FIG. 2.

Immunofluorescence imaging of cda12 and cda13 hypomorph phenotypes. (A) Wild-type cell undergoing mitosis. Blue is the result of fluorescence tagging of antibody to β-tubulin, and green is Sytox labeling of macronuclei (Mac) and micronuclei (mic). (B) cda12 antisense mutant showing blue anticentrin labeling, revealing cortical basal bodies, and green nuclear Sytox labeling. The cleavage furrow has formed but has not undergone abscission. Neither micronuclei nor macronuclei (which when viewed by through focusing, appeared to have divided) have segregated into division products and aggregated. (C) Later-stage cda12 mutant showing multiple (red) nuclei labeled with propidium iodide and multiple oral structures (basal bodies labeled with anticentrin antibody), indicating numerous rounds of karyokinesis and cortical predivision patterning coupled with numerous failed rounds of cytokinesis. Images in panels A to C involved the use of confocal scanning laser microscopy. (D) cda13 antisense mutants as failed exconjugants. Pink (originating from rabbit antiserum, obtained from the Millipore Corporation, specific to phosphorylated histone H3) indicates condensed micronuclear DNA, green indicates cortical basal bodies, and blue is DAPI labeling of vacuolated macronuclei. Pairs have failed to dissociate after mating and remain parabiotically fused. The cell on the left has entered mitosis, showing micronuclear division and replication of the cortical pattern with neither macronuclear fission nor cleavage furrow formation. (E) A second cda13 postconjugation cell fusion product showing multiple micronuclei (pink), as well as macronuclear and cytoplasmic fission failures. (At this stage of cortical development, wild-type cells show complete macronuclear fission and well-developed cytokinetic furrows.) Images in panels D and E involved the use of conventional fluorescence microscopy. (F) Confocal image showing cortical details of a large exconjugant fusion mass exhibiting the effects of numerous rounds of cytokinetic fission failure (basal bodies were labeled with antibody to centrin and fluorescein isothiocyanate secondary antiserum). Red arrows indicate supernumerary oral apparatuses. Scale bar, 10 μm. (G) Differential interference contrast image of a giant living postconjugation fission mass (scale bar, 20 μm). (H) Same parabiotic aggregate seen in panel G, showing nuclei (blue) with DAPI fluorescence. The mass possesses only two macronuclei, though there are nearly 20 micronuclei, many in anaphase configurations.