Kotynkova, K; (2016) Control of cleavage furrow formation by the RhoGEF Ect2. Doctoral thesis , UCL (University College London).

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Abstract

Cytokinesis is the final step of cell division that physically separates the cytoplasm of nascent daughter cells. The mitotic spindle plays a key role in positioning the cytokinetic furrow at the equator in animal cells but the exact mechanism is not yet understood. An important step during cleavage furrow formation is activation of small GTPase RhoA, which is brought about by the GEF factor Ect2. Our aim is to better understand the principles and regulation of cleavage furrow formation. Recent results in our lab have shown that the RhoGEF not only localizes to the spindle midzone after anaphase onset but also to the plasma membrane. Therefore we asked which lipids are involved in Ect2 membrane engagement and if the membrane translocation of Ect2 is an essential and rate-limiting step for cleavage furrow induction that confers spatial and temporal control of cytokinesis. Pharmacological interference with cellular lipids implicated PIP2 as an important anionic phospholipid for the association of Ect2 with the plasma membrane. We developed a chemical genetic system using hybrid proteins that allowed us to artificially target Ect2 to the plasma membrane. Our results demonstrate that the plasma membrane association of Ect2 is a prerequisite for cytokinesis in human cells. We also confirmed this finding by a complementary optogenetic approach of targeting Ect2 to the plasma membrane. Furthermore, light-induced membrane engagement of Ect2 highlighted the importance of local cortical Ect2 activity. Most current models for cytokinesis consider Ect2 recruitment to the spindle midzone as a key step in the furrow positioning in small animal cells. By replacing endogenous Ect2 with a mutated version that does not localize to the midzone, we have shown that this model cannot account for the placement and formation of the cleavage furrow at the cell equator. Unexpectedly, our results suggest that the midzone localization of Ect2 and the resulting equatorial gradient at the plasma membrane is dispensable for cytokinesis in mammalian cells. The equatorial concentration of Ect2 could still serve as a signal for furrow placement, but may be redundant with other not yet defined uncharacterized signals. In summary, our work firmly establishes plasma membrane engagement of Ect2 as a prerequisite for the execution of cytokinesis. It also reveals that prevailing models for how the cleavage furrow is placed in somatic cells are likely to be insufficient to explain the process.

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