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Visualization of Mad2 dynamics at kinetochores, along spindle fibers, and at spindle poles in living cells.

Howell BJ, Hoffman DB, Fang G, Murray AW, Salmon ED - J. Cell Biol. (2000)

Bottom Line: Third, ATP depletion resulted in microtubule-dependent depletion of Mad2 fluorescence at kinetochores and increased fluorescence at spindle poles.Finally, in normal cells, the half-life of Mad2 turnover at poles, 23 s, was similar to kinetochores.Thus, kinetochore-derived sites along spindle fibers and at spindle poles may also catalyze Mad2 inhibitory complex formation.

View Article: PubMed Central - PubMed

Affiliation: Department of Biology, CB#3280, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina 27599, USA. bhowell@email.unc.edu

ABSTRACT
The spindle checkpoint prevents errors in chromosome segregation by inhibiting anaphase onset until all chromosomes have aligned at the spindle equator through attachment of their sister kinetochores to microtubules from opposite spindle poles. A key checkpoint component is the mitotic arrest-deficient protein 2 (Mad2), which localizes to unattached kinetochores and inhibits activation of the anaphase-promoting complex (APC) through an interaction with Cdc20. Recent studies have suggested a catalytic model for kinetochore function where unattached kinetochores provide sites for assembling and releasing Mad2-Cdc20 complexes, which sequester Cdc20 and prevent it from activating the APC. To test this model, we examined Mad2 dynamics in living PtK1 cells that were either injected with fluorescently labeled Alexa 488-XMad2 or transfected with GFP-hMAD2. Real-time, digital imaging revealed fluorescent Mad2 localized to unattached kinetochores, spindle poles, and spindle fibers depending on the stage of mitosis. FRAP measurements showed that Mad2 is a transient component of unattached kinetochores, as predicted by the catalytic model, with a t(1/2) of approximately 24-28 s. Cells entered anaphase approximately 10 min after Mad2 was no longer detectable on the kinetochores of the last chromosome to congress to the metaphase plate. Several observations indicate that Mad2 binding sites are translocated from kinetochores to spindle poles along microtubules. First, Mad2 that bound to sites on a kinetochore was dynamically stretched in both directions upon microtubule interactions, and Mad2 particles moved from kinetochores toward the poles. Second, spindle fiber and pole fluorescence disappeared upon Mad2 disappearance at the kinetochores. Third, ATP depletion resulted in microtubule-dependent depletion of Mad2 fluorescence at kinetochores and increased fluorescence at spindle poles. Finally, in normal cells, the half-life of Mad2 turnover at poles, 23 s, was similar to kinetochores. Thus, kinetochore-derived sites along spindle fibers and at spindle poles may also catalyze Mad2 inhibitory complex formation.

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Release of fluorescent Mad2 particles from kinetochores. Fluorescent XMad2 particles were released from the kinetochore and transported towards the spindle pole via spindle microtubules. Arrows denote a release of fluorescent XMad2 particles. KT denotes the location of the kinetochore, whereas P denotes the location of the spindle pole. Time is shown in minutes:seconds. Bar, 2 μm. (See Video 4 available online at http://www.jcb.org/cgi/content/full/150/6/1233/DC1).
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Figure 8: Release of fluorescent Mad2 particles from kinetochores. Fluorescent XMad2 particles were released from the kinetochore and transported towards the spindle pole via spindle microtubules. Arrows denote a release of fluorescent XMad2 particles. KT denotes the location of the kinetochore, whereas P denotes the location of the spindle pole. Time is shown in minutes:seconds. Bar, 2 μm. (See Video 4 available online at http://www.jcb.org/cgi/content/full/150/6/1233/DC1).

Mentions: Interphase and mitotic PtK1 cells were injected with fluorescent XMad2 protein, and Mad2 localization and mitotic progression were observed using digital imaging fluorescence and phase-contrast microscopy. PtK1 cells were chosen as a model system because they are flat, typically contain 11 large chromosomes, and have been used extensively in studies of the spindle checkpoint (Rieder et al. 1994, Rieder et al. 1995; Gorbsky et al. 1998; Waters et al. 1998, Waters et al. 1999). After microinjection into the cytoplasm, we found that fluorescent XMad2 was diffuse throughout the cytoplasm but concentrated in the nucleus of interphase (data not shown) and prophase cells (Fig. 1 A). Consistent with previous anti-Mad2 immunofluorescence studies in PtK1 cells and other tissue culture lines (Chen et al. 1996; Waters et al. 1998; Gorbsky et al. 1998), fluorescent XMad2 localized strongly to kinetochores in late prophase (Fig. 1 A) and early prometaphase (Fig. 1 B) cells and remained localized to kinetochores until their chromosomes achieved bipolar attachment to the spindle during late prometaphase and early metaphase (Fig. 1 C and Fig. 2 D). In addition, fluorescent XMad2 protein localized to the spindle poles during prometaphase and early metaphase (Fig. 1 C, arrowheads), and was frequently observed along spindle microtubules (Fig. 1 B; see Fig. 8). In contrast to previous fixed cell studies (Chen et al. 1996; Li and Benezra 1996; Gorbsky et al. 1998; Waters et al. 1998), fluorescent XMad2 was not enhanced above background at either the nuclear envelope or prophase centrosomes in living PtK1 cells. Fluorescent XMad2 protein was not evident on kinetochores, spindle microtubules, or at the spindle poles during late metaphase, anaphase, and telophase (Fig. 1 D; also see Fig. 2E and Fig. F).


Visualization of Mad2 dynamics at kinetochores, along spindle fibers, and at spindle poles in living cells.

Howell BJ, Hoffman DB, Fang G, Murray AW, Salmon ED - J. Cell Biol. (2000)

Release of fluorescent Mad2 particles from kinetochores. Fluorescent XMad2 particles were released from the kinetochore and transported towards the spindle pole via spindle microtubules. Arrows denote a release of fluorescent XMad2 particles. KT denotes the location of the kinetochore, whereas P denotes the location of the spindle pole. Time is shown in minutes:seconds. Bar, 2 μm. (See Video 4 available online at http://www.jcb.org/cgi/content/full/150/6/1233/DC1).
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Related In: Results  -  Collection

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Figure 8: Release of fluorescent Mad2 particles from kinetochores. Fluorescent XMad2 particles were released from the kinetochore and transported towards the spindle pole via spindle microtubules. Arrows denote a release of fluorescent XMad2 particles. KT denotes the location of the kinetochore, whereas P denotes the location of the spindle pole. Time is shown in minutes:seconds. Bar, 2 μm. (See Video 4 available online at http://www.jcb.org/cgi/content/full/150/6/1233/DC1).
Mentions: Interphase and mitotic PtK1 cells were injected with fluorescent XMad2 protein, and Mad2 localization and mitotic progression were observed using digital imaging fluorescence and phase-contrast microscopy. PtK1 cells were chosen as a model system because they are flat, typically contain 11 large chromosomes, and have been used extensively in studies of the spindle checkpoint (Rieder et al. 1994, Rieder et al. 1995; Gorbsky et al. 1998; Waters et al. 1998, Waters et al. 1999). After microinjection into the cytoplasm, we found that fluorescent XMad2 was diffuse throughout the cytoplasm but concentrated in the nucleus of interphase (data not shown) and prophase cells (Fig. 1 A). Consistent with previous anti-Mad2 immunofluorescence studies in PtK1 cells and other tissue culture lines (Chen et al. 1996; Waters et al. 1998; Gorbsky et al. 1998), fluorescent XMad2 localized strongly to kinetochores in late prophase (Fig. 1 A) and early prometaphase (Fig. 1 B) cells and remained localized to kinetochores until their chromosomes achieved bipolar attachment to the spindle during late prometaphase and early metaphase (Fig. 1 C and Fig. 2 D). In addition, fluorescent XMad2 protein localized to the spindle poles during prometaphase and early metaphase (Fig. 1 C, arrowheads), and was frequently observed along spindle microtubules (Fig. 1 B; see Fig. 8). In contrast to previous fixed cell studies (Chen et al. 1996; Li and Benezra 1996; Gorbsky et al. 1998; Waters et al. 1998), fluorescent XMad2 was not enhanced above background at either the nuclear envelope or prophase centrosomes in living PtK1 cells. Fluorescent XMad2 protein was not evident on kinetochores, spindle microtubules, or at the spindle poles during late metaphase, anaphase, and telophase (Fig. 1 D; also see Fig. 2E and Fig. F).

Bottom Line: Third, ATP depletion resulted in microtubule-dependent depletion of Mad2 fluorescence at kinetochores and increased fluorescence at spindle poles.Finally, in normal cells, the half-life of Mad2 turnover at poles, 23 s, was similar to kinetochores.Thus, kinetochore-derived sites along spindle fibers and at spindle poles may also catalyze Mad2 inhibitory complex formation.

View Article: PubMed Central - PubMed

Affiliation: Department of Biology, CB#3280, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina 27599, USA. bhowell@email.unc.edu

ABSTRACT
The spindle checkpoint prevents errors in chromosome segregation by inhibiting anaphase onset until all chromosomes have aligned at the spindle equator through attachment of their sister kinetochores to microtubules from opposite spindle poles. A key checkpoint component is the mitotic arrest-deficient protein 2 (Mad2), which localizes to unattached kinetochores and inhibits activation of the anaphase-promoting complex (APC) through an interaction with Cdc20. Recent studies have suggested a catalytic model for kinetochore function where unattached kinetochores provide sites for assembling and releasing Mad2-Cdc20 complexes, which sequester Cdc20 and prevent it from activating the APC. To test this model, we examined Mad2 dynamics in living PtK1 cells that were either injected with fluorescently labeled Alexa 488-XMad2 or transfected with GFP-hMAD2. Real-time, digital imaging revealed fluorescent Mad2 localized to unattached kinetochores, spindle poles, and spindle fibers depending on the stage of mitosis. FRAP measurements showed that Mad2 is a transient component of unattached kinetochores, as predicted by the catalytic model, with a t(1/2) of approximately 24-28 s. Cells entered anaphase approximately 10 min after Mad2 was no longer detectable on the kinetochores of the last chromosome to congress to the metaphase plate. Several observations indicate that Mad2 binding sites are translocated from kinetochores to spindle poles along microtubules. First, Mad2 that bound to sites on a kinetochore was dynamically stretched in both directions upon microtubule interactions, and Mad2 particles moved from kinetochores toward the poles. Second, spindle fiber and pole fluorescence disappeared upon Mad2 disappearance at the kinetochores. Third, ATP depletion resulted in microtubule-dependent depletion of Mad2 fluorescence at kinetochores and increased fluorescence at spindle poles. Finally, in normal cells, the half-life of Mad2 turnover at poles, 23 s, was similar to kinetochores. Thus, kinetochore-derived sites along spindle fibers and at spindle poles may also catalyze Mad2 inhibitory complex formation.

Show MeSH