Probing the distribution of cortical components, including PtdIns(4,5)P2, during asymmetric division of C. elegans embryos

Asymmetric cell division is crucial for embryonic development and stem cell lineages. In the one-cell C. elegans embryo, a contractile cortical actomyosin network contributes to asymmetric division by segregating PAR proteins to discrete cortical domains. The anterior-posterior (AP) polarity established in this manner is maintained thereafter and translated into mechanisms that lead to asymmetric spindle positioning during mitosis, thus ensuring proper segregation of cell fate determinants and fate specification of the daughter cells.

Here, performing cortical live imaging using confocal spinning disk microscopy, we discovered that the plasma membrane lipid phosphatidylinositol 4,5-bisphosphate (PtdIns(4,5)P2; PIP2) forms polarized dynamic structures at the cell membrane of C. elegans zygotes, which distribute in a PAR-dependent manner along the AP embryonic axis. PIP2 cortical structures overlap with filamentous actin (F-actin), and coincide with the actin regulators RHO-1, CDC-42 as well as ECT-2. Particle image velocimetry analysis revealed that PIP2 and F-actin cortical movements are coupled, with PIP2 structures moving slightly ahead of polymerizing F-actin bundles. Importantly, we established that the formation of PIP2 cortical structure depends on F-actin and their movement on actin polymerization. Conversely, we found that decreasing or increasing the level of PIP2 results in severe F-actin disorganization, revealing interdependence between these components. Moreover, increasing or decreasing the levels of PIP2 revealed that the formation of CDC-42 and RHO-1 cortical structures depends on PIP2, while PIP2 structures formation is mostly independent of RHO-1 and CDC-42. Further-more, we uncovered that PIP2 and F-actin regulate the sizing of PAR cortical domains during the establishment and maintenance phases of polarization. Overall, our work establishes that a lipid membrane component, PIP2, modulates actin organization and cell polarity in C. elegans embryos.

Furthermore, we found that the force generation mediators GPR-1 and LIN-5, as well as the negative force regulators CSNK-1 and GPB-1, distribute in cortical structures. While CSNK-1 and GPB-1 form elongated cortical structures during pseudo-cleavage overlapping with PIP2 cortical structures, GPR-1 and LIN-5 form mostly cortical foci not overlapping with GPB-1 and PIP2 cortical structures. The formation of GPB-1 cortical structures is independent of GPR-1 and vice versa. However, the formation and localization of GPB-1 cortical structures depends on PIP2. Overexpressing GPR-1 increases the formation of GPR-1 cortical structures and PIP2/GPB-1 cortical structures, which overlap in large part. CSNK-1 regulates PPK-1 localization to the embryo posterior and is required for the formation of PIP2 cortical structures on the anterior side.

Overall, this work reveals that many important players of asymmetric cell division in the one-cell stage C. elegans embryo distribute unevenly on