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Figures
- Fig. 1.
Disruption of the svkA gene encoding severin kinase. (A) Two fragments of the kinase were amplified by PCR from genomic DNA, ligated to the blasticidin-resistance cassette and electroporated into AX2 wild-type cells. (B) Blasticidin-resistant clones were tested for knockout events by PCR1 (primers 1 and 2, 1385 bp for knockout) and PCR2 (primers 1 and 3, 2038 bp as a wild-type control). Under the conditions used, PCR2 did not ever amplify ∼3400 bp, including the blasticidin-resistance cassette, in the knockout, but the lack of a PCR product indicated a disruption of the svkA gene as well. (C) Western blot of 2×105 cells per lane, visualized with kinase-specific antibodies confirms the knockout at the level of SvkA protein and also shows the expression of the GFP fusion protein in the wild-type (first lane) and the mutant background (fourth lane). (D) GFP-SvkA-FL, immunoprecipitated from rescue cells, phosphorylated domains 2 and 3 of severin (DS211C, left), myelin basic protein (MyBP, right) and itself (both lanes).
- Fig. 2.
Expression of SvkA during development and induction of cytofission. (A) Western blot with antibodies against SvkA showing the presence of the kinase throughout the 24 hours of wild-type development (2×105 cells per lane). (B) The knockout cells grown on a plastic surface were considerably larger than the wild-type cells and the rescue cells. (C,D) SvkA-knockout cells were shifted from HL-5 medium to low-salt phosphate buffer (PB) and observed for several hours. The large and multinucleated cells separated only partially. Arrows indicate persistent connections between cells. Bars, 50 μm (B,C); 10 μm (D); see also supplementary material Movie 1.
- Fig. 3.
Developmental defects in the svkA-null mutant. (A) The development of starved svkA-null cells on phosphate agar showed a delay, whereas the wild-type and rescue strains developed as expected into loose and tight aggregates (6 hours/9 hours), slugs (12 hours) and mature fruiting bodies (24 hours). (B) Typical fruiting bodies obtained after three days on phosphate agar (wild-type and rescue strains: one each at the right; knockout cells: nine examples on the left). (C) Phototaxis of D. discoideum slugs on phosphate agar towards a light source at the right (*).
- Fig. 4.
Defective cytokinesis in svkA-null cells. (A,B) Cells grown on a solid surface or in a shaking culture (150 rpm) were fixed and stained with DAPI for quantification of nuclei per cell. More than 1000 cells were counted for each cell line. In the mutant, >50% of the total number of nuclei were found in multinucleated cells under both conditions.
- Fig. 5.
Asymmetric cytokinesis. (A,B) Time-lapse images of svkA-knockout cells dividing on a plastic surface. The increasingly asymmetric cleavage furrow inhibits complete separation of the daughter cells (bars, 20 μm). (C) Time-lapse series of projections calculated from stacks of confocal images with GFP-cortexillin I expressed in svkA-knockout cells. The dividing cell shows an asymmetric cleavage furrow and a remaining cytoplasmic bridge that eventually ruptures ∼8 minutes after the onset of cytokinesis (bar, 5 μm).
- Fig. 6.
Analysis of cytokinesis with marker proteins. (A-G) Confocal sections of fixed svkA-knockout cells representing different stages and the symmetrical population of SvkA-null cell division. All cells are stained for alpha-tubulin and in addition for F-actin (A,B) (the arrow in A indicates the dividing cell), cortexillin I (C,E), myosin II (D), DGAP1 (F) or coronin (G). Bars, 5 μm.
- Fig. 7.
Enrichment of SvkA around the centrosome, the spindle and the midzone. (A) SvkA-knockout cells expressing GFP-SvkA-FL were fixed and stained with antibodies against alpha-tubulin. Three confocal sections are shown. GFP-SvkA-FL is enriched diffusely around the centrosome of interphase cells (open arrow) and around the spindle in mitotic cells (closed arrows). (B) Confocal section of fixed wild-type cells expressing the C-terminal domain construct GFP-SvkA-CT. The arrow indicates the localization of GFP-SvkA-CT around the centrosome. (C) A confocal section of fixed wild-type cells expressing GFP-SvkA-FL shows colocalization with the DNA (upper panel) and, at later stages, around the spindle (lower panel). Bars, 5 μm.
- Fig. 8.
Live-cell recordings show SvkA in the midzone. (A) Wild-type cells expressing GFP-SvkA-FL accumulate the kinase only after the appearance of the cleavage furrow. The arrows indicate the localization of SvkA in the midzone during cell separation. (B) A svkA-knockout cell (confocal section of live cell, agar overlay) containing four nuclei (stars) and expressing the CT-terminal domain of SvkA. GFP-SvkA-CT determines localization to the midzone but is not sufficient to prevent incomplete cell division (see renewed fusion of partially separated cells in frame 11′). (C) SvkA-knockout cells expressing the dead kinase show that GFP-SvkA-K134A-FL localizes to the late cleavage furrow (arrows). (The stars indicate the approximate localization of the nuclei outside the focus plane. Bars, 5 μm.)
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