Study On The Function And Molecular Characteristics Of The Regulator Of The SIN Pathway Of The Initiation Network Produced By Aspergillus Nidulans

Aspergillus is one of the most widely distributed fungi in nature,which affects human life everywhere.Aspergillus consists of multicellular hyphae that are partitioned by septa and the conidiophore of filamentous hyphae could produce a large number of spores(conidia)through asexual development.Hyphae growth includes mycelial cell growth or elongation and cell division.And cell division is an important way of the asexual development in fungi.It is also necessary for the formation of fungi.The mitosis precedes after chromosome separation,mitotic exit and cytokinesis.Among them,cytokinesis is the process of cell division of the cytoplasm produces two daughter cells which is the final and key step in cell division.Therefore,the biologists focus on the studies of the regulation network of cytokinesis for a long time in the world.Since the filamentous fungus Aspergillus nidulans contains a mycelium of multinucleate cells that are partitioned by septa,it is able to endure more defects in cytokinesis than single-cell yeast.Thus,A.nidulans is an excellent model organism for allowing an unambiguous identification of investigating the regulation features of cytokinesis.The Septation Initiation Network(SIN)has been initiated after the conidia germination reaches a certain size,to form septum;one of these,the serine-threonine kinase SEPH,is a key player for trigger cytokinesis.The exact time and location of cytokinesis/septation is crucial for the growth of mycelia and conidiation.Many regulators are involved in regulation of septation process.Cell biologists and geneticists always focus on the function of those regulators,which can help us understand the cytokinesis process better.In this study,we have studied functions of Prs family,PP2A-ParA and PP2A-PabA related to the regulatory networks and mechanisms of SIN.At the same time,through RNA-Seq and phenotypic analysis in the background of ParA-overexpressed strain which complete abolished septation,we identified a new positive septation regulator,MztA.Moreover,the function of MztA that regulation of cytokinesis was studied in A.nidulans.The results of this study are summarized as follows:1.We identified that the filamentous fungus Aspergillus nidulans genome contains three PRPP synthase-homologous genes(AnprsA,AnprsB and AnprsC),among which AnprsB and AnprsC but not AnprsA are auxotrophic genes.Transcriptional expression profiles revealed that the mRNA levels of AnprsA,AnprsB and AnprsC are dynamic during germination,hyphal growth and sporulation and that they all showed abundant expression during the vigorous hyphal growth time point.Inhibiting the expression of AnprsB or AnprsC in conditional strains produced more effects on the total PRPP synthetase activity than did inhibiting AnprsA,thus indicating that different AnPrs proteins are unequal in their contributions to Prs enzyme activity.In addition,the constitutive overexpression of AnprsA or AnprsC could significantly rescue the defective phenotype of the AnprsB-absent strain,suggesting that the function of AnprsB is not a specific consequence of this auxotrophic gene but instead comes from the contribution of Prs proteins to PRPP synthetase activity.2.Reversible phosphorylation/dephosphorylation reactions play important roles in regulating protein activity and subcellular localization in the SIN pathway.Protein phosphatase 2A(PP2A)is a major intracellular protein phosphatase that regulates multiple aspects of cell growth and metabolism.The PP2A heterotrimeric protein complex,which consists of a cascade of three subunits,namely,a catalytic subunit(C)and a structural subunit(A)associated with a third,hypothetically competitive and variable regulatory subunit(B).Different activities of PP2A and subcellular localization are determined by its regulatory subunits.Here we identified and characterized the functions of two protein phosphatase regulatory subunit homologs,ParA and PabA,in Aspergillus nidulans.Our results demonstrate that ParA localizes to the septum site and that deletion of parA causes hyperseptation,while overexpression of parA abolishes septum formation;this suggests that ParA may function as a negative regulator of septation.In comparison,PabA displays a clear colocalization pattern with 4',6-diamidino-2-phenylindole(DAPI)-stained nuclei,and deletion of pabA induces a remarkable delayed-septation phenotype.Both parA and pabA are required for hyphal growth,conidiation,and self-fertilization,likely to maintain normal levels of PP2A activity.Most interestingly,parA deletion is capable of suppressing septation defects in pabA mutants,suggesting that ParA counteracts PabA during the septation process.In contrast,double mutants of parA and pabA led to synthetic defects in colony growth,indicating that ParA functions synthetically with PabA during hyphal growth.Moreover,unlike the case for PP2A-Par1 and PP2A-Pab1 in yeast(which are negative regulators that inactivate the septation initiation network[SIN]),loss of ParA or PabA fails to suppress defects of temperature-sensitive mutants of the SEPH kinase of the SIN.Thus,our findings support the previously unrealized evidence that the B-family subunits of PP2A have comprehensive functions as partners of heterotrimeric enzyme complexes of PP2A,both spatially and temporally,in.A.nidulans.3.The septation initiation network(SIN)componentjitis are a conserved spindle pole body(SPB)localized signaling cascade and the terminal kinase complex SidB-MobA,which must localize on the SPB in this pathway to trigger septation/cytokinesis.The regulatory subunit of phosphatase PP2A-ParA has been identified to be a negative regulator capable of inactivating the SIN.However,little is known about how ParA regulates the SIN pathway and whether ParA regulates the septum formation process through affecting the SPB-localized SIN proteins.In this study,through RNA-Seq and genetic approaches,we identified a new positive septation regulator,a putative mitotic-spindle organizing protein and a yeast Mztl homolog MztA,which acts antagonistically towards PP2A-ParA to coordinately regulate the SPB-localized SIN proteins SidB-MobA during septation.These findings imply that regulators,phosphatase PP2A-ParA and MztA counteract the septation function probably through balancing the polymerization and depolymerization of microtubules at the SPB.Findings in this project give a new sight about how regulators of SIN involve in cytokinesis and septation.And we also provide a theoretical basis of conidiation in A.nidulans.Meanwhile,this study can provide a theoretical basis for the study of cytokinesis in the clinical cancer.