The Functional Study Of Important Proteins PomA, PaxB And AcnA In Aspergillus Nidulans That Regulate Cytokinesis And Asexual Sporulation

Cell division,including nuclear division and cytokinesis,is an important process in cell.Cytokinesis,as the last step in the process of cell division,parted a mother cell into two daughter cells.In mammalian cells,abnormal cytokinesis may lead to a range of diseases and even to die.In yeast cells,abnormal cytokinesis result in the formation of non-nuclear or multinuclear cells.In filamentous fungi,cytokinesis is essential for conidiation,septation and hyphal growth.In the fungal cells,the invagination of the cell membrane caused by the contraction of the contractile actin ring(CAR)along with the septum formation to complete the process of cytokinesis.Which can be roughly divided into four steps: selecting a division site,assembling the actin ring,the contraction and disassembly of the actin ring.Previous studies have found that a septation initiation network(SIN)consisting of a series of conserved kinases positive regulates cytokinesis in the model filamentous fungus Aspergillus nidulans.Sep H is a core component of the SIN signaling pathway and plays an important role in cytokinesis and conidiation.Notably,Sep H mutant would abosish septation and conidiation,whereas hyper-activation would induce the formatiom of multiple septa.However,the current research on the regulation of cytokinesis is still not clear enough,and little is known about the regulation mechanism of cytokinesis.Through UV mutagenesis,our previous studies identified antagonizing components of the Sep H kinase cascade.Therefore,in this study,based on the above mutant library,the forward genetics and the reverse genetics were used to isolate the new regulator of cytokinesis.The main conclusions are as follows:1.Protein kinase Pom A is a suppressor of Sep H.The phenotypic analysis and microscopic observation revealed that the septation and conidiation were rescued to some extent in mutant strains compared to that of the sep H mutant with a completely abolished conidiophore structure.The results of hybridization indicated that the UV mutagenesis strains are extra-mutations from sep H.The next-generation sequencing and SNP analysis revealed mutations were harbored in the conserved domain of Pom A in the mutant strains.Next,the reverse genetics method was used to verify that the septation and conidiation were rescued in sep H and pom A double mutant strains.Those results indicate Pom A is a suppressor of Sep H.2.Pom A reverse regulates Sep H by negatively regulating the activity of the MAPK pathway.Quantitative phosphorylation proteomics experiments showed an increase activity of the MAPK pathway in Pom A deletion strain.Western blot analysis showed that MAPK activity was significantly decreased in Sep H mutant strain,while increased in Pom A mutant strain,compared with wild type strain,which suggested that Sep H is a positive regulator of MAPK pathway,while Pom A is a negative regulator.Microscopic observation revealed that the activated MAPK pathway caused by salt stress rescue the septation and conidiation in Sep H mutant strain.Those results suggested that Pom A suppress Sep H by negatively regulating the activity of the MAPK pathway.3.Paxillin protein PaxB involved in the regulation of septation and conidiation.According to bioinformatics analysis,two paxillin proteins Pax A and Pax B are contained in A.nidulans.The production of condia and the formation septa are disruptd in Pax B deletion strain,indicating that Pax B is involved in the regulation of septation and conidiation.4.Acn A and Pax B are required for cytokinesis by regulating the assembly of the Actin ring at the division site.Fluorescence microscopy observation revealed that actin could not assemble into a ring structure at the division site and cause cytokinesis defects in Acn A and Pax B deletion strains,which indicates that Acn A and Pax B are involved in the assembly of the Actin ring.In summary,this study found that Pom A acts as a negative regulator and Acn A and Pax B as positive regulators,to regulate the cytokinesis of A.nidulans.This study provides a new understanding for the regulation of cytokinesis.