Visualization of Mad2 dynamics at kinetochores, along spindle fibers, and at spindle poles in living cells - PubMed (original) (raw)

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

B J Howell et al. J Cell Biol. 2000.

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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Figures

Figure 1

Figure 1

Fluorescent and phase-contrast images of living mitotic PtK1 cells injected with Alexa 488-XMad2. Fluorescent XMad2 is diffusely distributed in the cytoplasm and accumulates in the interphase nucleus. It localizes to unattached kinetochores in prophase (A) and early prometaphase (B, large arrowhead) and to the spindle poles in prometaphase (B and C, small arrowheads). Fluorescent XMad2 depletes from kinetochores upon their attachment to the spindle in prometaphase (B, small arrows) and is undetectable on aligned chromosomes (C). Fluorescent XMad2 is not present at kinetochores or spindle poles during anaphase and telophase. (D). Bar, 3 μm.

Figure 2

Figure 2

Time-lapse of fluorescent XMad2 protein in a living mitotic PtK1 cell. Fluorescent XMad2 localizes to kinetochores in prophase (A) and early prometaphase (B), and to spindle poles in prometaphase (C, arrows). Kinetochores leading chromosome congression to the metaphase plate often retain Mad2 localization (D) and polar localization persists (D, arrows). Fluorescent XMad2 diminishes at kinetochores in prometaphase (B and C) and at kinetochores and spindle poles in early metaphase (D). After Mad2 has been depleted from all kinetochores at late metaphase (E), the cells enter anaphase (F). Time is shown in hours:minutes:seconds. (See Videos 1 and 2 available online at http://www.jcb.org/cgi/content/full/150/6/1233/DC1). Bar, 3 μm.

Figure 3

Figure 3

Characterization of purified XMad2 protein and analysis of its ability to inhibit premature anaphase onset in PtK1 cells injected with anti-Mad2 antibody. Unlabeled (A, inset lane 1) and Alexa 488–labeled XMad2 protein (A, inset lane 2) were run on a 12.5% SDS–polyacrylamide gel and purity was visualized by silver stain (A, inset). Sedimentation velocity ultracentrifugation was performed to determine the average molecular mass for the unlabeled XMad2. A graph of the concentration (absorbance units) as a function of rotor radius (r = cm2) is shown in A. The shape of the sedimentation curve corresponds to an average molecular mass of 107.5 kD, as determined by the XLA Origin software (see Materials and Methods). B is a time-lapse sequence of premature anaphase onset in a PtK1 cell ∼10 min after injection with anti-Mad2 antibody (black arrow). In C, a PtK1 cell is first injected with anti-Mad2 antibody (α-Mad2, black arrow), and then injected ∼4 min later with fluorescent XMad2 (Alexa 488-XMad2). Time-lapse phase and fluorescent imaging shows that the cell does not enter anaphase prematurely and that the fluorescent XMad2 localizes to unattached kinetochores (small white arrow) and spindle poles (white arrowheads). M r, markers are (top to bottom): 116, 66, 45, 31, 21.5, and 14 kD. Time is shown in minutes:seconds. Bar, 5 μm.

Figure 3

Figure 3

Characterization of purified XMad2 protein and analysis of its ability to inhibit premature anaphase onset in PtK1 cells injected with anti-Mad2 antibody. Unlabeled (A, inset lane 1) and Alexa 488–labeled XMad2 protein (A, inset lane 2) were run on a 12.5% SDS–polyacrylamide gel and purity was visualized by silver stain (A, inset). Sedimentation velocity ultracentrifugation was performed to determine the average molecular mass for the unlabeled XMad2. A graph of the concentration (absorbance units) as a function of rotor radius (r = cm2) is shown in A. The shape of the sedimentation curve corresponds to an average molecular mass of 107.5 kD, as determined by the XLA Origin software (see Materials and Methods). B is a time-lapse sequence of premature anaphase onset in a PtK1 cell ∼10 min after injection with anti-Mad2 antibody (black arrow). In C, a PtK1 cell is first injected with anti-Mad2 antibody (α-Mad2, black arrow), and then injected ∼4 min later with fluorescent XMad2 (Alexa 488-XMad2). Time-lapse phase and fluorescent imaging shows that the cell does not enter anaphase prematurely and that the fluorescent XMad2 localizes to unattached kinetochores (small white arrow) and spindle poles (white arrowheads). M r, markers are (top to bottom): 116, 66, 45, 31, 21.5, and 14 kD. Time is shown in minutes:seconds. Bar, 5 μm.

Figure 4

Figure 4

Fluorescent and phase images of GFP-hMad2 in living PtK1 cells. PtK1 cells were transfected with hGFP-MAD2 and observed for protein localization during mitosis. GFP-hMad2 accumulates in the nucleus and localizes to unattached kinetochores in prophase (A) and prometaphase (B, large arrowhead). During prometaphase, GFP-hMad2 is evident along some spindle fibers and at the spindle poles (B and C, small arrowheads). GFP-hMad2 fluorescence disappears from kinetochores as they became attached to the spindle in prometaphase (B, arrow) and aligned at the metaphase plate (C). GFP-hMad2 is not present at kinetochores, along spindle fibers, or at the spindle poles during anaphase (D) and telophase. Bar, 3 μm.

Figure 5

Figure 5

FRAP analysis of Mad2 turnover at kinetochores and spindle poles in untreated and nocodazole-treated mitotic PtK1 cells. Cells were fluorescently imaged before and after photobleaching of a single kinetochore (A and B) or spindle pole (C). Cells were either untreated (A and C) or treated with nocodazole for 20 min (B). The prephotobleach, postphotobleach, half recovery (two images), and full recovery time points are shown for each experiment. Arrows denote the photobleaching target. The time is shown in minutes:seconds. Also shown are the corresponding FRAP graphs of Mad2 turnover at bleached kinetochores (A′ and B′) and spindle poles (C′) in untreated (A′ and C′) and nocodazole-treated (B′) cells. A closed diamond is used to mark the photobleached kinetochores and spindle poles on the graph. Measurements of background fluorescence within the cytoplasm were also taken and are plotted on each graph (open triangles). Mad2 diffusion in the cytoplasm is rapid and the FRAP is too quick to record. Arrows denote time of photobleaching. (See Video 3 available online at http://www.jcb.org/cgi/content/full/150/6/1233/DC1). Bars, 3 μm.

Figure 5

Figure 5

FRAP analysis of Mad2 turnover at kinetochores and spindle poles in untreated and nocodazole-treated mitotic PtK1 cells. Cells were fluorescently imaged before and after photobleaching of a single kinetochore (A and B) or spindle pole (C). Cells were either untreated (A and C) or treated with nocodazole for 20 min (B). The prephotobleach, postphotobleach, half recovery (two images), and full recovery time points are shown for each experiment. Arrows denote the photobleaching target. The time is shown in minutes:seconds. Also shown are the corresponding FRAP graphs of Mad2 turnover at bleached kinetochores (A′ and B′) and spindle poles (C′) in untreated (A′ and C′) and nocodazole-treated (B′) cells. A closed diamond is used to mark the photobleached kinetochores and spindle poles on the graph. Measurements of background fluorescence within the cytoplasm were also taken and are plotted on each graph (open triangles). Mad2 diffusion in the cytoplasm is rapid and the FRAP is too quick to record. Arrows denote time of photobleaching. (See Video 3 available online at http://www.jcb.org/cgi/content/full/150/6/1233/DC1). Bars, 3 μm.

Figure 6

Figure 6

Photobleached kinetochores retain Mad2. PtK1 cells were injected with Alexa 488-XMad2 and imaged by fluorescence and phase-contrast microscopy immediately before (Prebleach) and after (Postbleach) photobleaching of a single kinetochore (arrows). Cells were quickly fixed (<15 s after photobleaching), lysed, and immunofluorescently processed for Mad2 using anti-Mad2 antibodies. Fixed cells show small amounts of exogenous Alexa 488-XMad2 fluorescence on photobleached kinetochores (Alexa 488-XMad2), however, anti-Mad2 staining revealed high concentrations of Mad2 protein on the photobleached kinetochore (α-Mad2). Bar, 3 μm.

Figure 7

Figure 7

Fluorescent Mad2 is extended from kinetochores. Fluorescent XMad2 streaks extended from kinetochores in either one (A and B) or two (C) directions. KT denotes the location of the kinetochore, whereas P denotes the location of the spindle pole. Time is shown in minutes:seconds. Bar, 1 μm.

Figure 8

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).

Figure 9

Figure 9

The loss of fluorescent XMad2 at spindle poles follows the loss of Mad2 localization to kinetochores. We observed the loss of fluorescent XMad2 at the kinetochore (small arrow) of a congressing chromosome and the subsequent loss of fluorescent XMad2 at the proximal spindle pole (B–E, large arrow). Interestingly, fluorescent XMad2 was only evident on the spindle pole proximal to the last monooriented chromosome (D–F). Time is shown in minutes:seconds. (See Video 5 available online at http://www.jcb.org/cgi/content/full/150/6/1233/DC1). Bar, 3 μm.

Figure 10

Figure 10

Mad2 fluorescence decreases at kinetochores and increases at spindle poles after ATP depletion in vivo if the spindle is present. Untreated (A–E) and nocodazole-treated (F–H) cells were injected with fluorescent XMad2 and imaged in saline with or without nocodazole (A, D, and F). Cells were incubated in medium containing 5 mM sodium azide and 1 mM 2-deoxy-glucose with or without nocodazole for 30 min, and reimaged for Mad2 localization (B, E, and G). Mad2 fluorescence concentrates strongly at the poles and is undetectable at kinetochores in prometaphase cells after 30 min treatment with inhibitors (B and B′), but remains in the cytoplasm in the metaphase cells (E and E′). After a saline rinse to wash out the inhibitors, Mad2 fluorescence quickly recovers on kinetochores and diminishes at spindle poles in prometaphase cells (C and C′). In contrast, however, Mad2 fluorescence remains at kinetochores and does not localize to spindle poles in nocodazole-treated cells after the 30-min incubation with the metabolic inhibitors (G and G′). Mad2 fluorescence in nocodazole-treated cells remains at the kinetochores after a brief washout of inhibitors with saline (H and H′). Bar, 5 μm.

Figure 11

Figure 11

The proposed model for spindle checkpoint inactivation. Unattached kinetochores serve as catalytic sites for forming Mad2 inhibitory complexes (Mad2*), as suggested previously by Kallio et al. 1998 and Chen et al. 1998. Mad2 inhibitory complexes are released into the cytoplasm at a rate of 0.025 s−1, where they prevent APC activation (A). Based on the 10-min delay between Mad2 depletion on the last chromosome and anaphase onset, inhibitory complexes are slowly inactivated (A). Interactions of kinetochores with spindle microtubules results in the depletion of coronal filaments containing Mad2 binding sites (x) from kinetochores and the translocation of some binding sites to the poles where they may also catalyze Mad2 inhibitory complexes before disassembly into the cytoplasm (B). Upon depletion of Mad2 binding sites from the kinetochores of the last congressing chromosome, inhibitory complex formation ceases at the kinetochores (and spindle poles), and the APC is activated.

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