Mechanisms Underlying The Cortical Septin Localization And The Role Of Septin In Regulating Cytokinesis

Septins are a family of highly conserved GTP-binding proteins in eukaryotes and were firstly discovered in the budding yeast Saccharomyces cerevisiae in a screen for the genes responsible for regulating septation.Septins are recognized as the fourth component of the cytosketelon because of their filamentous localization,similar to actin filaments,microtubules and intermediate filaments in mammalian cells.Septins interact with each other through their NC interfaces and the GTP-binding domain to form mirror symmetrical hetero-oligomers,and subsequently form filaments or highorder structures,such as hourglasses and rings.Most of Septins contain polybasic regions at their N terminus,which interact with the negatively charged lipids of the plasma membrane.Septins function as scaffolds and/or diffusion barrier to regulate cell migration,cytokinesis,biosynthesis of sperms and cilia,and cell polarity.However,it has remained unclear how Septins are localized to the plasma membrane and how Septins are involved in regulating cytokinesis.Using the fission yeast Schizosaccharomyces pombe as a model organism,we employed a combination of yeast genetics,molecular biology,biochemistry and livecell microscopy approaches to investigate the mechanism underlying the localization of Septin to the plasma membrane and the role of Septins in regulating cytokinesis.Live-cell microscopic analysis showed that Septin was enriched at the cortical region adjacent to the growing cell end during interphase.Therefore,we aimed to delineate the mechanim underlying the characteristic localization of Septin to the cortical membrane and to reveal the function of the cortical localization of Septin.In this study,we identified the Cdc42 GTPase-activating protein Rga6 as a crucial protein involved in promoting the cortical localization of Septin.The absence of Rga6 impariecd the cortical localization of Septin and abolished the enrichment of Septin at the cortical regions adjacent to the growing cell end.Microscopic data further showed that Rga6 partially co-localized with Septin on the cell cortex,and biochemical analysis showed that Rga6 physically interacted with the Septin complex.In addition,liposome reconstitution assays showed that the presence of Rga6 enhanced the affinity of Septin for the liposome membrane.These results suggest that Rga6 interacts with Septin to promote the cortical localization of Septin.Moreover,proper localization of Cdc42 to the growing cell end depended on Rga6 and the enrichment of Septin at the cortical regions adjacent to the growing cell end.Hence,our findings suggest a model that Rga6 regulates cell polarity by direct inhibition of Cdc42 on the cortical membrane and/or by the confinement of Cdc42 to the growing cell end.Live-cell microscopic analysis also revealed that the absence of the core septin component Spnl advanced the assembly of CAR(contracticle actomyosin ring)and decreased the constriction rate of CAR.Hence,we aimed to delineate the mechanim of Septin regulating the cytokinesis.In fission yeast,the SIN(Septation Initiation Network)and the septum biosynthesis are involved in regulating the maturation and constriction of CAR.Therefore,we tested whether the absence of Spn 1 alters SIN and the formation of septa.We found that the absence of Spnl caused the SIN downstream kinase Sid2 and the glucan synthases Bgsl and Agsl to form an equatorial ring prematurely and that the absence of Spn1 impaired the maintainenace of Sid2,Bgs1,and Ags 1 at the the queatorial region.These findings suggest that Septin regulates cytokinesis through mediating proper localization of the SIN components and the glucan synthases.In summary,we have identified a crucial regulator of Septin,i.e.,Rga6 and have delinated the new mechanism underlying the Rga6-dependent cortical localization of Septin.In addition,we have demonstrated the roles of Septin in regulating cytokinesis.