Crosslinking by ZapD drives the assembly of short, discontinuous FtsZ filaments into ring-like structures in solution

In most bacteria, division depends on a cytoskeletal structure, the FtsZ ring, that functions as a scaffold to recruit additional proteins, with which it forms the machinery responsible for division, the divisome. The detailed architecture of the ring, in particular the mechanisms of assembly, stabilization, and disassembly, are still largely unknown. Here, we highlight the role of FtsZ-associated proteins (Zaps) that stabilize the FtsZ ring by crosslinking the filaments. Among Zap proteins, ZapD binds the C-terminal domain of FtsZ, which serves as a hub for its regulation. We demonstrate that ZapD crosslinks FtsZ filaments into ring-like structures formed by a discontinuous arrangement of short filaments. Using cryo-electron tomography combined with biochemical analysis, we reveal the three-dimensional organization of the ring-like structures and shed light on the mechanism of FtsZ filament crosslinking by ZapD. Together, our data provide a model of how FtsZ-associated proteins can stabilize FtsZ filaments into discontinuous ring-like structures reminiscent of that existing in the bacterial cell.This project has been supported by the Max Planck-Bristol Centre for Minimal Biology (A.M.-S.) and the Deutsche Forschungsgemeinschaft (P. S.). J.S. and M. Jasnin are supported by the French Agence Nationale de la Recherche (ANR) and the Deutsche Forschungsgemeinschaft (DFG) call ANR-DFG 2020 NLE for the project no. JA-3038/2-1 (to M. Jasnin). J.-H.K. is funded by the Germany’s Excellence Strategy – EXC-2094 – 390783311. This work was also supported by the Spanish Government through Grant PID2019-104544GB-I00/AEI/10.13039/501100011033 (M.Jimenez, C.A. and G.R.). M.S.-S. was supported by European Social Fund through Grant PTA2020-018219-I/AEI/10.13039/501100011033. J.R.L.-O and M S.-S. acknowledge support from the Molecular Interactions Facility at the CIB Margarita Salas-CSIC. A.M.-S and J.-H.K. are part of IMPRS-LS, J.-H.K. is also a CeNS Center for NanoScience associate.Peer reviewe

A highly specific and sensitive serological assay detects SARS‑CoV‑2 antibody levels in COVID‑19 patients that correlate with neutralization

Objective The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pandemic challenges national health systems and the global economy. Monitoring of infection rates and seroprevalence can guide public health measures to combat the pandemic. This depends on reliable tests on active and former infections. Here, we set out to develop and validate a specific and sensitive enzyme linked immunosorbent assay (ELISA) for detection of anti SARS-CoV-2 antibody levels. Methods In our ELISA, we used SARS-CoV-2 receptor-binding domain (RBD) and a stabilized version of the spike (S) ectodomain as antigens. We assessed sera from patients infected with seasonal coronaviruses, SARS-CoV-2 and controls. We determined and monitored IgM-, IgA- and IgG-antibody responses towards these antigens. In addition, for a panel of 22 sera, virus neutralization and ELISA parameters were measured and correlated. Results The RBD-based ELISA detected SARS-CoV-2-directed antibodies, did not cross-react with seasonal coronavirus antibodies and correlated with virus neutralization (R2 = 0.89). Seroconversion started at 5 days after symptom onset and led to robust antibody levels at 10 days after symptom onset. We demonstrate high specificity (99.3%; N = 1000) and sensitivity (92% for IgA, 96% for IgG and 98% for IgM; > 10 days after PCR-proven infection; N = 53) in serum. Conclusions With the described RBD-based ELISA protocol, we provide a reliable test for seroepidemiological surveys. Due to high specificity and strong correlation with virus neutralization, the RBD ELISA holds great potential to become a preferred tool to assess thresholds of protective immunity after infection and vaccination

Liquid-liquid phase separation of germline proteins

Spatial organization of chemical processes is a widely accepted requirement for living systems. Cells have developed a great variety of mechanisms to achieve spatial and temporal organization of which many rely on lipid membranes to separate biochemical processes from bulk phases. However, studies in recent years have shown that membraneless organelles are highly dynamic entities that can organize and facilitate chemical reactions. Membraneless organelles are known to consist of a diverse population of different proteins and nucleic acids. Many studies focus on the phase behaviour and properties of single protein regions. We therefore investigated the phase behaviour of more complex systems using the well-characterized germ granule protein Ddx4 and the uncharacterized N-terminal regions of Piwi proteins. We show that the interplay of these disordered regions has differential effects on the phase properties and the propensity to phase separate. Furthermore, we find that liquidliquid phase separation of Piwi protein N-terminal regions is dependent on folded domains. The evolution of disordered proteins is not well understood and experimental data is scarce. Therefore, we compared the phase behaviour of human germ granule proteins to Drosophila melanogaster proteins. We show that the propensity to phase separate, phase properties and interactions of composite systems are evolutionarily conserved between Homo sapiens and Drosophila melanogaster homologues, even though sequence similarity is remarkably low.</p

Liquid-liquid phase separation of germline proteins

Spatial organization of chemical processes is a widely accepted requirement for living systems. Cells have developed a great variety of mechanisms to achieve spatial and temporal organization of which many rely on lipid membranes to separate biochemical processes from bulk phases. However, studies in recent years have shown that membraneless organelles are highly dynamic entities that can organize and facilitate chemical reactions. Membraneless organelles are known to consist of a diverse population of different proteins and nucleic acids. Many studies focus on the phase behaviour and properties of single protein regions. We therefore investigated the phase behaviour of more complex systems using the well-characterized germ granule protein Ddx4 and the uncharacterized N-terminal regions of Piwi proteins. We show that the interplay of these disordered regions has differential effects on the phase properties and the propensity to phase separate. Furthermore, we find that liquidliquid phase separation of Piwi protein N-terminal regions is dependent on folded domains. The evolution of disordered proteins is not well understood and experimental data is scarce. Therefore, we compared the phase behaviour of human germ granule proteins to Drosophila melanogaster proteins. We show that the propensity to phase separate, phase properties and interactions of composite systems are evolutionarily conserved between Homo sapiens and Drosophila melanogaster homologues, even though sequence similarity is remarkably low.</p

Regulating the dynamic folding of a DNA hairpin at the expense of a small, molecular fuel

Molecular machines, such as ATPases or motor proteins, couple the catalysis of a chemical reaction, most commonly hydrolysis of nucleotide triphosphates, to their conformational change. In essence, they continuously convert a chemical fuel to drive their motion. An outstanding goal of nanotechnology remains to synthesize a nanomachine with similar functions, precision, and speed. The field of DNA nan- otechnology has given rise to the engineering precision required for such a device. Simultaneously, the field of systems chemistry developed fast chemical reaction cycles that convert fuel to change the function of molecules. In this work, we thus combined a fast, chemical reaction cycle with the precision of DNA nanotechnology to yield kinetic control over the conformational state of a DNA hairpin. Future work on such systems will result in fast and precise DNA nanodevices

Ion binding with charge inversion combined with screening modulates DEAD box helicase phase transitions

Membraneless organelles, or biomolecular condensates, enable cells to compartmentalize material and processes into unique biochemical environments. While specific, attractive molecular interactions are known to stabilize biomolecular condensates, repulsive interactions, and the balance between these opposing forces, are largely unexplored. Here, we demonstrate that repulsive and attractive electrostatic interactions regulate condensate stability, internal mobility, interfaces, and selective partitioning of molecules both in vitro and in cells. We find that signaling ions, such as calcium, alter repulsions between model Ddx3 and Ddx4 condensate proteins by directly binding to negatively charged amino acid sidechains and effectively inverting their charge, in a manner fundamentally dissimilar to electrostatic screening. Using a polymerization model combined with generalized stickers and spacers, we accurately quantify and predict condensate stability over a wide range of pH, salt concentrations, and amino acid sequences. Our model provides a general quantitative treatment for understanding how charge and ions reversibly control condensate stability

Diagnostic Yield of Initial and Consecutive Blood Cultures in Children With Cancer and Febrile Neutropenia

BACKGROUND: The timing and necessity of repeated blood cultures (BCs) in children with cancer and febrile neutropenia (FN) are unknown. We evaluated the diagnostic yield of BCs collected pre- and post-empiric FN antibiotics. METHODS: Data collected prospectively from the Australian Predicting Infectious ComplicatioNs in Children with Cancer (PICNICC) study were used. Diagnostic yield was calculated as the number of FN episodes with a true bloodstream infection (BSI) detected divided by the number of FN episodes that had a BC taken. RESULTS: A BSI was identified in 13% of 858 FN episodes. The diagnostic yield of pre-antibiotic BCs was higher than of post-antibiotic cultures (12.3% vs 4.4%, P < .001). Two-thirds of the post-antibiotic BSIs were associated with a new episode of fever or clinical instability, and only 2 new BSIs were identified after 48 hours of empiric antibiotics and persistent fever. A contaminated BC was identified more frequently in post-antibiotic cultures. CONCLUSIONS: In the absence of new fever or clinical instability, BCs beyond 48 hours of persistent fever have limited yield. Opportunity exists to optimize BC collection in this population and reduce the burden of unnecessary tests on patients, healthcare workers, and hospitals