Analysis of mitotic microtubule-associated proteins using mass spectrometry identifies astrin, a spindle-associated protein - PubMed (original) (raw)

Analysis of mitotic microtubule-associated proteins using mass spectrometry identifies astrin, a spindle-associated protein

G J Mack et al. Proc Natl Acad Sci U S A. 2001.

Abstract

We purified microtubules from a mammalian mitotic extract and obtained an amino acid sequence from each microtubule-associated protein by using mass spectrometry. Most of these proteins are known spindle-associated components with essential functional roles in spindle organization. We generated antibodies against a protein identified in this collection and refer to it as astrin because of its association with astral microtubule arrays assembled in vitro. Astrin is approximately 134 kDa, and except for a large predicted coiled-coil domain in its C-terminal region it lacks any known functional motifs. Astrin associates with spindle microtubules as early as prophase where it concentrates at spindle poles. It localizes throughout the spindle in metaphase and anaphase and associates with midzone microtubules in anaphase and telophase. Astrin also localizes to kinetochores but only on those chromosomes that have congressed. Deletion analysis indicates that astrin's primary spindle-targeting domain is at the C terminus, although a secondary domain in the N terminus can target some of the protein to spindle poles. Thus, we have generated a comprehensive list of major mitotic microtubule-associated proteins, among which is astrin, a nonmotor spindle protein.

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Figures

Figure 1

Figure 1

Identification of mitotic microtubule-associated proteins. (A) Equivalent quantities of the total extract and the insoluble fractions obtained after incubation with (+) and without (−) taxol were separated by size on 7.5% SDS/PAGE and stained with Coomassie blue. Protein identities revealed by mass spectrometry are indicated on the right of each protein band. The lower panels show immunoblots of each of these three samples for NuMA and Eg5 to verify the enrichment of microtubule-associated proteins by using this technique. Molecular masses in kDa are indicated on the left. (B) Immunoblots of various microtubule-associated proteins (as indicated) identified by mass spectrometry of soluble (S) and insoluble (P) fractions obtained after microtubule aster assembly in the mitotic extract.

Figure 2

Figure 2

Alignment of the human astrin protein sequence with mouse S17 and rat Spag5 protein sequences. Identical amino acids residues are shaded, and predicted coiled-coil domains spanning amino acids 550–857 and 970–1,175 are in boxes.

Figure 3

Figure 3

Cell cycle distribution of astrin. HeLa cells were fixed in cold methanol and stained with antibodies against astrin, tubulin, and the DNA-specific dye 4′,6-diamidino-2-phenylindole (DAPI) as indicated. Cells were classified as being in interphase (A), prophase (B), prometaphase (C), metaphase (D), anaphase (E), and telophase (F) on the basis of the morphology and alignment of chromosomes and spindle organization. (Bar, 10 μm.)

Figure 4

Figure 4

Localization of astrin to kinetochores. CFPAC-1 cells were fixed in glutaraldehyde and stained with antibodies against astrin, anti-centromere antibodies (ACA), and the DNA-specific dye 4′,6-diamidino-2-phenylindole (DAPI) as indicated. Cells were classified as being in prometaphase (A) and metaphase (B) on the basis of chromosome alignment. Arrowheads highlight sister kinetochores in metaphase that stain for astrin, and the arrow highlights the kinetochore of an unaligned chromosome in prometaphase that lacks astrin staining. (Bar, 10 μm.)

Figure 5

Figure 5

Expression of GFP-astrin fusion proteins in HeLa cells. (A) Schematic diagram of astrin fragments that were tested for their ability to bind the mitotic spindle. +++, strong binding; ++, moderate binding; +, weak binding; −, no binding; shaded boxes, regions of astrin predicted to form a coiled-coil structure. (B) Immunoblot of transfected HeLa cell lysates expressing the various GFP-astrin fusion proteins. Molecular masses in kDa are indicated on the left. (C) Immunofluorescent images of metaphase mitotic cells expressing the various GFP-astrin fusion proteins. Cells were fixed in cold methanol, and GFP was detected with a rabbit anti-GFP antibody. (Bar, 10 μm.)

References

    1. Compton D A. Annu Rev Biochem. 2000;69:95–114. - PubMed
    1. McIntosh J R, Koonce M P. Science. 1989;246:622–628. - PubMed
    1. Mitchison T J. Curr Opin Cell Biol. 1989;1:69–74. - PubMed
    1. Rieder C L. Curr Opin Cell Biol. 1991;3:59–66. - PubMed
    1. Hyman A A, Karsenti E. Cell. 1996;84:401–410. - PubMed

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