Cerium oxide capping on Y-doped HfO2 films for ferroelectric phase stabilization with endurance improvement

The effects of 1-nm-thick CeOx capping on 7.5-nm-thick Y-doped HfO2 films on the ferroelectric characteristics are investigated. From the ferroelectric characteristics of the samples annealed at different temperatures from 450 to 600oC and annealing durations, the time (τ) required to stabilize the ferroelectric phase at each temperature was shortened by the capping. The identical activation energy (Ea) of 2.65 eV for ferroelectric stabilization without and with capping suggests the same kinetics for phase transformation. However, an increase in the remnant polarization (Pr) was obtained. Only a few Ce atoms diffused into the underlying HfO2 film even after 600oC annealing. Ferroelectric switching tests revealed an improvement in endurance from 107 to 1010 by the capping, presumably owing to the suppression of conductive filament formation. Therefore, CeOx capping is effective in promoting the ferroelectric phase in HfO2 with high switching endurance.

Chromosome remodelling by SMC/Condensin in B. subtilis is regulated by Soj/ParA during growth and sporulation

SMC complexes, loaded at ParB-parS sites, are key mediators of chromosome organization in bacteria. ParA/Soj proteins interact with ParB/Spo0J in a pathway involving ATP-dependent dimerization and DNA binding, leading to chromosome segregation and SMC loading. In Bacillus subtilis, ParA/Soj also regulates DNA replication initiation, and along with ParB/Spo0J is involved in cell cycle changes during endospore formation. The first morphological stage in sporulation is the formation of an elongated chromosome structure called an axial filament. We now show that a major redistribution of SMC complexes drives axial filament formation, in a process regulated by ParA/Soj. Unexpectedly, this regulation is dependent on monomeric forms of ParA/Soj that cannot bind DNA or hydrolyse ATP. These results reveal a new role for ParA/Soj proteins in the regulation of SMC dynamics in bacteria, and yet further complexity in the web of interactions involving chromosome replication, segregation, and organization, controlled by ParAB and SMC.

The Ability of CBFβ-SMMHC to Increase RUNX1 Binding Affinity to Target DNA Correlates with Its Leukemogenic Potential

Inversion of chromosome 16 is a consistent finding in patients with acute myeloid leukemia subtype M4 with eosinophilia (AML M4Eo), which generates a CBFB-MYH11 fusion gene. We recently showed that RUNX1 is indispensable for Cbfb-MYH11-induced leukemogenesis in a mouse model. We found that RUNX1 interacted with CBFβ-SMMHC, the fusion protein encoded by CBFB-MYH11, to directly regulate critical genes for leukemogenesis (Zhen et al., Blood, 2020). However, our current understanding of the interaction between CBFβ-SMMHC and RUNX1 does not provide adequate explanation on how the RUNX1-CBFβ-SMMHC complex forms and how the complex interacts with DNA for leukemogenesis as CBFβ-SMMHC without the RUNX1 high-affinity-binding-domain (CBFβ-SMMHC-ΔHABD) is also able to induce leukemia while CBFβ-SMMHC with mutations in the C-terminal multimerization domain (CBFβ-SMMHC-mDE) is not able to induce leukemia in mice. To address this question, we used RHD domain of RUNX1, CBFβ, CBFβ-SMMHC, CBFβ-SMMHC-ΔHABD and CBFβ-SMMHC-mDE proteins, which were purified from E. coli overexpressing these proteins, to explore how the HABD and DE domains affect the interactions between CBFβ-SMMHC, RHD and RUNX1-target DNA Bio-Layer Interferometry (BLI) and negative staining. As expected, deletion of the HABD domain significantly reduced CBFβ-SMMHC's binding affinity to RHD by BLI assay. Interestingly, differences in binding affinity between RHD and different versions of CBFβ-SMMHC did not correlate with their leukemogenic capability. On the other hand, the binding affinity between RHD and its target oligo was more significantly enhanced by CBFβ-SMMHC and CBFβ-SMMHC-ΔHABD that can induce leukemia than CBFβ-SMMHC-DE, which cannot. We also found that both CBFβ-SMMHC and CBFβ-SMMHC-ΔHABD, but not CBFβ-SMMHC-mDE, could form a filament structure by negative staining, suggesting the filament formation ability is important for leukemogenesis by CBFβ-SMMHC. In addition, RHD reduces filament formation by CBFβ-SMMHC, which was overcome when target oligo was added. In contrast, RHD could not inhibit filament formation by CBFβ-SMMHC-ΔHABD, suggesting that HABD interaction is required for RHD to disrupt filament formation by CBFβ-SMMHC. Overall, we found that leukemogenic capability of CBFβ-SMMHC correlates with its ability to enhance binding between RHD and its target DNA and to form multimerized filaments. The results also suggest that HABD and DE domains of CBFβ-SMMHC are required for the formation of the RUNX1-CBFβ-SMMHC complex with higher binding affinity to target DNA. Disclosures No relevant conflicts of interest to declare.

Multiple Roles of Flagellar Export Chaperones for Efficient and Robust Flagellar Filament Formation in Salmonella

FlgN, FliS, and FliT are flagellar export chaperones specific for FlgK/FlgL, FliC, and FliD, respectively, which are essential component proteins for filament formation. These chaperones facilitate the docking of their cognate substrates to a transmembrane export gate protein, FlhA, to facilitate their subsequent unfolding and export by the flagellar type III secretion system (fT3SS). Dynamic interactions of the chaperones with FlhA are thought to determine the substrate export order. To clarify the role of flagellar chaperones in filament assembly, we constructed cells lacking FlgN, FliS, and/or FliT. Removal of either FlgN, FliS, or FliT resulted in leakage of a large amount of unassembled FliC monomers into the culture media, indicating that these chaperones contribute to robust and efficient filament formation. The ∆flgN ∆fliS ∆fliT (∆NST) cells produced short filaments similarly to the ∆fliS mutant. Suppressor mutations of the ∆NST cells, which lengthened the filament, were all found in FliC and destabilized the folded structure of FliC monomer. Deletion of FliS inhibited FliC export and filament elongation only after FliC synthesis was complete. We propose that FliS is not involved in the transport of FliC upon onset of filament formation, but FliS-assisted unfolding of FliC by the fT3SS becomes essential for its rapid and efficient export to form a long filament when FliC becomes fully expressed in the cytoplasm.

Stress-dependent dynamic and reversible formation of cytoskeleton-like filaments and gel-transition by tardigrade tolerance proteins

Tardigrades are able to tolerate almost complete dehydration by entering a reversible ametabolic state called anhydrobiosis and resume their animation upon rehydration. Dehydrated tardigrades are exceptionally stable and withstand various physical extremes. Although trehalose and late embryogenesis abundant (LEA) proteins have been extensively studied as potent protectants against dehydration in other anhydrobiotic organisms, tardigrades produce high amounts of tardigrade-unique protective cytoplasmic-abundant heat-soluble (CAHS) proteins which are essential for the anhydrobiotic survival of tardigrades. However, the precise mechanisms of their action in this protective role are not fully understood. In the present study, we first postulated the presence of tolerance proteins that form protective condensates via phase separation in a stress-dependent manner and searched for tardigrade proteins that reversibly form condensates upon dehydration-like stress. Through comprehensive analysis, we identified 336 such proteins, collectively dubbed “dehydration-induced reversibly condensing proteins (DRPs)”. Unexpectedly, we rediscovered CAHS proteins as highly enriched in DRPs, 3 of which were major components of DRPs. We revealed that these CAHS proteins reversibly polymerize into many cytoskeleton-like filaments depending on hyperosmotic stress in cultured cells and undergo reversible gel-transition in vitro, which increases the mechanical strength of cell-like microdroplets. The conserved putative helical C-terminal region is necessary and sufficient for filament formation by CAHS proteins, and mutations disrupting the secondary structure of this region impaired both the filament formation and the gel transition. On the basis of these results, we propose that CAHS proteins are novel cytoskeletal proteins that form filamentous networks and undergo gel-transition in a stress-dependent manner to provide on-demand physical stabilization of cell integrity against deformative forces during dehydration and contribute to the exceptional stability of dehydrated tardigrades.