Study On The Cell Division And Cell Cycle Regulation Of Saccharolobus Islandicus

Cell division is a key biological process in the cell cycle of all organisma.A specialized set of proteins and complicated cell signaling processe are required to ensure that the mother cell divides faithfully into two daughter cells.Eukaryotes use the endosomal sorting transporter complex ESCRT to drive the process of membrane cleavage during the final stages of cell division,resulting in the separation of two daughter cells.In almost all bacteria,the process of cytokinesis is driven by the microtubule-like protein FtsZ,which directs the cell wall synthesis machinery to build the cell wall and cell membrane invagination until the cell divides in two.Archaea,although prokaryotes,possess both ESCRT and FtsZ machineries of cell division,demonstrating the diverse nature of Archaea cell division.Recent growing evidences support that eukaryotes may have originated from archaea,particularly Asgard archaea,suggesting that studying archaea can further deepen our understanding of the cellular ancestry of eukaryotes.However,many archaeal biological processes remain largely unexplored.One of the most striking features of Sulfolobales such as Saccharolobus islandicus is that they have a cell cycle similar to that of eukaryotic cells,i.e.G1、S、G2 and M periods.In eukaryotic cells,cell cycle regulation is mainly mediated by the Cyclin/Cdk complexes,but in archaea only Eukaryotic-like protein kinases are present,and it remains unknown whether the kinases are actually involved in cell cycle regulation.The fact that the proteasome regulates certain key periodic proteins at the protein level and thus influences cell cycle progression,a phenomenon found in eukaryotic cells,has been confirmed in archaea,providing further evidence that eukaryotic cells originated in archaea.In this study,we first explored the process of cytokinesis and investigated the function of ESCRT-Ⅲ and its homologues during cytokinesis.Through endogenous CRISPR-based gene editing techniques,we analyzed the in vivo roles of ESCRT-Ⅲand its homologues ESCRT-Ⅲ-1、2、3 in Saccharolobus islandicus.We found that ESCRT-Ⅲ-1 and ESCRT-Ⅲ-3 are non-essential and can be knocked out simultaneously.Based on cell cycle synchronization techniques,we found that both ESCRT-Ⅲ and the homologues ESCRT-Ⅲ-1、ESCRT-Ⅲ-2 were expressed cyclically at protein level,and that the expression peaks of ESCRT-Ⅲ-1 and ESCRT-Ⅲ-2 were later than that of ESCRT-Ⅲ.Immunofluorescence localization ESCRT-Ⅲ in the cells of Saccharolobus islandicus REY15A and the Vps4 dominant mutant overexpression strain Sis/pSeSD-Vps4-T148A showed that ESCRT-Ⅲ can form rings of different diameters during cytokinesis,implying that ESCRT-Ⅲ drive the cell membrane to contract.In ESCRT-Ⅲ-1-deficient,ESCRT-Ⅲ-2 knockdown strain,the contraction of the cells still proceeded normally,except that at the final stage the membrane could not excise properly,resulting in bead-like cells.This suggests that during cytokinesis in Saccharolobus islandicus REY15A,ESCRT-Ⅲ forms a divisive ring that can contract,whereas ESCRT-Ⅲ-1 and ESCRT-Ⅲ-2 act during the final membrane excision of cytokinesis.Next,during the studies of the cell cycle regulation,we identified a conserved important cell cycle-specific transcription factor(named aCcrl)and its viral homologues which control cell division in Saccharolobus islandicus.We found that the transcript levels of aCcr1 peaked during active cell division(M phase)following expression of CdvA,an archaea-specific cytokinesis protein.Cells overexpressing the 58-aa long RHH(Ribbon-helix-helix)family of cellular transcription factors accrl and homologues encoded by the large spindle-shaped viruses Acidianus two-tailed virus(ATV)and Sulfolobus monocaudavirus 3(SMV3)showed significant growth retardation and cytokinesis failure,manifested as large cells with multiple chromosomes.Transcriptome analysis identified aCcrl overexpression resulting in the down-regulation(>4-fold)of 17 genes,including cdvA.13 of the 17 highly repressed genes had a conserved motif between the TATA-binding box and the translation initiation site,the aCcrl-box,which is essential for aCcrl binding.aCcrl-box is present throughout the Sulfolobales cdvA gene in the promoter and 5’UTR,suggesting that aCcrl-mediated cdvA repression is an evolutionarily conserved mechanism by which Saccharolobus islandicus determines the course of cell division and which their viruses use to manipulate the cell cycle of their hosts.Most archaeal viruses do not have their own replication machinery and can only proliferate through the host’s replication machinery,so manipulation of the host’s cell cycle is particularly critical for viruses.The interaction between the large spindle-shaped virus STSV2 and the Saccharolobus islandicus was studied.We found that STSV2 infection leads to significant cell enlargement in Saccharolobus islandicus cells,forming large cells up to 20 times the size of normal cells in diameter.During this process,cell division is blocked,cell division-related genes are severely down-regulated,and cell division takes on an asymmetric form.In short,the virus infects the host and turns it into a giant virus manufacturing factory for its own proliferation.Our study provides important insights into both eukaryotic origins and virus-host interactions.