The process of cytokinesis ultimately results in the partitioning of a mother cell into two daughter cells. Cytokinesis is initiated through the formation of an actomyosin ring at the equatorial plane of the mother cell, which drives the ingression of a cleavage furrow. At this stage, the scaffolding protein anillin stabilizes the actomyosin ring, and recruits septins. Once the ingression of the cleavage furrow is completed, the furrow membrane anchors to the bundled microtubules of the cytokinetic bridge. This is achieved through the centralspindlin complex, which resides at the nascent midbody organelle. Subsequently, anillin and septins further regulate the maturation of an intercellular bridge (ICB), which will ultimately be resolved by the abscission machinery. The phospholipid phosphatidylinositol 4,5-bisphosphate [PI(4,5)P2] plays a crucial role in mammalian cytokinesis. It is essential for the anillin-dependent stabilization of the actomyosin ring at the cleavage furrow, and it is required for the centralspindlin-dependent tethering of bridge microtubules to the plasma membrane. Nevertheless, the molecular mechanisms orchestrating PI(4,5)P2 synthesis in space and time during cytokinesis have remained elusive so far. In this study we investigated the contribution of type I PIP-kinases (phosphatidylinositol-4-phosphate 5-kinases) to the progression of cytokinesis and unveiled a crucial role for PIPKIy at the ICB. We found that septins interact with a common splice insert of PIPKIy isoforms 3 and 5 (PIPKIy-i3/i5), and that both septins and PIPKIy form a complex with centralspindlin. SiRNA-mediated depletion of PIPKIy-i3/i5 resulted in the loss of septin association with ICB microtubules, scattering of anillin away from the ICB, reduction of centralspindlin at the midbody, and a shorter cytokinetic bridge. Notably, these defects were rescued by wild-type PIPKIy, but not by septin binding-deficient or catalytically inactive mutants. These data support a model wherein septins recruit specific PIPKIy isoforms to the nascent midbody and, at this locale, PIPKIy-i3/i5 synthetize a pool of PI(4,5)P2 required for maintaining anillin, centralspindlin, and septins in place. Super-resolution analysis via ultrastructure-expansion microscopy further confirmed the above-mentioned defects at cytokinetic bridges in cells depleted of PIPKIy-i3/i5. Furthermore, live cell imaging of endogenously tagged eGFP-SEPT6 revealed that the hindered association of septins with microtubules observed at cytokinesis persisted within the daughter cells. Notably, siRNA-mediated depletion of centralspindlin phenocopied the loss of septin association to microtubules in non-mitotic cells, pointing towards a role of centralspindlin in coordinating the translocation of septins to bridge microtubules and, thereby, the inheritance of microtubule-associated septin fibers by daughter cells. Taken together, our findings establish septin-associated PIPKIy isoforms as novel regulators of late cytokinesis and demonstrate that both septins and PIPKIy are essential during midbody assembly and maturation.
