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. 1998 May 18;141(4):849-62.
doi: 10.1083/jcb.141.4.849.

Evidence for a role of CLIP-170 in the establishment of metaphase chromosome alignment

D Dujardin  1 , U I Wacker, A Moreau, T A Schroer, J E Rickard, J R De Mey
Affiliations

Evidence for a role of CLIP-170 in the establishment of metaphase chromosome alignment

D Dujardin et al. J Cell Biol. .

Abstract

CLIPs (cytoplasmic linker proteins) are a class of proteins believed to mediate the initial, static interaction of organelles with microtubules. CLIP-170, the CLIP best characterized to date, is required for in vitro binding of endocytic transport vesicles to microtubules. We report here that CLIP-170 transiently associates with prometaphase chromosome kinetochores and codistributes with dynein and dynactin at kinetochores, but not polar regions, during mitosis. Like dynein and dynactin, a fraction of the total CLIP-170 pool can be detected on kinetochores of unattached chromosomes but not on those that have become aligned at the metaphase plate. The COOH-terminal domain of CLIP-170, when transiently overexpressed, localizes to kinetochores and causes endogenous full-length CLIP-170 to be lost from the kinetochores, resulting in a delay in prometaphase. Overexpression of the dynactin subunit, dynamitin, strongly reduces the amount of CLIP-170 at kinetochores suggesting that CLIP-170 targeting may involve the dynein/dynactin complex. Thus, CLIP-170 may be a linker for cargo in mitosis as well as interphase. However, dynein and dynactin staining at kinetochores are unaffected by this treatment and further overexpression studies indicate that neither CLIP-170 nor dynein and dynactin are required for the formation of kinetochore fibers. Nevertheless, these results strongly suggest that CLIP-170 contributes in some way to kinetochore function in vivo.

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Figures

Figure 1
Figure 1
Subcellular distribution of CLIP-170 throughout the mitotic cycle of human A431 cells. (a, e, i, m, and q) CREST autoimmune staining; (b, f, j, n, and r) anti–CLIP-170 staining (pAb rα55); (c, g, k, and o) superimposition of the CREST (red) and CLIP-170 (green) signals. (d, h, l, and p) DAPI DNA staining. (a–d) Prophase cells. (e–h) Early prometaphase cells. The inset in g shows the relative location of CREST antigens and CLIP-170. (i–l) Late prometaphase. (m–p) Metaphase cell (top) and anaphase cell (bottom). (q and r) Caco2 cell treated with vinblastine. Note the strong CLIP-170 signal at centromeric regions. All the images are maximal projections of optical section stacks. (s) thin section of a vinblastine-treated Caco2 cell labeled for CLIP-170 with pAb rα55 and silver-enhanced immunogold staining. Silver particles accumulate at the kinetochore. The cells were fixed in formaldehyde followed by detergent extraction (or cold methanol treatment in s). Bars: (a–r) 5 μm; (s) 0.05 μm.
Figure 2
Figure 2
CLIP-170, cytoplasmic dynein and dynactin components transiently colocalize at kinetochores of nonaligned chromosomes. (a and c) Anti-dynactin (Arp1) staining; (b, d, and f) Anti–CLIP-170 staining (monoclonal anti– CLIP-170, 4D3 in b and d, rabbit anti–CLIP-170 [rα55] in f); (g) anti-cytoplasmic dynein intermediate chain staining. (e) CREST autoimmune staining. (a–d) Cells were fixed in paraformaldehyde and permeabilized in cold methanol. a and b show the colocalization of Arp1 and CLIP-170 at kinetochores of a prometaphase cell. Staining is no longer seen at aligned chromosomes in a metaphase cell (c and d). The color insets show the overlay of CLIP-170 (red) and Arp1 signals (green). Note the relative absence of CLIP-170 concentration in the polar regions. (e–g) cells were fixed in −20°C methanol. Arrows show the same kinetochores labeled for cytoplasmic dynein and CLIP-170 in a prometaphase cell. Similar results were obtained with a monoclonal against p150GLUED (not shown). a–d are complete maximal projections of optical section stacks. e–g were taken using a cooled CCD camera. Bar, 5 μm.
Figure 3
Figure 3
(a–b) CLIP-170 staining of isolated HeLa chromosomes. Chromosomes were isolated from colcemid-treated HeLa cells, then labeled with anti–CLIP-170 (pAb rα55) (a) and CREST auto-immune serum (b) and imaged by confocal microscopy (a and b) and differential interference contrast microscopy (c). In a and b, a single optical section is shown. (d–f) Coomassie blue stained gel (d) and Western blots (e and f), of cell fractions in the course of chromosome purification. Lane 1, total cell extract; lane 2, cytosol; lane 3, chromosomes purified on a glycerol gradient; lane 4, chromosomes purified on a Percoll gradient. The amounts of the samples used were adjusted to yield comparative immune signals and do not reflect the quantitative distribution of CLIP-170 in the various fractions. (e) Labeling with anti–CLIP-170 serum rα55; (f) labeling with anti–CLIP-170 mAb 4D3. The lower band in f, lane 4 is a COOH-terminal proteolytic fragment of CLIP-170, that is not recognized by rα55. Bar, 2 μm.
Figure 4
Figure 4
CLIP-170 species that possess the COOH-terminal domain can bind kinetochores. a–i show COS-7 cells transiently transfected by standard techniques. j–o show A431 cells seeded on CELLocate coverslips, synchronized and intranuclearly microinjected with plasmids, and then treated with nocodazole before fixation and processing for immunofluorescence. The fixation in 3% paraformaldehyde was preceded by extraction in Triton X-100, in order to render kinetochore-bound CLIP-170 detectable. The cells were double labeled with CREST autoimmune serum (a, d, g, j, and m) and anti-myc (b, e, h, k, and n). c, f, i, l, and o are superimpositions of CREST (red) and myc (green) signals. The A431 cells were relocated on the coverslip. Myc staining is seen at kinetochores of nonaligned chromosomes in cells transiently overexpressing wt-CLIP-170 (a–c), ΔN (d–f), and ΔNΔR (g–i). CLIP-170 species lacking the COOH-terminal domain (ΔC [j–l] and ΔNΔC [m–o]) do not appear to bind kinetochores. The panels at the left of the figure are diagrams representing the constructs used. All the panels, except d–i are complete maximal projections of x/y optical section stacks. d–i were taken using a cooled CCD camera. Bar, 5 μm.
Figure 5
Figure 5
Effect of overexpressing dynamitin on anti–CLIP-170 staining of kinetochores. COS-7 cells transfected with chicken dynamitin were nocodazole-treated and double stained with anti-dynamitin (a and d), monoclonal anti–CLIP-170 (b and e), and DAPI for DNA staining (c and f). (a–c) A mitotic cell, representative of the majority of the cells overexpressing dynamitin. No CLIP-170 signal is visible at kinetochores. (d–f) mitotic cell to the right, not overexpressing dynamitin: normal CLIP-170 staining at the kinetochores. Mitotic cell to the left, overexpressing dynamitin: diminished CLIP-170 at the kinetochores, a phenotype seen in a small subpopulation of cells. The cells were fixed in formaldehyde followed by detergent extraction. The images were taken using a cooled CCD camera. Bar, 10 μm.
Figure 6
Figure 6
Effect of overexpression of CLIP-170 deletion mutants on mitotic index (A) and mitotic progression (B). COS-7 cells were transfected with constructs of CLIP-170 or dynamitin. (A) The mitotic indexes of cells overexpressing dynamitin and the CLIP-170 species addressed to the kinetochore were significantly different from the control value in transfected, nonoverexpressing cells, whereas that for ΔNΔC–CLIP-170 indicates a block of entry into M-phase. (B) Mitotic phase indexes of nonoverexpressing cells in the transfected population (control, black bars), and of cells overexpressing dynamitin or various CLIP-170 deletion mutants. Cultures stained with anti–CLIP-170, anti-tubulin, and DAPI were scored for mitotic phase on the basis of chromosome configurations and spindle morphology. All values are means from three independent experiments. At least 3,000 transfected cells (mitotic and nonmitotic) were counted for each construct. For ΔNΔC, more than 50,000 cells were counted because of the very low mitotic index (O.19%). Values for the dynamitin construct were comparable to those of a previous study (Echeverri et al., 1996). Those for wt-CLIP-170, ΔN and ΔNΔR were found to be significantly different from those for ΔNΔC and for transfected nonoverexpressing control cells (P < 0.05, according to Student's t test). The latter were not significantly different from the values for untransfected cultures (not shown).
Figure 7
Figure 7
Analysis of the phenotypic effects of ΔNΔR and dynamitin overexpression on spindle morphology. (a–c) ΔNΔR transfected COS-7 cells were stained for tubulin (green) and overexpressed CLIP-170 (pAb rα55, red); (a and a′) show the spindle of two nonexpressing early prometaphase cells in the transfected population. (b and c) show the spindles of two ΔNΔR overexpressing cells that were scored as being in pseudoprometaphase. The images show complete maximal projections of optical section stacks. The ΔNΔR staining is provided as a single optical section to allow for the localization of chromosomes. The cells were fixed in 4% paraformaldehyde, 0.05% glutaraldehyde, and 0.05% Triton X-100. (d–f) Analysis of the phenotypic effects of ΔNΔR and dynamitin overexpression on chromosome/spindle interactions. COS-7 cells were transfected with ΔNΔR (d and e) or dynamitin (f) then labeled with antibodies for tubulin, the CREST antigen and the transfected protein (d and f), or with antibodies to tubulin, the transfected protein and DAPI for the chromosomes (e). d and f are superimpositions of selected single optical x/y sections of the CREST (red) and tubulin signals (green). Labeling for the transfected proteins is not shown but was used to identify transfected cells. The kinetochores are apparently correctly attached to kinetochore fibers despite the overexpression of ΔNΔR or dynamitin. e represents a superimposition of three single optical sections showing tubulin (green), ΔNΔR (blue), and DNA (red) signals. The labeling of the chromosomes shows that their arms are correctly oriented outward. The cells in e were fixed in 4% paraformaldehyde, 0.05% glutaraldehyde, and 0.05% Triton X-100. The cells in d and f were fixed with the same mixture, except that 0.01% glutaraldehyde was used. Bar: (a–d) 5 μm; (e and f), 2.5 μm.
Figure 8
Figure 8
Overexpression of ΔNΔR displaces endogenous CLIP-170 from kinetochores, but has no effect on the transient kinetochore localization of dynactin (Arp1) and ΔNΔR. COS-7 cells were transfected with ΔNΔR and fixed in 3% paraformaldehyde, followed by permeabilization with detergent (a–f and k–m) or cold methanol (g–j). Cells were labeled for the transfected ΔNΔR with the anti–CLIP-170 pAb H2A (a and d) or with monoclonal anti–c-myc (g, i, and l), for endogenous CLIP-170 with a monoclonal, 4D3 (b and e), which does not recognize the transfected protein, for chromosomes with DAPI (c, f, and m) and for Arp1 (h and j). Overexpression of ΔNΔR leads to diminished staining of nonaligned kinetochores for the endogenous protein with 4D3 (a–c), whereas in nonexpressing cells, 4D3 strongly labels such kinetochores (d–f). (g–j) Arp1 labeling of cells expressing ΔNΔR shows that the targeting of this protein to prometaphase kinetochores (g and h) as well as its disappearance from aligned chromosomes (i and j) is unaffected by overexpression of ΔNΔR. Panels (a–f) are images taken using a cooled CCD camera. g and i are single optical sections of the ΔNΔR signal and h and j are maximal projections of the Arp1 signal. Bars, 5 μm.
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References

    1. Aniento F, Gruenberg J. Membrane transport from early to late endosomes. Cold Spring Harbor Symp Quant Biol. 1995;60:205–209. - PubMed
    1. Barton NR, Goldstein LS. Going mobile: microtubule motors and chromosome segregation. Proc Natl Acad Sci USA. 1996;93:1735–1742. - PMC - PubMed
    1. Berlin V, Styles CA, Fink GR. BIK1, a protein required for microtubule function during mating and mitosis in Saccharomyces cerevisiae, colocalizes with tubulin. J Cell Biol. 1990;111:2573–2586. - PMC - PubMed
    1. Bernat RL, Delannoy MR, Rothfield NF, Earnshaw WC. Disruption of centromere assembly during interphase inhibits kinetochore morphogenesis and function in mitosis. Cell. 1991;66:1229–1238. - PubMed
    1. Clark SW, Meyer DI. ACT3: a putative centractin homologue is S. cerevisiaeis required for proper orientation of the mitotic spindle. J Cell Biol. 1994;127:129–138. - PMC - PubMed

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