Microinjection of antibody to Mad2 protein into mammalian cells in mitosis induces premature anaphase

Abstract

In yeast, the Mad2 protein is required for the M phase arrest induced by microtubule inhibitors, but the protein is not essential under normal culture conditions. We tested whether the Mad2 protein participates in regulating the timing of anaphase onset in mammalian cells in the absence of microtubule drugs. When microinjected into living prophase or prometaphase PtK1 cells, anti-Mad2 antibody induced the onset of anaphase prematurely during prometaphase, before the chromosomes had assembled at the metaphase plate. Anti-Mad2 antibody-injected cells completed all aspects of anaphase including chromatid movement to the spindle poles and pole-pole separation. Identical results were obtained when primary human keratinocytes were injected with anti-Mad2 antibody. These studies suggest that Mad2 protein function is essential for the timing of anaphase onset in somatic cells at each mitosis. Thus, in mammalian somatic cells, the spindle checkpoint appears to be a component of the timing mechanism for normal mitosis, blocking anaphase onset until all chromosomes are aligned at the metaphase plate.

Figure 1

Anti-Mad2 antibody recognizes a 24-kD band in whole cell extracts prepared from Ptk1 cells and LLC-Pk cells. Cells were dissolved in SDS sample buffer, electrophoresed on polyacrylamide gradient gels under denaturing conditions, transferred to Immobilon-P paper, and probed with anti-Mad2 antibody.

Figure 2

Mad2 protein is distributed on kinetochores, spindle poles, and in the cytoplasm of early prometaphase LLC-Pk cells. Cells were fixed with formaldehyde and then permeabilized with detergent. In early prometaphase cells, immunofluorescence with anti-Mad2 antibody reveals a high concentration of label at the spindle poles (arrows) and at the kinetochores (arrowheads) with some granular label in the cytoplasm. Kinetochore labeling with a human autoimmune serum is shown in the second column. A phase contrast image is shown in the third column, and a merged fluorescence and phase contrast image is shown in the fourth column. The kinetochores appear yellow because of superimposition of the red anti-Mad2 antibody label and the green antikinetochore label. When anti-Mad2 antibody was blocked by coincubation with bacterially expressed Mad2 protein, no anti-Mad2 fluorescence is detected (B), though kinetochores were still labeled with the antikinetochore antiserum. Bar, 5 μm.

Figure 3

Cell cycle distribution of Mad2 protein in detergent-extracted LLC-Pk cells. Immunofluorescence labeling with anti-Mad2 antibody is shown in the first column, phase contrast of the same field is shown in the second column, and a merged image is shown in the third column. In interphase (A), the Mad2 protein is concentrated in the nucleus. In the cytoplasm, Mad2 is present diffusely and is concentrated in small granules, many of which are clustered in the region of the centrosome (arrow). In prophase (B), the Mad2 protein remains in the nucleus with concentrations of the label continues to be associated with the separating centrosomes (arrows). (The lower centrosome is in a different plane of focus.) Note the indentation of the nuclear envelope (curved arrow), presumably due to ingrowth of microtubules nucleated from the nearby centrosome. At early prometaphase (C), most chromosomes show concentrations of Mad2 at the kinetochores (arrowheads). Anti-Mad2 label is also found associated with the centrosomes (arrow) and in the cytoplasm. At late prometaphase (D), anti-Mad2 labeling is weak or absent at the kinetochores of fully congressed chromosomes. However, noncongressed chromosomes continue to show labeling of their kinetochores. Chromosomes located between the spindle pole and the metaphase plate often exhibit asymmetric labeling of the two sister kinetochores (arrowheads). At metaphase (E), labeling in the region of the chromosomes is weak, but considerable Mad2 protein remains near the centrosomes and distributed through the cytoplasm. At anaphase (F), the overall level of anti-Mad2 labeling appears to diminish. The centrosomes continue to exhibit accumulations of Mad2 protein, and some labeled granules are also present in the cytoplasm. At telophase (G), diffuse anti-Mad2 label appears to concentrate in the vicinity of the reforming nuclei. Bar, 5 μm.

Figure 4

Mad2 protein is found at spindle poles, kinetochores, and in the cytoplasm of Ptk1 cells. Ptk1 cells were fixed in formaldehyde and then permeabilized with detergent (A) or preextracted with detergent before fixation (B). With both specimen preparations, immunofluorescence of mid-prometaphase cells reveals that the Mad2 protein is concentrated near spindle poles (arrows) and at kinetochores (arrowheads) of chromosomes before their congression to the metaphase plate. Granular labeling is also detected in the cytoplasm. Bar, 5 μm.

Figure 5

Injection of anti-Mad2 antibody induces premature anaphase. A Ptk1 cell in mid-prometaphase that had undergone partial congression of the chromosomes was injected with anti-Mad2 antibody (time of injection = time 0). At 0.5 min, a low-resolution image was recorded (A). High-resolution phase contrast optics were used for subsequent images. Congression of chromosomes in prometaphase continued at 5.5 min (B). By 7 min after injection (C), many chromosomes were at the metaphase plate (brackets), but others were only partially congressed (arrows). The cell entered premature anaphase between 7 and 8 min. At 8 min (D), separation of chromatids can be clearly visualized (small arrows). The positions of the spindle poles (centrosomes) are indicated by arrowheads. Anaphase A movements occur with many chromatids undergoing normal poleward movement to the poles, but others, presumably those that lacked stable attachment to the pole, drift in the cytoplasm with little evidence of directed movement (arrow) (E). Anaphase A chromatid separation continues with chromosomes nearing the poles (F). By 17 min (G), anaphase A movements have finished, but anaphase B, the separation of the spindle poles, continues. (Compare distance between poles [arrowheads] in D versus G). Some chromatids, which presumably lacked stable attachment to microtubules at anaphase onset, remained at the metaphase plate (curved arrow). Bar, 5 μm.

Figure 6

Injection of anti-Mad2 antibody in prophase induces premature anaphase onset. A Ptk1 cell in mid-prophase was injected in the cytoplasm with anti-Mad2 antibody at time 0. Prophase continued unaffected (A), and nuclear envelope breakdown occurred ∼29 min after injection (B). Chromosomes attached to the spindle during prometaphase (C–E), and some moved to the metaphase plate. One chromosome (arrowhead) moved above the upper spindle pole and remained monopolarly oriented. Anaphase onset occurred ∼42 min after injection (F) while many chromosomes were still far from the metaphase plate. During anaphase (G–I) chromatids moved to the spindle poles, and the spindle poles moved apart. In some cases (curved arrow), both sister chromatids from a single chromosome moved to the same spindle pole. Bar, 5 μm.

Figure 7

Normal progression of mitosis in a human keratinocyte injected with control, preimmune IgG. A keratinocyte in prophase was injected with preimmune IgG at time 0. The cell progressed through prophase (A) and underwent nuclear envelope breakdown. During prometaphase (B–D), a mitotic spindle formed with discrete spindle poles (D, arrows). During prometaphase, the chromosomes congressed to the spindle equator assemble the metaphase plate (D, arrowhead). At metaphase (E), the chromosomes were assembled into a tight plate (E, arrowhead). Anaphase onset (F) ensued with chromatid separation (F, arrowhead). The chromatids (arrowheads) separated normally as the cell progressed through anaphase, and cytokinesis was initiated (G and H). During telophase (I and J), cytokinesis was completed. Bar, 5 μm.

Figure 8

Injection of a keratinocyte with anti-Mad2 antibody induces premature anaphase. A keratinocyte was injected in prometaphase at time 0. At 6 min after injection (A), the cell had assembled a bipolar spindle, and the chromosomes began to congress. The approximate locations of the spindle poles are indicated by the arrows. At 14 min (B), the cell had initiated anaphase before congression was complete. Anaphase continued (C and D). The cleavage furrow (arrowhead) appeared in late anaphase (E). At 50 min, the culture was fixed (G). Images of the fixed cells showed that cytokinesis had been completed. Bar, 5 μm.

Figure 9

Injection of a keratinocyte with anti-Mad2 antibody induces premature anaphase before assembly of a bipolar spindle. A keratinocyte was injected with anti-Mad2 antibody just before nuclear envelope breakdown (A). As happens spontaneously in these cells, the spindle poles had not separated appreciably before nuclear envelope breakdown. The chromosomes clustered around the unseparated spindle poles (arrow) (B). Anaphase initiated while the chromosomes were still clustered around the unseparated spindle poles (C). The chromatids separated but moved very little during “anaphase” (D). The chromosomes then decondensed and the nucleus reformed (E). The culture was later fixed. The cell was relocated, showing that it had progressed into interphase. In the absence of a bipolar spindle, the cell did not undergo cytokinesis. Bar, 5 μm.

Figure 10

Injected anti-Mad2 antibody collects in cytoplasmic aggregates that also label with 3F3/2 antiphosphoepitope antibody. A Ptk1 cell at mid-prometaphase was microinjected with anti-Mad2 antibody at time 0. At 12 min after microinjection, the cell was still in prometaphase (A). The cell entered anaphase at 14 min (B) with clear separation of the chromatids (arrowhead). The spindle poles are indicated by the straight arrows. Anaphase continued (C and D). The final image of the live cell was obtained in late anaphase (E). The cell was then extracted with detergent and fixed. The phase contrast image of the fixed cell reveals that the chromosomes have swollen somewhat during the extraction and fixation protocol, but morphology is preserved (F). The microinjected antibody was localized by applying a fluorescent anti-rabbit IgG secondary antibody (G). The 3F3/2 phosphoepitope was immunolocalized in the same cell (H). Comparison of the phase and fluorescent images reveals that microinjected anti-Mad2 (F) antibody accumulated in aggregates (curved arrows) that also labeled with the 3F3/2 phosphoepitope antibody (G). These aggregates are also visible in the phase contrast image of the fixed cell, though the phase density may be at least partially due to the primary and secondary antibodies used for immunolabeling. Bars, 5 μm.

Figure 11

Anti-Mad2 antibody injected in nocodazole-treated Ptk1 cells induces exit from M phase and formation of Mad2 aggregates in the cytoplasm. Anti-Mad2 antibody was injected into a Ptk1 cell arrested in M phase by pretreatment with nocodazole. The cell at low magnification at the time of injection is shown in A. After incubation at 37°C for an additional 25 min in the presence of nocodazole, the cell was lysed with detergent and fixed. A phase contrast image at high magnification (B) showing a portion of the cell reveals that the chromosomes have partially decondensed and the nuclear envelope has begun to reform (B, arrow). These indicators show that the cell was exiting M phase at the time of fixation. Immunolabeling with anti–rabbit secondary antibody (C) shows that detergent-insoluble anti-Mad2 antibody aggregates are present in the cytoplasm (C, curved arrows). Colabeling with 3F3/2 antibody (D) shows that these same aggregates contain the 3F3/2 phosphoepitope. Bars, 5 μm.

Figure 12

A proposed model for Mad2 protein dynamics and inhibition of anaphase onset. The unattached kinetochore (black) catalyzes the assembly and/or activation of a Mad2-containing complex (black shading indicates active complex). This complex is released from the kinetochore and blocks the ability of the APC to ubiquitinylate target anaphase inhibitor proteins such as Pds1 in budding yeast and Cut2 in fission yeast. The Mad2-containing complex undergoes spontaneous inactivation (indicated by shading change from black to gray) perhaps by dephosphorylation or disassembly. However, as long as unattached kinetochores persist, active Mad2-containing inhibitor complex is continuously regenerated, perhaps by recycling inactivated subunits. Attachment of kinetochores halts the assembly/activation of the Mad2-complex (indicated by gray color of kinetochores attached to microtubule bundles). Residual inhibitor complex is slowly inactivated, thus allowing time for the final attaching chromosome to move to the metaphase plate. The APC, released from inhibition, then catalyzes ubiquitinylation of the Pds1/Cut2-type anaphase inhibitor proteins. These proteins are then degraded by the proteasome, and anaphase is initiated.