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. 2001 Jul;12(7):1995-2009.
doi: 10.1091/mbc.12.7.1995.

Microtubule-dependent Changes in Assembly of Microtubule Motor Proteins and Mitotic Spindle Checkpoint Proteins at PtK1 Kinetochores

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Free PMC article

Microtubule-dependent Changes in Assembly of Microtubule Motor Proteins and Mitotic Spindle Checkpoint Proteins at PtK1 Kinetochores

D B Hoffman et al. Mol Biol Cell. .
Free PMC article

Abstract

The ability of kinetochores to recruit microtubules, generate force, and activate the mitotic spindle checkpoint may all depend on microtubule- and/or tension-dependent changes in kinetochore assembly. With the use of quantitative digital imaging and immunofluorescence microscopy of PtK1 tissue cells, we find that the outer domain of the kinetochore, but not the CREST-stained inner core, exhibits three microtubule-dependent assembly states, not directly dependent on tension. First, prometaphase kinetochores with few or no kinetochore microtubules have abundant punctate or oblate fluorescence morphology when stained for outer domain motor proteins CENP-E and cytoplasmic dynein and checkpoint proteins BubR1 and Mad2. Second, microtubule depolymerization induces expansion of the kinetochore outer domain into crescent and ring morphologies around the centromere. This expansion may enhance recruitment of kinetochore microtubules, and occurs with more than a 20- to 100-fold increase in dynein and relatively little change in CENP-E, BubR1, and Mad2 in comparison to prometaphase kinetochores. Crescents disappear and dynein decreases substantially upon microtubule reassembly. Third, when kinetochores acquire their full metaphase complement of kinetochore microtubules, levels of CENP-E, dynein, and BubR1 decrease by three- to sixfold in comparison to unattached prometaphase kinetochores, but remain detectable. In contrast, Mad2 decreases by 100-fold and becomes undetectable, consistent with Mad2 being a key factor for the "wait-anaphase" signal produced by unattached kinetochores. Like previously found for Mad2, the average amounts of CENP-E, dynein, or BubR1 at metaphase kinetochores did not change with the loss of tension induced by taxol stabilization of microtubules.

Figures

Figure 1
Figure 1
Immunofluorescence colocalization of BubR1, CENP-E, and Mad2 with cytoplasmic dynein to the kinetochore outer domain. Kinetochore outer domain crescents were induced by incubation of mitotic cells for 4 h in 20 μM nocodazole to completely depolymerize spindle and kinetochore microtubules. Rows contain images of the same cell. (A–C) Left column shows DIC images of the cells, the center column shows fluorescein isothiocyanate fluorescence of cytoplasmic dynein, and the right column shows rhodamine fluorescence staining of BubR1, CENP-E, or Mad2. (D) Left frame is CREST staining, the right is Mad2 staining, and the lower is a color overlay for the same cell, with 4,6-diaminidino-2-phenylindole (DAPI) staining for DNA. Note again that Mad2, like BubR1, CENP-E, and cytoplasmic dynein, localizes to the outer domain crescents. CREST staining is restricted to the kinetochore inner core. Scale, 10 μm.
Figure 2
Figure 2
Immunofluorescence staining for BubR1, CENP-E, cytoplasmic dynein, Mad2, and the 3F3/2 antigen in control interphase, mid-to-late prophase, and early prometaphase cells. Phase contrast and rhodamine fluorescence images are shown for interphase cells (left), mid-to-late-prophase cells (center), and early prometaphase cells (right). Only CENP-E, Mad2, and 3F3/2 antigens appear at kinetochores during prophase, whereas BubR1 and cytoplasmic dynein do not become evident at kinetochores until after nuclear envelope breakdown. No crescent or ring morphologies were seen for prometaphase kinetochores. Scale: 10 μm = 0.33 in.
Figure 3
Figure 3
Analysis used to determine the integrated intensity of a given immunofluorescently stained kinetochore as diagrammed in the figure and described in MATERIALS AND METHODS. The great majority of kinetochore fluorescence is contained within the central 9 × 9 pixel square, which corresponds to a 0.9- × 0.9-μm square region of the specimen.
Figure 4
Figure 4
(A) Diagram showing relative numbers of kinetochore microtubules for unattached kinetochores on mono-oriented chromosomes (1; no kinetochore microtubules), leading kinetochores on congressing chromosomes (2; according to McEwen et al., 1997, these generally possess a few microtubules and can be grouped with unattached kinetochores), and fully attached kinetochores on chromosomes aligned at the metaphase plate (3; these are bound to mature kinetochore fibers, which contain ∼25 microtubules [McEwen et al., 1997]) in prometaphase cells. Nonkinetochore spindle microtubules are not shown for clarity. (B) Immunofluorescence images comparing the fluorescence intensity of unattached or leading kinetochores of unaligned chromosomes and fully attached kinetochores of metaphase-aligned chromosomes for the proteins Bub1R, CENP-E, CREST, cytoplasmic dynein, and Mad2. CREST remains unchanged, BubR1 and CENP-E deplete to moderate levels, cytoplasmic dynein depletes to a low level, and Mad2 becomes undetectable. The pixel density in the photographic images is higher than in the original micrographs. Scale = 1 μm.
Figure 5
Figure 5
Quantitative comparisons of the relative changes in fluorescence of unattached or leading kinetochores in prometaphase (Figure 4A) labeled with CENP-E, and BubR1 antibodies in (A) and cytoplasmic dynein and Mad2 antibodies in (B) relative to kinetochores on chromosomes at the metaphase plate. Average values taken from Table 1 are normalized to those values for metaphase control cells. Note that unattached or leading kinetochores exhibit integrated fluorescence intensity levels relative to kinetochores of metaphase-aligned chromosomes of 4.4 for BubR1 (A), 2.9 for CENP-E (A), 5.6 for cytoplasmic dynein (B), and 97 for Mad2 (B).
Figure 6
Figure 6
Kinetochore immunofluorescence and corresponding chromosome DIC image overlays of PtK1 cells treated with nocodazole, taxol, or high Ca2+ buffer. Changes in kinetochore protein localization were compared between control metaphase cells and cells treated with 20 μM nocodazole for 30 min or 4 h to induce prolonged microtubule disassembly or the same treatments followed by nocodazole washout for 30 min to induce microtubule reassembly and kinetochore microtubule formation (A); 10 μM taxol for 45 min to induce loss of kinetochore tension without major changes in kinetochore microtubule number (B); and high Ca2+ buffer at 4°C after cell lysis to induce disassembly of kinetochore microtubules and test for microtubule steric hindrance of antibody labeling (C). For each protein tested, both immunofluorescence and DIC images were recorded. The left column contains black-and-white rhodamine-fluorescence images of immunofluorescence all printed at the same contrast to show relative differences in immunostaining brightness levels. The right column contains color overlays of rhodamine fluorescence on the corresponding DIC images of the chromosomes. Color images were artificially contrast-enhanced to show location of kinetochores on the chromosomes. Arrowheads in the Mad2, BubR1, CENP-E, and cytoplasmic dynein images of cells treated with nocodazole for 30 min or 4 h indicate kinetochore crescent (white arrowheads) or ring-shaped (orange arrowheads) morphologies. Under all conditions tested, kinetochores stained with CREST maintained the same punctuate or oblate morphology. Scale, 10 μm.
Figure 7
Figure 7
Quantitative comparisons of relative changes in fluorescence staining intensity of kinetochores labeled with CREST, CENP-E, and BubR1 antibodies in (A) and cytoplasmic dynein and Mad2 antibodies in (B) relative to kinetochores on metaphase chromosomes in control cells for the various experimental protocols listed along the x-axis. Average values taken from Table 2 are normalized to those values for metaphase control cells. Values underestimate total crescent fluorescence as described in RESULTS. Note in (A) that both CENP-E and BubR1 fluorescence at kinetochores in cells fixed after 30-min or 4-h nocodazole incubations showed increase in intensity in the range of 2.5- to 4-fold beyond control metaphase kinetochores. When subjected to taxol treatment to test for tension effects, or incubation in high Ca2+ buffer to test for microtubule steric hindrance, both CENP-E and BubR1 fluorescence at kinetochores was not changed from values for control metaphase cells. Note in (B) that both cytoplasmic dynein and Mad2 fluorescence at kinetochores in cells fixed after 30-min nocodazole incubations showed increase in intensity in the range of 55- to 65-fold increase beyond the control. After 4-h nocodazole incubation, both intensities increased further to 100- to 110-fold. Like CENP-E and BubR1, cytoplasmic dynein-stained kinetochores in metaphase cells that underwent taxol treatment or incubation in high Ca2+ buffer after cell lysis showed fluorescence that was similar to control metaphase kinetochores.
Figure 8
Figure 8
(A) Schematic diagram of the vertebrate kinetochore. Structural domains are labeled according to the model prese nted by Rieder and Salmon (1998). Two major domains are delineated for the purposes of this article: the inner core, consisting of the inner plate proximal centromere region, and the outer domain, which extends from the inner plate through the interzone, outer plate, and fibrous corona. (B) Illustration of the reversible morphological changes in the kinetochore outer domain between metaphase kinetochores with their full complement of kinetochore microtubules (I) and kinetochores depleted of kinetochore microtubules in cells where microtubule polymerization is blocked for prolonged periods of time with 20 μM nocodazole (II and III). We have taken a perpendicular cross section of a chromosome through the centromere region (top, center). Electron microscopy has shown that coronal fibers are difficult to detect at kinetochores of metaphase chromosomes (Rieder, 1982; Cassimeris et al., 1990), and by immunofluorescence the outer domain remains compact, appearing as punctate or oblate points of fluorescence (I). After depletion of kinetochore microtubules in nocodazole, the outer plate region appears extended and the coronal fibers dense in electron micrographs (Rieder, 1982; Cassimeris et al., 1990), whereas our fluorescence analysis shows that the outer domain can expand outward around the centromere forming crescent or ring morphologies (III) in prolonged nocodazole incubations. These morphological changes in the kinetochore outer domain are reversible with microtubule reassembly and kinetochore microtubule formation.

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