Fig. 6: Positive feedback mechanism between Myosin X and F-actin is required for efficient pMTOC sorting and clustering.

A Schematic diagrams showing the domains of Myosin X constructs: full-length Myosin X (upper), microtubule binding domain (MyTH4, K1651D/K1654D) Myosin X mutant (MyoX MyTH-mut, middle), and headless Myosin X mutant (lacking the motor head, 1–739 deletion, MyoX MH-mut, lower). B Representative confocal images of metaphase I oocytes microinjected with mRuby-tagged versions of MyoX, MyoX MyTH-mut, or MyoX MH-mut cRNA at the germinal vesicle stage. The oocytes were labeled with phalloidin (F-actin, gray) and pricentrin antibody (magenta). DNA was labeled with 4′,6-diamidino-2-phenylindole, dihydrochloride (DAPI, cyan). Scale bar represents 10 μm. C Average spindle-localized F-actin fluorescence intensity of oocytes microinjected with MyoX, MyoX MyTH-mut, or MyoX MH-mut cRNA. Each dot in the plot represents the value obtained from an individual biological replicate. D Plot profile of pMTOCs (pericentrin fluorescence) along the longitudinal axis of the spindle in the groups indicated in (B). E Quantification of pMTOC volume at the spindle poles to the total volume of all pMTOCs in the spindle. F Average pMTOC number quantification after 3D reconstruction of pericentrin from oocytes in (B). G Average pMTOC volume quantification after 3D reconstruction of pericentrin from oocytes in (B). H Schematic diagram depicting the positive feedback mechanism by which pMTOCs are clustered, sorted, and tethered/maintained. Data are represented as means ± SEM. Asterisks represent significant differences, * p < 0.05, ** p < 0.01, and **** p < 0.0001. The total number of analyzed oocytes per each experimental group (from 3 independent replicates) is specified above each graph.