Figure 2.
Injection of anti-xNdc80 and anti-xNuf2 antibodies into mitotic XTC cells induces a “cut” phenotype, defects in chromosome alignment, and diminution of kinetochore fiber microtubule bundles. (A) An XTC cell was injected with anti-xNuf2 antibody at early prometaphase, incubated for ∼60 min, fixed, immunolabeled to visualize the injected antibody, and stained with DAPI to visualize DNA. Cell cleavage resulted in a “cut” phenotype with unequally sized daughter nuclei connected by a chromatin bridge (arrow). (B) XTC cells injected with anti-xNuf2 antibody at early mitosis were incubated for 2 h in the presence of MG132, fixed, immunolabeled to visualize the injected antibody, and stained with DAPI. Many of the antibody-injected cells contained several unaligned chromosomes (arrowheads), whereas the majority of noninjected, mitotic cells (yellow arrow) in the same population had arrested with all chromosomes aligned at the metaphase plate. The inset shows a higher magnification of an anti-xNuf2-antibody-injected cell to reveal that the injected antibody concentrates at presumptive kinetochores. (C) Microinjection of anti-xNdc80 antibody also blocked chromosome alignment in cells maintained in the M phase with MG132. Labeling with anti-tubulin antibody reveals changes in spindle structure compared with an uninjected control cell on the same coverslip. Note the decrease in organized bundles of kinetochore microtubules extending from the spindle poles to the chromosomes. The inset shows the injected antibody labeled with secondary anti-rabbit IgG. Bar, 10 μm.