Figure 3.

Phosphorylation of Merlin is necessary for mitotic progression. A, the induced expression of a double alanine mutant of Merlin (S518A/T581A) strongly increases the interval between NEB (black arrowhead) and anaphase (white arrowhead) as observed by DIC microscopy. In contrast, the double aspartic mutant (S518D/T581D) does not. Scale bar = 30 μm. The statistical analysis was done using Mann-Whitney test on R Software. B, the quantification of experiments such as presented in A shows that Merlin dual alanine mutant doubles the duration of NEB to anaphase. The induction of the phosphomimetic mutant has only a minor impact. C, phase contrast microscopy analysis reveals that mitotic delay is the result of a prolonged metaphase. D, the angle of the plan going through the centrosome to the horizontal was measured during metaphase by combining α-tubulin staining and confocal microscopy on Merlin AA and DD overexpressing HeLa cells. E, the expression of Merlin AA but not Merlin DD mutant results in an increased frequency of higher angles. F, quantification of the average angle of the mitotic spindle plan to the horizontal in HeLa cells shows a significant increase upon Merlin AA mutant expression. One-way analysis of variance (ANOVA); ns, p > 0.05; *, p < 0.05; **, p < 0.01.