During bacterial cell division, the tubulin-homolog FtsZ forms a ring-like structure at the center of the cell. This Z-ring not only organizes the division machinery, but treadmilling of FtsZ filaments was also found to play a key role in distributing proteins at the division site. What regulates the architecture, dynamics and stability of the Z-ring is currently unknown, but FtsZ-associated proteins are known to play an important role. Here, using an in vitro reconstitution approach, we studied how the well-conserved protein ZapA affects FtsZ treadmilling and filament organization into large-scale patterns. Using high-resolution fluorescence microscopy and quantitative image analysis, we found that ZapA cooperatively increases the spatial order of the filament network, but binds only transiently to FtsZ filaments and has no effect on filament length and treadmilling velocity. Together, our data provides a model for how FtsZ-associated proteins can increase the precision and stability of the bacterial cell division machinery in a switch-like manner.
DNA oxidation by ten-eleven translocation (TET) family enzymes is essential for epigenetic reprogramming. The conversion of 5-methylcytosine (5mC) into 5-hydroxymethylcytosine (5hmC) initiates developmental and cell-type-specific transcriptional programs through mechanisms that include changes in the chromatin structure. Here, we show that the presence of 5hmC in the transcribed gene promotes the annealing of the nascent RNA to the template DNA strand, leading to the formation of an R-loop. Depletion of TET enzymes reduced global R-loops in absence of gene expression changes, whereas CRISPR-mediated tethering of TET to an active gene promoted the formation of R-loops. The genome-wide distribution of 5hmC and R-loops show a positive correlation in mouse and human stem cells and overlap in half of all active genes. Moreover, R-loop resolution leads to differential expression of a subset of genes that are involved in crucial events during stem cell proliferation. Altogether, our data reveal that epigenetic reprogramming via TET activity promotes co-transcriptional R-loop formation, disclosing new mechanisms of gene expression regulation.
The actin-homologue FtsA is essential for E. coli cell division, as it links FtsZ filaments in the Z-ring to transmembrane proteins. FtsA is thought to initiate cell constriction by switching from an inactive polymeric to an active monomeric conformation, which recruits downstream proteins and stabilizes the Z-ring. However, direct biochemical evidence for this mechanism is missing. Here, we use reconstitution experiments and quantitative fluorescence microscopy to study divisome activation in vitro. By comparing wild-type FtsA with FtsA R286W, we find that this hyperactive mutant outperforms FtsA WT in replicating FtsZ treadmilling dynamics, FtsZ filament stabilization and recruitment of FtsN. We could attribute these differences to a faster exchange and denser packing of FtsA R286W below FtsZ filaments. Using FRET microscopy, we also find that FtsN binding promotes FtsA self-interaction. We propose that in the active divisome FtsA and FtsN exist as a dynamic copolymer that follows treadmilling filaments of FtsZ.
13For bacterial cell division, treadmilling filaments of FtsZ organize into a ring-like structure at the 14 center of the cell. What governs the architecture and stability of this dynamic Z-ring is currently 15 unknown, but FtsZ-associated proteins have been suggested to play an important role. Here, we 16 used an in vitro reconstitution approach combined with fluorescence microscopy to study the 17 influence of the well-conserved protein ZapA on the organization and dynamics of FtsZ filaments 18 recruited to a supported membrane. We found that ZapA increases the spatial order and stabilizes 19 the steady-state architecture of the FtsZ filament network in a highly cooperative manner. Despite 20 its strong influence on their large-scale organization, ZapA binds only transiently to FtsZ filaments 21 and has no effect on their treadmilling velocity. Together, our data explains how FtsZ-associated 22 proteins can contribute to the precision and stability of the Z-ring without compromising 23 treadmilling dynamics. 24 25 26 ZapC or ZapD are usually longer than wild-type cells with a more heterogeneous cell length 12 distribution. Furthermore, they show mislocalized or misaligned Z-rings and skewed division 13 septae 7-10 . Despite not being structurally related, all these proteins have similar functions in vivo 14 with their corresponding phenotype becoming even more severe in cells missing more than one 15 Zap protein. However, the mechanism by which FtsZ-associated proteins contribute to the stability 16 of the Z-ring and precision of cell division is currently not known. 17The best characterized of those proteins is ZapA, a widely conserved protein critical for the 18 positioning and stability of the division machinery 7,8,11 . Structural studies on ZapA from E. coli and 19Pseudomonas aeruginosa revealed that ZapA has two distinct domains. A globular N-terminal 20 portion that forms the FtsZ-binding domain, and a C-terminal part that forms a 14-turn alpha helix, 21 responsible to promote the oligomerization of the protein into a pseudosymmetric tetramer 12-14 . 22 In vitro experiments using purified proteins showed that ZapA promotes the formation of stable 23FtsZ protofilament bundles and reduces the GTPase activity of FtsZ in solution 15-17 . These two 24 properties, however, would potentially interfere with the function of FtsZ treadmilling to distribute 25 cell wall synthases. Accordingly, it is currently not clear how ZapA can contribute to the stability 26 of the cell division machinery without having a negative effect on the polymerization dynamics of 27 its main organizer, FtsZ. 28Here, we used an in vitro reconstitution approach recapitulating Z-ring assembly and early events 29 during divisome maturation. Our biomimetic system combines purified proteins, supported 30 bilayers and total internal reflection fluorescence (TIRF) microscopy. This allowed us to directly 31 visualize the polymerization of FtsZ with its membrane anchor FtsA into filament networks and 32 the influence of ZapA on their archite...
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