Figure 4. Organization of the MT cytoskeleton is perturbed in differentiated Sun2−/− keratinocytes. (A) Localization of β-tubulin, phosphorylated myosin light chain (p-MLC), keratin 14 (K14), actin (phalloidin), and nuclei (Hoechst) in WT MKCs. (B) Insets of β-tubulin localization in white boxes in A and D. Arrowheads indicate MT organization at the cell periphery. (C) Quantification of the percentage of WT or Sun2−/− MKCs in the indicated media with cortical accumulation of MTs. n > 200 cells per condition or genotype from three experiments. Asterisks denote indicated significance (**, P < 0.01; ANOVA with Dunnett’s posttest). Error bars indicate SDs. (D) Staining as in A for Sun2−/− MKCs in high Ca2+ medium after 24 h. Arrows in β-tubulin images indicate a perinuclear β-tubulin cage in WT cells that is often lacking in Sun2−/− MKCs. Arrows in K14 images indicate a perinuclear K14 cage in both WT and Sun2−/− MKCs. (E) Localization of β-tubulin, E-cad, and nuclei (Hoechst) after 5 h of taxol treatment in WT or Sun2−/− MKCs. Arrows indicate intercellular junctions (β-tubulin images), cell periphery (I and II), or NE (III and IV). Note the lack of MT accumulation at these sites in Sun2−/− MKCs. Dashed lines indicate the colony periphery defined by E-cad staining. (F) Quantification of the percentage of WT and Sun2−/− MKCs with a stabilized pool of NE-associated MTs. n > 200 cells from three experiments. Error bars indicate SDs. Statistical significance determined by unpaired, two-tailed t test. (G) Actin (phalloidin), E-cad, and nuclei (Hoechst) localization in Sun2−/− MKCs in high Ca2+ medium for 24 h treated with the indicated drug for 5 h. Diagrams illustrate the effect of drug treatment on nuclear position. (H) Plots of nuclear centroid to cell centroid distance normalized to cell radius. Latrunculin A partially rescued the excessive nuclear movement of Sun2−/− MKCs. n > 50 cells (representative of three experiments). Asterisks denote indicated significance (**, P = 0.0087; ANOVA with Dunnett’s posttest). The bottom and top of the box display the 25th and 75th percentiles, whereas the central band represents the median. The whiskers indicate the minimum and maximum values, and the plus signs indicate the mean. AU, arbitrary unit; cal, calcium; Blebb, blebbistatin; Lat A, Latrunculin A.
Figure 4.

Organization of the MT cytoskeleton is perturbed in differentiated Sun2−/− keratinocytes. (A) Localization of β-tubulin, phosphorylated myosin light chain (p-MLC), keratin 14 (K14), actin (phalloidin), and nuclei (Hoechst) in WT MKCs. (B) Insets of β-tubulin localization in white boxes in A and D. Arrowheads indicate MT organization at the cell periphery. (C) Quantification of the percentage of WT or Sun2−/− MKCs in the indicated media with cortical accumulation of MTs. n > 200 cells per condition or genotype from three experiments. Asterisks denote indicated significance (**, P < 0.01; ANOVA with Dunnett’s posttest). Error bars indicate SDs. (D) Staining as in A for Sun2−/− MKCs in high Ca2+ medium after 24 h. Arrows in β-tubulin images indicate a perinuclear β-tubulin cage in WT cells that is often lacking in Sun2−/− MKCs. Arrows in K14 images indicate a perinuclear K14 cage in both WT and Sun2−/− MKCs. (E) Localization of β-tubulin, E-cad, and nuclei (Hoechst) after 5 h of taxol treatment in WT or Sun2−/− MKCs. Arrows indicate intercellular junctions (β-tubulin images), cell periphery (I and II), or NE (III and IV). Note the lack of MT accumulation at these sites in Sun2−/− MKCs. Dashed lines indicate the colony periphery defined by E-cad staining. (F) Quantification of the percentage of WT and Sun2−/− MKCs with a stabilized pool of NE-associated MTs. n > 200 cells from three experiments. Error bars indicate SDs. Statistical significance determined by unpaired, two-tailed t test. (G) Actin (phalloidin), E-cad, and nuclei (Hoechst) localization in Sun2−/− MKCs in high Ca2+ medium for 24 h treated with the indicated drug for 5 h. Diagrams illustrate the effect of drug treatment on nuclear position. (H) Plots of nuclear centroid to cell centroid distance normalized to cell radius. Latrunculin A partially rescued the excessive nuclear movement of Sun2−/− MKCs. n > 50 cells (representative of three experiments). Asterisks denote indicated significance (**, P = 0.0087; ANOVA with Dunnett’s posttest). The bottom and top of the box display the 25th and 75th percentiles, whereas the central band represents the median. The whiskers indicate the minimum and maximum values, and the plus signs indicate the mean. AU, arbitrary unit; cal, calcium; Blebb, blebbistatin; Lat A, Latrunculin A.

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