Fig. 3: Myosin X regulates spindle-localized F-actin and pMTOC organization in mouse oocytes. | Nature Communications

Fig. 3: Myosin X regulates spindle-localized F-actin and pMTOC organization in mouse oocytes.

From: Spindle-localized F-actin regulates polar MTOC organization and the fidelity of meiotic spindle formation

Fig. 3

A, F Representative confocal images of metaphase I oocytes microinjected with control siRNA or Myosin X siRNA at the germinal vesicle stage. The oocytes were stained with phalloidin (F-actin) and 4′,6-diamidino-2-phenylindole, dihydrochloride (DAPI) in (A) or immunostained with α-tubulin and pericentrin antibodies in (F). Scale bar represents 10 μm. B Quantification of average spindle-localized F-actin. C Quantification of average cytoplasmic F-actin. D Quantification of average cortical F-actin. E Quantification of F-actin angle dispersion in relation to the longitudinal axis of the spindle. G Plot profile of pMTOCs (pericentrin fluorescence) along the longitudinal axis of the spindle. H Quantification of pMTOC volume at the spindle poles to the total volume of all pMTOCs in the spindle. I Average pMTOC number quantification. J Average pMTOC volume quantification. K Metaphase I oocytes microinjected with control siRNA, Myosin X siRNA or Myosin VIIb siRNA were stained with SiR-DNA followed by time-lapse confocal microscopy. Scale bar represents 20 μm. Chromosomes were tracked throughout live imaging by using the autotracking function of Imaris software. Yellow arrowheads represent lagging chromosomes. Time points represent the timing from the start of confocal imaging. L Quantification of the average percentage of misaligned chromosomes at metaphase I. M Quantification of the average percentage of misaligned chromosomes at metaphase I. N Average percentage of lagging chromosomes of control, Myosin X knockdown and Myosin VIIb knockdown oocytes. O Representative confocal images of metaphase I oocytes expressing mRuby (control), mRuby-Myosin X microtubule binding domain mutant (MyoX MyTH-mut) or MyoX MyTH-mut + MAP4-UtrCH-GFP cRNA. Metaphase I oocytes were immunostained with pericentrin to label MTOCs and stained with DAPI to label DNA. Scale bar represents 10 μm. P Plot profile of pMTOCs (pericentrin fluorescence) along the longitudinal axis of the spindle. Q Quantification of pMTOC volume at the spindle poles to the total volume of all pMTOCs in the spindle. R Average pMTOC number quantification after 3D reconstruction of pericentrin. S Average pMTOC volume quantification. Data are represented as means ± SEM. Asterisks represent significant differences, * p < 0.05, *** p < 0.001, and **** p < 0.0001. The total number of analyzed oocytes (from 3 independent replicates) is specified above each graph. Each dot in the plots shown in panels (L, M and N) represents the value obtained from an individual biological replicate.

Back to article page