears to be a 200,000-dalton polypeptide. Both immunoglobulin preparations stain interphase and mitotic apparatus microtubules in cultured human cells. One of the clones (N-3B4.3.10) secretes antibody that reacts only with cells of human origin, while antibody from the other hybridoma (N-2B5.11.2) cross-reacts with BSC and PtK1 cells, but not with 3T3 cells. In PtK1 cells the N-2B5 antigen is associated with the microtubules of the mitotic apparatus, but there is no staining of the interphase microtubule array; rather, the antibody stains an ill-defined juxtanuclear structure. Further, neither antibody stains vinblastine crystals in either human or marsupial cells at any stage of the cell cycle. N-2B5 antibody microinjected into living PtK1 cells binds to the mitotic spindle, but does not cause a rapid dissolution of either mitotic or interphase microtubule structures. When injected before the onset of anaphase, however, the N- 2B5 antibody inhibits proper chromosome partition in mitotic PtK1 cells. N-2B5 antibody injected into interphase cells causes a redistribution of MAP antigen onto the microtubule network.
Full Text
The Full Text of this article is available as a PDF (4.6 MB).
Selected References
These references are in PubMed. This may not be the complete list of references from this article.
- Bensch K. G., Malawista S. E. Microtubular crystals in mammalian cells. J Cell Biol. 1969 Jan;40(1):95–107. doi: 10.1083/jcb.40.1.95. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Brinkley B. R., Fuller E. M., Highfield D. P. Cytoplasmic microtubules in normal and transformed cells in culture: analysis by tubulin antibody immunofluorescence. Proc Natl Acad Sci U S A. 1975 Dec;72(12):4981–4985. doi: 10.1073/pnas.72.12.4981. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Browne C. L., Lockwood A. H., Su J. L., Beavo J. A., Steiner A. L. Immunofluorescent localization of cyclic nucleotide-dependent protein kinases on the mitotic apparatus of cultured cells. J Cell Biol. 1980 Nov;87(2 Pt 1):336–345. doi: 10.1083/jcb.87.2.336. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Bulinski J. C., Borisy G. G. Immunofluorescence localization of HeLa cell microtubule-associated proteins on microtubules in vitro and in vivo. J Cell Biol. 1980 Dec;87(3 Pt 1):792–801. doi: 10.1083/jcb.87.3.792. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Bulinski J. C., Borisy G. G. Microtubule-associated proteins from cultured HeLa cells. Analysis of molecular properties and effects on microtubule polymerization. J Biol Chem. 1980 Dec 10;255(23):11570–11576. [PubMed] [Google Scholar]
- Bulinski J. C., Borisy G. G. Self-assembly of microtubules in extracts of cultured HeLa cells and the identification of HeLa microtubule-associated proteins. Proc Natl Acad Sci U S A. 1979 Jan;76(1):293–297. doi: 10.1073/pnas.76.1.293. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Burridge K. Direct identification of specific glycoproteins and antigens in sodium dodecyl sulfate gels. Methods Enzymol. 1978;50:54–64. doi: 10.1016/0076-6879(78)50007-4. [DOI] [PubMed] [Google Scholar]
- Connolly J. A., Kalnins V. I., Cleveland D. W., Kirschner M. W. Immunoflourescent staining of cytoplasmic and spindle microtubules in mouse fibroblasts with antibody to tau protein. Proc Natl Acad Sci U S A. 1977 Jun;74(6):2437–2440. doi: 10.1073/pnas.74.6.2437. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Connolly J. A., Kalnins V. I., Cleveland D. W., Kirschner M. W. Intracellular localization of the high molecular weight microtubule accessory protein by indirect immunofluorescence. J Cell Biol. 1978 Mar;76(3):781–786. doi: 10.1083/jcb.76.3.781. [DOI] [PMC free article] [PubMed] [Google Scholar]
- De Brabander M., Bulinski J. C., Geuens G., De Mey J., Borisy G. G. Immunoelectron microscopic localization of the 210,000-mol wt microtubule-associated protein in cultured cells of primates. J Cell Biol. 1981 Nov;91(2 Pt 1):438–445. doi: 10.1083/jcb.91.2.438. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Haskins K. M., Donoso J. A., Himes R. H. Spirals and paracrystals induced by Vinca alkaloids: evidence that microtubule-associated proteins act as polycations. J Cell Sci. 1981 Feb;47:237–247. doi: 10.1242/jcs.47.1.237. [DOI] [PubMed] [Google Scholar]
- Izant J. G., McIntosh J. R. Microtubule-associated proteins: a monoclonal antibody to MAP2 binds to differentiated neurons. Proc Natl Acad Sci U S A. 1980 Aug;77(8):4741–4745. doi: 10.1073/pnas.77.8.4741. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Izant J. G., Weatherbee J. A., McIntosh J. R. A microtubule-associated protein in the mitotic spindle and the interphase nucleus. Nature. 1982 Jan 21;295(5846):248–250. doi: 10.1038/295248a0. [DOI] [PubMed] [Google Scholar]
- Kim H., Binder L. I., Rosenbaum J. L. The periodic association of MAP2 with brain microtubules in vitro. J Cell Biol. 1979 Feb;80(2):266–276. doi: 10.1083/jcb.80.2.266. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Laemmli U. K. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature. 1970 Aug 15;227(5259):680–685. doi: 10.1038/227680a0. [DOI] [PubMed] [Google Scholar]
- Lin J. J., Feramisco J. R. Disruption of the in vivo distribution of the intermediate filaments in fibroblasts through the microinjection of a specific monoclonal antibody. Cell. 1981 Apr;24(1):185–193. doi: 10.1016/0092-8674(81)90514-6. [DOI] [PubMed] [Google Scholar]
- Ludueña R. F., Fellous A., Francon J., Nunez J., McManus L. Effect of tau on the vinblastine-induced aggregation of tubulin. J Cell Biol. 1981 Jun;89(3):680–683. doi: 10.1083/jcb.89.3.680. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Lydersen B. K., Pettijohn D. E. Human-specific nuclear protein that associates with the polar region of the mitotic apparatus: distribution in a human/hamster hybrid cell. Cell. 1980 Nov;22(2 Pt 2):489–499. doi: 10.1016/0092-8674(80)90359-1. [DOI] [PubMed] [Google Scholar]
- Mabuchi I., Okuno M. The effect of myosin antibody on the division of starfish blastomeres. J Cell Biol. 1977 Jul;74(1):251–263. doi: 10.1083/jcb.74.1.251. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Manson L. A., Verastegui-Cerdan E., Sporer R. A quantitative disc radioimmunoassay for antibodies directed against membrane-associated antigens. Curr Top Microbiol Immunol. 1978;81:232–234. doi: 10.1007/978-3-642-67448-8_38. [DOI] [PubMed] [Google Scholar]
- Matus A., Bernhardt R., Hugh-Jones T. High molecular weight microtubule-associated proteins are preferentially associated with dendritic microtubules in brain. Proc Natl Acad Sci U S A. 1981 May;78(5):3010–3014. doi: 10.1073/pnas.78.5.3010. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Murphy D. B., Borisy G. G. Association of high-molecular-weight proteins with microtubules and their role in microtubule assembly in vitro. Proc Natl Acad Sci U S A. 1975 Jul;72(7):2696–2700. doi: 10.1073/pnas.72.7.2696. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Nagle B. W., Doenges K. H., Bryan J. Assembly of tubulin from cultured cells and comparison with the neurotubulin model. Cell. 1977 Nov;12(3):573–586. doi: 10.1016/0092-8674(77)90258-6. [DOI] [PubMed] [Google Scholar]
- Sherline P., Schiavone K. Immunofluorescence localization of proteins of high molecular weight along intracellular microtubules. Science. 1977 Dec 9;198(4321):1038–1040. doi: 10.1126/science.337490. [DOI] [PubMed] [Google Scholar]
- Sloboda R. D., Dentler W. L., Rosenbaum J. L. Microtubule-associated proteins and the stimulation of tubulin assembly in vitro. Biochemistry. 1976 Oct 5;15(20):4497–4505. doi: 10.1021/bi00665a026. [DOI] [PubMed] [Google Scholar]
- Weatherbee J. A., Luftig R. B., Weihing R. R. In vitro polymerization of microtubules from HeLa cells. J Cell Biol. 1978 Jul;78(1):47–57. doi: 10.1083/jcb.78.1.47. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Weatherbee J. A., Luftig R. B., Weihing R. R. Purification and reconstitution of HeLa cell microtubules. Biochemistry. 1980 Aug 19;19(17):4116–4123. doi: 10.1021/bi00558a033. [DOI] [PubMed] [Google Scholar]
- Weatherbee J. A., Sherline P., Mascardo R. N., Izant J. G., Luftig R. B., Weihing R. R. Microtubule-associated proteins of HeLa cells: heat stability of the 200,000 mol wt HeLa MAPs and detection of the presence of MAP-2 in HeLa cell extracts and cycled microtubules. J Cell Biol. 1982 Jan;92(1):155–163. doi: 10.1083/jcb.92.1.155. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Weingarten M. D., Lockwood A. H., Hwo S. Y., Kirschner M. W. A protein factor essential for microtubule assembly. Proc Natl Acad Sci U S A. 1975 May;72(5):1858–1862. doi: 10.1073/pnas.72.5.1858. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Welsh M. J., Dedman J. R., Brinkley B. R., Means A. R. Calcium-dependent regulator protein: localization in mitotic apparatus of eukaryotic cells. Proc Natl Acad Sci U S A. 1978 Apr;75(4):1867–1871. doi: 10.1073/pnas.75.4.1867. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Wiche G., Cole R. D. Reversible in vitro polymerization of tubulin from a cultured cell line (rat glial cell clone C6). Proc Natl Acad Sci U S A. 1976 Apr;73(4):1227–1231. doi: 10.1073/pnas.73.4.1227. [DOI] [PMC free article] [PubMed] [Google Scholar]