Figure 1.
DscI is required for normal growth, cytokinesis, and cortical integrity. (A) The CKs pre-assemble in the cytoplasm and accumulate quickly to the cortex upon mechanical stimuli. (B and C)dsc1 null mutant clones generated in the KAx3 background were verified using Western analysis. Western blot showed deletion of DscI bands, quantified and normalized to WT level in C, in all dsc1 null mutant clones. Dynacortin (Dyn) is provided as a loading control. (D) Representative growth curves show that dsc1 null mutants have mild growth defects in suspension culture. Cell densities, as indicated, are plotted as a function of time. Growth curves for control cell lines are shown in black and those for dsc1 null mutant clones are shown in blue. Number of samples = 3 per growth curve; bars represent the SEM. Some SEM bars are smaller than the symbols. (E) Growth rates quantified during exponential growth and normalized to WT reveal significant reduction in suspension culture growth in different dsc1 mutant clones. Growth rate of each cell line was normalized to that of WT control. Number of samples analyzed, n = 6 from two independent experiments. (F) Quantification of the number of nuclei per cell when cells were 3 d in suspension culture. All but one of dsc1 mutants became more multinucleated, especially within 3–5 nuclei/cell. n = 4 per cell line, with 100–200 cells per cell line quantified per replicate. P values were calculated using the ANOVA followed by Fisher’s LSD test based on the single nuclei/cell fraction subset (**, P < 0.001). (G) Images of DIC channel and nuclear staining by Hoescht reveal a slight increase in cell size as well as number of nuclei in each cell in several dsc1 clones. DIC and Hoescht images for WT are reproduced in Fig. 4 C. (H) Graph shows fraction of WT and dsc1 cells having a WT-like U-shaped cleavage furrow, an aberrant V-shaped furrow, or an intermediate furrow during cytokinesis. An intermediate furrow is one that shows both a U-shaped and V-shaped furrow along different stages of cytokinesis. n = 28 for WT and n = 20 for dsc1. P value was calculated using the Comparison of Proportions test. (I) Images of different stages of cytokinesis of a WT cell and a dsc1 cell having a V-shaped furrow. Images were collected from cells at 70% confluency, and t = 0 is defined as the time when each video begins. Full video of WT and dsc1 dividing cells are provided in the Supplemental materials (Videos 1 and 2). (J) The diagram shows the manipulation of cells by MPA for cortical tension and mechanoresponsiveness analysis. For cortical tension measurements, the pressure is increased until the length (Lp) of the region of the cell pulled into the micropipette equals the radius of the pipette (Rp). At this equilibrium pressure (ΔPc), the radius of the cell at can be quantified, and the effective cortical tension Teff is calculated from the equation shown in the panel. (K)dsc1 mutant cells have a higher degree of deformation when aspirated at the same negative pressure compared to WT cells. Red arrows show the front of the cell inside the micropipette. Scale bar, 10 μm and applies to all panels. (L) The effective cortical tension values of WT, dsc1, and dsc1-complemented cells were quantified as detailed in the Materials and methods. Gray background indicates non-transformed cell lines. P values were calculated using Kruskal-Wallis followed by Wilcoxon test. Number of cells measured, n = 7–10 from three independent experiments. (M) When compressed using agarose overlay, which introduces mechanical stress to the cortex, dsc1 mutant cells expressing only GFP displayed a bleb-like morphology. This morphology was observed in both DIC and GFP channels. This phenotype was rescued to WT levels in dsc1:: GFP-DscIA cells. (N)dsc1 mutants displayed a significant reduction in solidity, a measure of smoothness of the cell periphery, when subjected to mechanical stress, as compared to WT control and rescue control (Kruskal-Wallis, P < 0.0001; Wilcoxon, P < 0.0001). Number of cells analyzed, n = 17–28 from two independent experiments. Solidity measurement was analyzed using ImageJ and calculated as the area of a particle divided by its convex hull area. Source data are available for this figure: SourceData F1.

DscI is required for normal growth, cytokinesis, and cortical integrity. (A) The CKs pre-assemble in the cytoplasm and accumulate quickly to the cortex upon mechanical stimuli. (B and C)dsc1 null mutant clones generated in the KAx3 background were verified using Western analysis. Western blot showed deletion of DscI bands, quantified and normalized to WT level in C, in all dsc1 null mutant clones. Dynacortin (Dyn) is provided as a loading control. (D) Representative growth curves show that dsc1 null mutants have mild growth defects in suspension culture. Cell densities, as indicated, are plotted as a function of time. Growth curves for control cell lines are shown in black and those for dsc1 null mutant clones are shown in blue. Number of samples = 3 per growth curve; bars represent the SEM. Some SEM bars are smaller than the symbols. (E) Growth rates quantified during exponential growth and normalized to WT reveal significant reduction in suspension culture growth in different dsc1 mutant clones. Growth rate of each cell line was normalized to that of WT control. Number of samples analyzed, n = 6 from two independent experiments. (F) Quantification of the number of nuclei per cell when cells were 3 d in suspension culture. All but one of dsc1 mutants became more multinucleated, especially within 3–5 nuclei/cell. n = 4 per cell line, with 100–200 cells per cell line quantified per replicate. P values were calculated using the ANOVA followed by Fisher’s LSD test based on the single nuclei/cell fraction subset (**, P < 0.001). (G) Images of DIC channel and nuclear staining by Hoescht reveal a slight increase in cell size as well as number of nuclei in each cell in several dsc1 clones. DIC and Hoescht images for WT are reproduced in Fig. 4 C. (H) Graph shows fraction of WT and dsc1 cells having a WT-like U-shaped cleavage furrow, an aberrant V-shaped furrow, or an intermediate furrow during cytokinesis. An intermediate furrow is one that shows both a U-shaped and V-shaped furrow along different stages of cytokinesis. n = 28 for WT and n = 20 for dsc1. P value was calculated using the Comparison of Proportions test. (I) Images of different stages of cytokinesis of a WT cell and a dsc1 cell having a V-shaped furrow. Images were collected from cells at 70% confluency, and t = 0 is defined as the time when each video begins. Full video of WT and dsc1 dividing cells are provided in the Supplemental materials (Videos 1 and 2). (J) The diagram shows the manipulation of cells by MPA for cortical tension and mechanoresponsiveness analysis. For cortical tension measurements, the pressure is increased until the length (Lp) of the region of the cell pulled into the micropipette equals the radius of the pipette (Rp). At this equilibrium pressure (ΔPc), the radius of the cell at can be quantified, and the effective cortical tension Teff is calculated from the equation shown in the panel. (K)dsc1 mutant cells have a higher degree of deformation when aspirated at the same negative pressure compared to WT cells. Red arrows show the front of the cell inside the micropipette. Scale bar, 10 μm and applies to all panels. (L) The effective cortical tension values of WT, dsc1, and dsc1-complemented cells were quantified as detailed in the Materials and methods. Gray background indicates non-transformed cell lines. P values were calculated using Kruskal-Wallis followed by Wilcoxon test. Number of cells measured, n = 7–10 from three independent experiments. (M) When compressed using agarose overlay, which introduces mechanical stress to the cortex, dsc1 mutant cells expressing only GFP displayed a bleb-like morphology. This morphology was observed in both DIC and GFP channels. This phenotype was rescued to WT levels in dsc1:: GFP-DscIA cells. (N)dsc1 mutants displayed a significant reduction in solidity, a measure of smoothness of the cell periphery, when subjected to mechanical stress, as compared to WT control and rescue control (Kruskal-Wallis, P < 0.0001; Wilcoxon, P < 0.0001). Number of cells analyzed, n = 17–28 from two independent experiments. Solidity measurement was analyzed using ImageJ and calculated as the area of a particle divided by its convex hull area. Source data are available for this figure: SourceData F1.

This Feature Is Available To Subscribers Only

or Create an Account

Close Modal
Close Modal