During determination of the cell division plane, an actomyosin contractile ring is induced at the equatorial cell cortex by signals from the mitotic apparatus and contracts to cause cleavage furrow progression. Although the small GTPase RhoA is known to regulate the progression, probably by controlling actin filament assembly and enhancing actomyosin interaction, any involvement of RhoA in division plane determination is unknown. In this study, using a trichloroacetic acid (TCA) fixation protocol we recently developed, we show that RhoA accumulates at the equatorial cortex before furrow initiation and continues to concentrate at the cleavage furrow during cytokinesis. We also demonstrate that both Rho activity and microtubule organization are required for RhoA localization and proper furrowing. Selective disruption of microtubule organization revealed that both astral and central spindle microtubules can recruit RhoA at the equatorial cortex. We find that centralspindlin and ECT2 are required for RhoA localization and furrowing. Centralspindlin is localized both to central spindle microtubules and at the tips of astral microtubules near the equatorial cortex and recruits ECT2. Positional information for division plane determination from microtubules is transmitted to the cell cortex to organize actin cytoskeleton through a mechanism involving these proteins.
Cell polarization enables zygotes to acquire spatial asymmetry, which in turn patterns cellular and tissue axes during development. Local modification in the actomyosin cytoskeleton mediates spatial segregation of partitioning-defective (PAR) proteins at the cortex, but how mechanical changes in the cytoskeleton are transmitted to PAR proteins remains elusive. Here we uncover a role of actomyosin contractility in the remodelling of PAR proteins through cortical clustering. During embryonic polarization in Caenorhabditis elegans, actomyosin contractility and the resultant cortical tension stimulate clustering of PAR-3 at the cortex. Clustering of atypical protein kinase C (aPKC) is supported by PAR-3 clusters and is antagonized by activation of CDC-42. Cortical clustering is associated with retardation of PAR protein exchange at the cortex and with effective entrainment of advective cortical flows. Our findings delineate how cytoskeleton contractility couples the cortical clustering and long-range displacement of PAR proteins during polarization. The principles described here would apply to other pattern formation processes that rely on local modification of cortical actomyosin and PAR proteins.
Microtubule filaments form ubiquitous networks. However, quantitative analysis of this structure is difficult due to its complex architecture. A tool is given for the automated retrieval of microtubule filaments from superresolution microscopy images and used for a quantitative analysis of microtubule network architecture phenotypes in fibroblasts.
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