Fig. 5: Spindle-localized F-actin regulates pMTOC sorting and clustering.

A Chemical structure of Optojasp-1. B Metaphase I oocytes expressing CEP192-mCherry and labeled with SiR-actin (F-actin) were treated with Optojasp-1 and exposed to 405 nm laser at one spindle pole (spindle pole-localized F-actin dynamic perturbation). Bleaching was performed 1 h post-405 nm laser exposure at the two spindle poles using 100% 647 nm laser power (100 ms laser pulse) followed by imaging for an additional 5 min to monitor the fluorescence recovery after photobleaching (FRAP). C Quantification of SiR-actin fluorescence recovery after photobleaching (FRAP). D Schematic diagram shows the experimental design of selectively disrupting spindle-localized F-actin using Optojasp-1. Created in BioRender. Londono, A. (2025) https://BioRender.com/tfvjgd8. E Time-lapse live imaging confocal microscopy of pMTOCs (Cep192-mCherry; pseudo green) and SiR-tubulin (spindle; magenta) in prometaphase I mouse oocytes treated with DMSO and exposed to 405 nm laser at the spindle (control 1), treated with Optojasp-1 and exposed to 405 nm laser at the cytoplasm (control 2), or treated with Optojasp-1 and exposed to 405 nm laser at the spindle (spindle-localized F-actin perturbation). Images were taken every 30 min. Scale bar represents 20 μm. F Average pMTOC number quantification after 3D reconstruction of pMTOCs for the oocytes illustrated in (E). G Average pMTOC volume quantification after 3D reconstruction of pMTOCs for the oocytes illustrated in (E). Data are represented as means ± SEM. Asterisk represents significant differences, * p < 0.05 and ** p < 0.01. The total number of analyzed oocytes per each experimental group (from 3 independent replicates) is specified above each graph.