Recommendations for accelerating open preprint peer review to improve the culture of science.

Peer review is an important part of the scientific process, but traditional peer review at journals is coming under increased scrutiny for its inefficiency and lack of transparency. As preprints become more widely used and accepted, they raise the possibility of rethinking the peer-review process. Preprints are enabling new forms of peer review that have the potential to be more thorough, inclusive, and collegial than traditional journal peer review, and to thus fundamentally shift the culture of peer review toward constructive collaboration. In this Consensus View, we make a call to action to stakeholders in the community to accelerate the growing momentum of preprint sharing and provide recommendations to empower researchers to provide open and constructive peer review for preprints.

Tracking changes between preprint posting and journal publication during a pandemic.

Amid the Coronavirus Disease 2019 (COVID-19) pandemic, preprints in the biomedical sciences are being posted and accessed at unprecedented rates, drawing widespread attention from the general public, press, and policymakers for the first time. This phenomenon has sharpened long-standing questions about the reliability of information shared prior to journal peer review. Does the information shared in preprints typically withstand the scrutiny of peer review, or are conclusions likely to change in the version of record? We assessed preprints from bioRxiv and medRxiv that had been posted and subsequently published in a journal through April 30, 2020, representing the initial phase of the pandemic response. We utilised a combination of automatic and manual annotations to quantify how an article changed between the preprinted and published version. We found that the total number of figure panels and tables changed little between preprint and published articles. Moreover, the conclusions of 7.2% of non-COVID-19-related and 17.2% of COVID-19-related abstracts undergo a discrete change by the time of publication, but the majority of these changes do not qualitatively change the conclusions of the paper.

The evolving role of preprints in the dissemination of COVID-19 research and their impact on the science communication landscape.

The world continues to face a life-threatening viral pandemic. The virus underlying the Coronavirus Disease 2019 (COVID-19), Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2), has caused over 98 million confirmed cases and 2.2 million deaths since January 2020. Although the most recent respiratory viral pandemic swept the globe only a decade ago, the way science operates and responds to current events has experienced a cultural shift in the interim. The scientific community has responded rapidly to the COVID-19 pandemic, releasing over 125,000 COVID-19-related scientific articles within 10 months of the first confirmed case, of which more than 30,000 were hosted by preprint servers. We focused our analysis on bioRxiv and medRxiv, 2 growing preprint servers for biomedical research, investigating the attributes of COVID-19 preprints, their access and usage rates, as well as characteristics of their propagation on online platforms. Our data provide evidence for increased scientific and public engagement with preprints related to COVID-19 (COVID-19 preprints are accessed more, cited more, and shared more on various online platforms than non-COVID-19 preprints), as well as changes in the use of preprints by journalists and policymakers. We also find evidence for changes in preprinting and publishing behaviour: COVID-19 preprints are shorter and reviewed faster. Our results highlight the unprecedented role of preprints and preprint servers in the dissemination of COVID-19 science and the impact of the pandemic on the scientific communication landscape.

Technical and social issues influencing the adoption of preprints in the life sciences.

Preprints are gaining visibility in many fields. Thanks to the exponential growth in submissions to bioRxiv, an online server for preprints in biology, versions of manuscripts prior to the completion of journal-organized peer review are poised to become a standard component of the publishing experience in the life sciences. Here, we provide an overview of current challenges facing preprints, both technical and social, and a vision for their future development.

Cryo-EM structure of the bacterial actin AlfA reveals unique assembly and ATP-binding interactions and the absence of a conserved subdomain.

Bacterial actins are an evolutionarily diverse family of ATP-dependent filaments built from protomers with a conserved structural fold. Actin-based segregation systems are encoded on many bacterial plasmids and function to partition plasmids into daughter cells. The bacterial actin AlfA segregates plasmids by a mechanism distinct from other partition systems, dependent on its unique dynamic properties. Here, we report the near-atomic resolution electron cryo-microscopy structure of the AlfA filament, which reveals a strikingly divergent filament architecture resulting from the loss of a subdomain conserved in all other actins and a mode of ATP binding. Its unusual assembly interfaces and nucleotide interactions provide insight into AlfA dynamics, and expand the range of evolutionary variation accessible to actin quaternary structure.

Non-model model organisms.

Model organisms are widely used in research as accessible and convenient systems to study a particular area or question in biology. Traditionally only a handful of organisms have been widely studied, but modern research tools are enabling researchers to extend the set of model organisms to include less-studied and more unusual systems. This Forum highlights a range of 'non-model model organisms' as emerging systems for tackling questions across the whole spectrum of biology (and beyond), the opportunities and challenges, and the outlook for the future.

Bacterial Tubulins A and B Exhibit Polarized Growth, Mixed-Polarity Bundling, and Destabilization by GTP Hydrolysis.

Bacteria of the genus Prosthecobacter express homologs of eukaryotic α- and ß-tubulin, called BtubA and BtubB (BtubA/B), that have been observed to assemble into filaments in the presence of GTP. BtubA/B polymers are proposed to be composed in vitro by two to six protofilaments in contrast to that in vivo, where they have been reported to form 5-protofilament tubes named bacterial microtubules (bMTs). The btubAB genes likely entered the Prosthecobacter lineage via horizontal gene transfer and may be derived from an early ancestor of the modern eukaryotic microtubule (MT). Previous biochemical studies revealed that BtubA/B polymerization is reversible and that BtubA/B folding does not require chaperones. To better understand BtubA/B filament behavior and gain insight into the evolution of microtubule dynamics, we characterized in vitro BtubA/B assembly using a combination of polymerization kinetics assays and microscopy. Like eukaryotic microtubules, BtubA/B filaments exhibit polarized growth with different assembly rates at each end. GTP hydrolysis stimulated by BtubA/B polymerization drives a stochastic mechanism of filament disassembly that occurs via polymer breakage and/or fast continuous depolymerization. We also observed treadmilling (continuous addition and loss of subunits at opposite ends) of BtubA/B filament fragments. Unlike MTs, polymerization of BtubA/B requires KCl, which reduces the critical concentration for BtubA/B assembly and induces it to form stable mixed-orientation bundles in the absence of any additional BtubA/B-binding proteins. The complex dynamics that we observe in stabilized and unstabilized BtubA/B filaments may reflect common properties of an ancestral eukaryotic tubulin polymer.IMPORTANCE Microtubules are polymers within all eukaryotic cells that perform critical functions; they segregate chromosomes, organize intracellular transport, and support the flagella. These functions rely on the remarkable range of tunable dynamic behaviors of microtubules. Bacterial tubulin A and B (BtubA/B) are evolutionarily related proteins that form polymers. They are proposed to be evolved from the ancestral eukaryotic tubulin, a missing link in microtubule evolution. Using microscopy and biochemical approaches to characterize BtubA/B assembly in vitro, we observed that they exhibit complex and structurally polarized dynamic behavior like eukaryotic microtubules but differ in how they self-associate into bundles and how this bundling affects their stability. Our results demonstrate the diversity of mechanisms through which tubulin homologs promote filament dynamics and monomer turnover.

Accessory factors promote AlfA-dependent plasmid segregation by regulating filament nucleation, disassembly, and bundling.

In bacteria, some plasmids are partitioned to daughter cells by assembly of actin-like proteins (ALPs). The best understood ALP, ParM, has a core set of biochemical properties that contributes to its function, including dynamic instability, spontaneous nucleation, and bidirectional elongation. AlfA, an ALP that pushes plasmids apart in Bacillus, relies on a different set of underlying properties to segregate DNA. AlfA elongates unidirectionally and is not dynamically unstable; its assembly and disassembly are regulated by a cofactor, AlfB. Free AlfB breaks up AlfA bundles and promotes filament turnover. However, when AlfB is bound to the centromeric DNA sequence, parN, it forms a segrosome complex that nucleates and stabilizes AlfA filaments. When reconstituted in vitro, this system creates polarized, motile comet tails that associate by antiparallel filament bundling to form bipolar, DNA-segregating spindles.

Microtubule nucleating gamma-TuSC assembles structures with 13-fold microtubule-like symmetry.

Microtubules are nucleated in vivo by gamma-tubulin complexes. The 300-kDa gamma-tubulin small complex (gamma-TuSC), consisting of two molecules of gamma-tubulin and one copy each of the accessory proteins Spc97 and Spc98, is the conserved, essential core of the microtubule nucleating machinery. In metazoa multiple gamma-TuSCs assemble with other proteins into gamma-tubulin ring complexes (gamma-TuRCs). The structure of gamma-TuRC indicated that it functions as a microtubule template. Because each gamma-TuSC contains two molecules of gamma-tubulin, it was assumed that the gamma-TuRC-specific proteins are required to organize gamma-TuSCs to match 13-fold microtubule symmetry. Here we show that Saccharomyces cerevisiae gamma-TuSC forms rings even in the absence of other gamma-TuRC components. The yeast adaptor protein Spc110 stabilizes the rings into extended filaments and is required for oligomer formation under physiological buffer conditions. The 8-A cryo-electron microscopic reconstruction of the filament reveals 13 gamma-tubulins per turn, matching microtubule symmetry, with plus ends exposed for interaction with microtubules, implying that one turn of the filament constitutes a microtubule template. The domain structures of Spc97 and Spc98 suggest functions for conserved sequence motifs, with implications for the gamma-TuRC-specific proteins. The gamma-TuSC filaments nucleate microtubules at a low level, and the structure provides a strong hypothesis for how nucleation is regulated, converting this less active form to a potent nucleator.

Architecture and assembly of a divergent member of the ParM family of bacterial actin-like proteins.

Eubacteria and archaea contain a variety of actin-like proteins (ALPs) that form filaments with surprisingly diverse architectures, assembly dynamics, and cellular functions. Although there is much data supporting differences between ALP families, there is little data regarding conservation of structure and function within these families. We asked whether the filament architecture and biochemical properties of the best-understood prokaryotic actin, ParM from plasmid R1, are conserved in a divergent member of the ParM family from plasmid pB171. Previous work demonstrated that R1 ParM assembles into filaments that are structurally distinct from actin and the other characterized ALPs. They also display three biophysical properties thought to be essential for DNA segregation: 1) rapid spontaneous nucleation, 2) symmetrical elongation, and 3) dynamic instability. We used microscopic and biophysical techniques to compare and contrast the architecture and assembly of these related proteins. Despite being only 41% identical, R1 and pB171 ParMs polymerize into nearly identical filaments with similar assembly dynamics. Conservation of the core assembly properties argues for their importance in ParM-mediated DNA segregation and suggests that divergent DNA-segregating ALPs with different assembly properties operate via different mechanisms.

The structure and assembly dynamics of plasmid actin AlfA imply a novel mechanism of DNA segregation.

Bacterial cytoskeletal proteins participate in a variety of processes, including cell division and DNA segregation. Polymerization of one plasmid-encoded, actin-like protein, ParM, segregates DNA by pushing two plasmids in opposite directions and forms the current paradigm for understanding active plasmid segregation. An essential feature of ParM assembly is its dynamically instability, the stochastic switching between growth and disassembly. It is unclear whether dynamic instability is an essential feature of all actin-like protein-based segregation mechanisms or whether bacterial filaments can segregate plasmids by different mechanisms. We expressed and purified AlfA, a plasmid-segregating actin-like protein from Bacillus subtilis, and found that it forms filaments with a unique structure and biochemistry; AlfA nucleates rapidly, polymerizes in the presence of ATP or GTP, and forms highly twisted, ribbon-like, helical filaments with a left-handed pitch and protomer nucleotide binding pockets rotated away from the filament axis. Intriguingly, AlfA filaments spontaneously associate to form uniformly sized, mixed-polarity bundles. Most surprisingly, our biochemical characterization revealed that AlfA does not display dynamic instability and is relatively stable in the presence of diphosphate nucleotides. These results (i) show that there is remarkable structural diversity among bacterial actin filaments and (ii) indicate that AlfA filaments partition DNA by a novel mechanism.

Superresolution microscopy of the ß-carboxysome reveals a homogeneous matrix.

Carbon fixation in cyanobacteria makes a major contribution to the global carbon cycle. The cyanobacterial carboxysome is a proteinaceous microcompartment that protects and concentrates the carbon-fixing enzyme ribulose-1,5-bisphosphate carboxylase/oxygenase (RuBisCO) in a paracrystalline lattice, making it possible for these organisms to fix CO2 from the atmosphere. The protein responsible for the organization of this lattice in beta-type carboxysomes of the freshwater cyanobacterium Synechococcus elongatus, CcmM, occurs in two isoforms thought to localize differentially within the carboxysome matrix. Here we use wide-field time-lapse and three-dimensional structured illumination microscopy (3D-SIM) to study the recruitment and localization of these two isoforms. We demonstrate that this superresolution technique is capable of distinguishing the localizations of the outer protein shell of the carboxysome and its internal cargo. We develop an automated analysis pipeline to analyze and quantify 3D-SIM images and generate a population-level description of the carboxysome shell protein, RuBisCO, and CcmM isoform localization. We find that both CcmM isoforms have similar spatial and temporal localization, prompting a revised model of the internal arrangement of the ß-carboxysome.

A Tunable Protein Piston That Breaks Membranes to Release Encapsulated Cargo.

Movement of molecules across membranes in response to a stimulus is a key component of cellular programming. Here, we characterize and manipulate the response of a protein-based piston capable of puncturing membranes in a pH-dependent manner. Our protein actuator consists of modified R bodies found in a bacterial endosymbiont of paramecium. We express and purify R bodies from in E. coli; these pistons undergo multiple rounds of rapid extension and retraction. We developed a high throughput screen for mutants with altered pH sensitivity for tuning of the extension process. We show that the R bodies are capable of acting as synthetic pH-dependent pistons that can puncture E. coli membranes to release the trapped content. As such, these protein machines present a novel way to selectively rupture membrane compartments and will be important for programming cellular compartmentalization.

Building Spatial Synthetic Biology with Compartments, Scaffolds, and Communities.

Traditional views of synthetic biology often treat the cell as an unstructured container in which biological reactions proceed uniformly. In reality, the organization of biological molecules has profound effects on cellular function: not only metabolic, but also physical and mechanical. Here, we discuss a variety of perturbations available to biologists in controlling protein, nucleotide, and membrane localization. These range from simple tags, fusions, and scaffolds to heterologous expression of compartments and other structures that confer unique physical properties to cells. Next, we relate these principles to those guiding the spatial environments outside of cells such as the extracellular matrix. Finally, we discuss new directions in building intercellular organizations to create novel symbioses.

Synthetic Lipid-Containing Scaffolds Enhance Production by Colocalizing Enzymes.

Subcellular organization is critical for isolating, concentrating, and protecting biological activities. Natural subcellular organization is often achieved using colocalization of proteins on scaffold molecules, thereby enhancing metabolic fluxes and enabling coregulation. Synthetic scaffolds extend these benefits to new biological processes and are typically constructed from proteins or nucleic acids. To expand the range of available building materials, we use a minimal set of components from the lipid-encapsulated bacteriophage ϕ6 to form synthetic lipid-containing scaffolds (SLSs) in E. coli. Analysis of diffusive behavior by particle tracking in live cells indicates that SLSs are >20 nm in diameter; furthermore, density measurements demonstrate that SLSs contain a mixture of lipids and proteins. The fluorescent proteins mCitrine and mCerulean can be colocalized to SLSs. To test for effects on enzymatic production, we localized two enzymes involved in indigo biosynthesis to SLSs. We observed a scaffold-dependent increase in indigo production, showing that SLSs can enhance the production of a commercially relevant metabolite.

A call for transparency in tracking student and postdoc career outcomes.

There is a common misconception that the United States is suffering from a "STEM shortage," a dearth of graduates with scientific, technological, engineering, and mathematical backgrounds. In biomedical science, however, we are likely suffering from the opposite problem and could certainly better tailor training to actual career outcomes. At the Future of Research Symposium, various workshops identified this as a key issue in a pipeline traditionally geared toward academia. Proposals for reform all ultimately come up against the same problem: there is a shocking lack of data at institutional and national levels on the size, shape, and successful careers of participants in the research workforce. In this paper, we call for improved institutional reporting of the number of graduate students and postdocs and their training and career outcomes.

Induced sensitivity of Bacillus subtilis colony morphology to mechanical media compression.

Bacteria from several taxa, including Kurthia zopfii, Myxococcus xanthus, and Bacillus mycoides, have been reported to align growth of their colonies to small features on the surface of solid media, including anisotropies created by compression. While the function of this phenomenon is unclear, it may help organisms navigate on solid phases, such as soil. The origin of this behavior is also unknown: it may be biological (that is, dependent on components that sense the environment and regulate growth accordingly) or merely physical. Here we show that B. subtilis, an organism that typically does not respond to media compression, can be induced to do so with two simple and synergistic perturbations: a mutation that maintains cells in the swarming (chained) state, and the addition of EDTA to the growth media, which further increases chain length. EDTA apparently increases chain length by inducing defects in cell separation, as the treatment has only marginal effects on the length of individual cells. These results lead us to three conclusions. First, the wealth of genetic tools available to B. subtilis will provide a new, tractable chassis for engineering compression sensitive organisms. Second, the sensitivity of colony morphology to media compression in Bacillus can be modulated by altering a simple physical property of rod-shaped cells. And third, colony morphology under compression holds promise as a rapid, simple, and low-cost way to screen for changes in the length of rod-shaped cells or chains thereof.