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Journal of Cell Science, Vol 100, Issue 3 533-540, Copyright © 1991 by Company of Biologists
JOURNAL ARTICLES |
T Maekawa and R Kuriyama
Department of Cell Biology and Neuroanatomy, University of Minnesota, Minneapolis 55455.
As cells enter mitosis, centrosomes undergo many transformations and become associated with different molecules in a stage-specific manner. We have developed a protocol for immunofluorescence staining with four antibody probes that can help us to follow the interaction of centrosomal components during mitosis. The cells were first stained with a human autoimmune serum (5051); a monoclonal anti-phosphocentrosomal antibody (CHO3); and an antitubulin antibody. Localization of the antibodies was detected using rhodamine-, fluorescein- and AMCA-conjugated second antibodies, respectively. After photographing marked mitotic cells, coverslips were soaked with 0.2 M glycine-HCl at pH 1.0 for 1 h to release all antibodies bound to the structures. The same cells were re-stained with a human autoantibody (SP-H) specific for spindle poles and a fluorescein-conjugated second antibody. This allowed us to compare the subcellular distribution of three kinds of centrosomal antigens in a single cell. Mitotic PtK1 cells treated with either nocodazole or taxol included microtubule-containing cytoplasmic foci and parallel bundles of short microtubules at the cell periphery. All the centrosomal antibodies stained the same one or two dots corresponding to structures labeled by the tubulin antibody. CHO3 also revealed extra cytoplasmic foci, whereas the SP-H antigen was additionally localized at one end of the free microtubule bundles. As the microtubules reorganized into bipolar spindles during the recovery from drug treatment, the CHO3 and SP-H antigens coalesced into the spindle poles where the 5051 antigen was located, suggesting that centrosomal antigens become associated with spindle poles through very different recruitment pathways.
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  R. Kuriyama, Y. Terada, K. S. Lee, and C. L. C. Wang Centrosome replication in hydroxyurea-arrested CHO cells expressing GFP-tagged centrin2 J. Cell Sci., July 15, 2007; 120(14): 2444 - 2453. [Abstract] [Full Text] [PDF]
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H Masuda and T Shibata Role of gamma-tubulin in mitosis-specific microtubule nucleation from the Schizosaccharomyces pombe spindle pole body J. Cell Sci., January 1, 1996; 109(1): 165 - 177. [Abstract] [PDF]
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A Vassilev, M Kimble, C. Silflow, M LaVoie, and R Kuriyama Identification of intrinsic dimer and overexpressed monomeric forms of gamma-tubulin in Sf9 cells infected with baculovirus containing the Chlamydomonas gamma-tubulin sequence J. Cell Sci., January 3, 1995; 108(3): 1083 - 1092. [Abstract] [PDF]
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T. Tang, C. Tang, Y. Chao, and C. Wu Nuclear mitotic apparatus protein (NuMA): spindle association, nuclear targeting and differential subcellular localization of various NuMA isoforms J. Cell Sci., January 6, 1994; 107(6): 1389 - 1402. [Abstract] [PDF]
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T Maekawa and R Kuriyama Primary structure and microtubule-interacting domain of the SP-H antigen: a mitotic MAP located at the spindle pole and characterized as a homologous protein to NuMA J. Cell Sci., January 6, 1993; 105(2): 589 - 600. [Abstract] [PDF]
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M Kallajoki, J Harborth, K Weber, and M Osborn Microinjection of a monoclonal antibody against SPN antigen, now identified by peptide sequences as the NuMA protein, induces micronuclei in PtK2 cells J. Cell Sci., January 1, 1993; 104(1): 139 - 150. [Abstract] [PDF]
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