Tetrameric UvrD Helicase Is Located at the E. Coli Replisome due to Frequent Replication Blocks.

DNA replication in all organisms must overcome nucleoprotein blocks to complete genome duplication. Accessory replicative helicases in Escherichia coli, Rep and UvrD, help remove these blocks and aid the re-initiation of replication. Mechanistic details of Rep function have emerged from recent live cell studies; however, the division of UvrD functions between its activities in DNA repair and role as an accessory helicase remain unclear in live cells. By integrating super-resolved single-molecule fluorescence microscopy with biochemical analysis, we find that UvrD self-associates into tetrameric assemblies and, unlike Rep, is not recruited to a specific replisome protein despite being found at approximately 80% of replication forks. Instead, its colocation with forks is likely due to the very high frequency of replication blocks composed of DNA-bound proteins, including RNA polymerase and factors involved in repairing DNA damage. Deleting rep and DNA repair factor genes mutS and uvrA, and inhibiting transcription through RNA polymerase mutation and antibiotic inhibition, indicates that the level of UvrD at the fork is dependent on UvrD's function. Our findings show that UvrD is recruited to sites of nucleoprotein blocks via different mechanisms to Rep and plays a multi-faceted role in ensuring successful DNA replication.

Community-developed checklists for publishing images and image analyses.

Images document scientific discoveries and are prevalent in modern biomedical research. Microscopy imaging in particular is currently undergoing rapid technological advancements. However, for scientists wishing to publish obtained images and image-analysis results, there are currently no unified guidelines for best practices. Consequently, microscopy images and image data in publications may be unclear or difficult to interpret. Here, we present community-developed checklists for preparing light microscopy images and describing image analyses for publications. These checklists offer authors, readers and publishers key recommendations for image formatting and annotation, color selection, data availability and reporting image-analysis workflows. The goal of our guidelines is to increase the clarity and reproducibility of image figures and thereby to heighten the quality and explanatory power of microscopy data.

The role of BST4 in the pyrenoid of Chlamydomonas reinhardtii.

In many eukaryotic algae, CO2 fixation by Rubisco is enhanced by a CO2-concentrating mechanism, which utilizes a Rubisco-rich organelle called the pyrenoid. The pyrenoid is traversed by a network of thylakoid-membranes called pyrenoid tubules, proposed to deliver CO2. In the model alga Chlamydomonas reinhardtii (Chlamydomonas), the pyrenoid tubules have been proposed to be tethered to the Rubisco matrix by a bestrophin-like transmembrane protein, BST4. Here, we show that BST4 forms a complex that localizes to the pyrenoid tubules. A Chlamydomonas mutant impaired in the accumulation of BST4 (bst4) formed normal pyrenoid tubules and heterologous expression of BST4 in Arabidopsis thaliana did not lead to the incorporation of thylakoids into a reconstituted Rubisco condensate. Chlamydomonas bst4 mutant did not show impaired growth at air level CO2. By quantifying the non-photochemical quenching (NPQ) of chlorophyll fluorescence, we show that bst4 displays a transiently lower thylakoid lumenal pH during dark to light transition compared to control strains. When acclimated to high light, bst4 had sustained higher NPQ and elevated levels of light-induced H2O2 production. We conclude that BST4 is not a tethering protein, but rather is an ion channel involved in lumenal pH regulation possibly by mediating bicarbonate transport across the pyrenoid tubules.

Community-developed checklists for publishing images and image analyses.

Images document scientific discoveries and are prevalent in modern biomedical research. Microscopy imaging in particular is currently undergoing rapid technological advancements. However for scientists wishing to publish the obtained images and image analyses results, there are to date no unified guidelines. Consequently, microscopy images and image data in publications may be unclear or difficult to interpret. Here we present community-developed checklists for preparing light microscopy images and image analysis for publications. These checklists offer authors, readers, and publishers key recommendations for image formatting and annotation, color selection, data availability, and for reporting image analysis workflows. The goal of our guidelines is to increase the clarity and reproducibility of image figures and thereby heighten the quality and explanatory power of microscopy data is in publications.

Working at the interface of physics and biology: An early career researcher perspective.

We are a network of Early Career Researchers (ECRs) and a Project Manager who are working on UKRI's "Physics of Life" grants which aim to merge ideas and techniques predominantly used in physics and apply them to biological questions. We have been collaborating since early 2021 to share research, experiences, and provide peer to peer support. Interdisciplinary projects are known for presenting challenges, bringing together disparate subjects and people with not only different knowledge bases, methods, and equipment but also varying ways of working and common languages. This has been the subject of commentary by researchers and funders from a management perspective, and we wanted to add to this discourse, using our experience to share the lessons and challenges we have encountered, from an ECR perspective.

Single-molecule and super-resolved imaging deciphers membrane behavior of onco-immunogenic CCR5.

The ability of tumors to establish a pro-tumorigenic microenvironment is an important point of investigation in the search for new therapeutics. Tumors form microenvironments in part by the "education" of immune cells attracted via chemotactic axes such as that of CCR5-CCL5. Further, CCR5 upregulation by cancer cells, coupled with its association with pro-tumorigenic features such as drug resistance and metastasis, has suggested CCR5 as a therapeutic target. However, with several conformational "pools" being reported, phenotypic investigations must be capable of unveiling conformational heterogeneity. Addressing this challenge, we performed super-resolution structured illumination microscopy (SIM) and single molecule partially TIRF-coupled HILO (PaTCH) microscopy of CCR5 in fixed cells. SIM data revealed a non-random spatial distribution of CCR5 assemblies, while Intensity-tracking of CCR5 assemblies from PaTCH images indicated dimeric sub-units independent of CCL5 perturbation. These biophysical methods can provide important insights into the structure and function of onco-immunogenic receptors and many other biomolecules.

RecA and RecB: probing complexes of DNA repair proteins with mitomycin C in live Escherichia coli with single-molecule sensitivity.

The RecA protein and RecBCD complex are key bacterial components for the maintenance and repair of DNA. RecBCD is a helicase-nuclease that uses homologous recombination to resolve double-stranded DNA breaks. It also facilitates coating of single-stranded DNA with RecA to form RecA filaments, a vital step in the double-stranded break DNA repair pathway. However, questions remain about the mechanistic roles of RecA and RecBCD in live cells. Here, we use millisecond super-resolved fluorescence microscopy to pinpoint the spatial localization of fluorescent reporters of RecA or RecB at physiological levels of expression in individual live Escherichia coli cells. By introducing the DNA cross-linker mitomycin C, we induce DNA damage and quantify the resulting steady state changes in stoichiometry, cellular protein copy number and molecular mobilities of RecA and RecB. We find that both proteins accumulate in molecular hotspots to effect repair, resulting in RecA stoichiometries equivalent to several hundred molecules that assemble largely in dimeric subunits before DNA damage, but form periodic subunits of approximately 3-4 molecules within mature filaments of several thousand molecules. Unexpectedly, we find that the physiologically predominant forms of RecB are not only rapidly diffusing monomers, but slowly diffusing dimers.

Critical roles for EGFR and EGFR-HER2 clusters in EGF binding of SW620 human carcinoma cells.

Epidermal growth factor (EGF) signalling regulates normal epithelial and other cell growth, with EGF receptor (EGFR) overexpression reported in many cancers. However, the role of EGFR clusters in cancer and their dependence on EGF binding is unclear. We present novel single-molecule total internal reflection fluorescence microscopy of (i) EGF and EGFR in living cancer cells, (ii) the action of anti-cancer drugs that separately target EGFR and human EGFR2 (HER2) on these cells and (iii) EGFR-HER2 interactions. We selected human epithelial SW620 carcinoma cells for their low level of native EGFR expression, for stable transfection with fluorescent protein labelled EGFR, and imaged these using single-molecule localization microscopy to quantify receptor architectures and dynamics upon EGF binding. Prior to EGF binding, we observe pre-formed EGFR clusters. Unexpectedly, clusters likely contain both EGFR and HER2, consistent with co-diffusion of EGFR and HER2 observed in a different model CHO-K1 cell line, whose stoichiometry increases following EGF binding. We observe a mean EGFR : EGF stoichiometry of approximately 4 : 1 for plasma membrane-colocalized EGFR-EGF that we can explain using novel time-dependent kinetics modelling, indicating preferential ligand binding to monomers. Our results may inform future cancer drug developments.

Single-Molecular Quantification of Flowering Control Proteins Within Nuclear Condensates in Live Whole Arabidopsis Root.

Here we describe the coupled standardization of two complementary fluorescence imaging techniques and apply it to liquid-liquid phase-separated condensates formed from an EGFP fluorescent reporter of flowering control locus A (FCA), a protein that associates with chromosomal DNA in plants during epigenetic regulation of the flowering process. First, we use home-built single-molecule Slimfield microscopy to establish a fluorescent protein standard. This sample comprises live yeast cells expressing Mig1 protein, a metabolic regulator which localizes to the nucleus under conditions of high glucose, fused to the same type of EGFP label as for the FCA fusion construct. Then we employ commercial confocal AiryScan microscopy to study the same standard. Finally, we demonstrate how to quantify FCA-EGFP nuclear condensates in intact root tips at rapid timescales and apply this calibration. This method is a valuable approach to obtaining single-molecule precise stoichiometry and copy number estimates of protein condensates that are integrated into the chromosome architecture of plants, using confocal instrumentation that lacks de facto single-molecule detection sensitivity.

Sperm selection with hyaluronic acid improved live birth outcomes among older couples and was connected to sperm DNA quality, potentially affecting all treatment outcomes.

STUDY QUESTION: What effects did treatment using hyaluronic acid (HA) binding/selection prior to ICSI have on clinical outcomes in the Hyaluronic Acid Binding sperm Selection (HABSelect) clinical trial? SUMMARY ANSWER: Older women randomized to the trial's experimental arm (selection of sperm bound to immobilized (solid-state) HA) had the same live birth rates as younger women, most likely a result of better avoidance of sperm with damaged DNA. WHAT IS KNOWN ALREADY: Recent randomized controlled trials (RCTs) investigating the efficacy of HA-based sperm selection prior to ICSI, including HABSelect, have consistently reported reductions in the numbers of miscarriages among couples randomized to the intervention, suggesting a pathological sperm-mediated factor mitigated by prior HA-binding/selection. The mechanism of that protection is unknown. STUDY DESIGN, SIZE, DURATION: The original HABSelect Phase 3 RCT ran from 2014 to 2017 and included 2752 couples from whom sperm samples used in control (ICSI) and intervention (Physiological IntraCytoplasmic Sperm Injection; PICSI) arms of the trial were stored frozen for later assessment of DNA quality (DNAq). The trial overlapped with its mechanistic arm, running from 2016 to 2018. PARTICIPANTS/MATERIALS, SETTING, METHODS: As miscarriage reduction was a significant secondary outcome of the trial, samples (n = 1247) selected for the mechanistic analysis were deliberately enriched for miscarriage outcomes (n = 92 or 7.4%) from a total of 154 miscarriages (5.6%) among all (n = 2752) couples randomized by stratified random sampling. Values from fresh semen samples for sperm concentration (mml), percentage forward progressive motility and percentage HA-binding score (HBS) were obtained before being processed by differential density gradient centrifugation or (rarely) by swim-up on the day of treatment. Surplus sperm pellets were recovered, aliquoted and cryopreserved for later analysis of DNAq using slide-based Comet, TUNEL, acridine orange (AO) and the sperm chromatin dispersion (SCD) assays. Following their classification into normal and abnormal sample subcategories based on reference values for sperm concentration and motility, relationships with HBS and DNAq were examined by Spearman correlation, Student's t-tests, Mann Whitney U tests, and logistic regression (univariable and multivariable). Parsimonious selection enabled the development of models for exploring and explaining data trends. Potential differences in future cumulative pregnancy rates relating to embryo quality were also explored. MAIN RESULTS AND THE ROLE OF CHANCE: Results from the 1247 sperm samples assayed for HBS and/or DNAq, generated data that were considered in relation to standard physiological measures of (sperm) vitality and to treatment outcomes. All measures of HBS and DNAq discriminated normal from abnormal sperm samples (P < 0.001). SCD correlated negatively with the Comet (r = -0.165; P < 0.001) and TUNEL assays (r = -0.200; P < 0.001). HBS correlated negatively with AO (r = -0.211; P < 0.001), Comet (r = -0.127; P < 0.001) and TUNEL (r = -0.214; P < 0.001) and positively with SCD (r = 0.255; P < 0.001). A model for predicting live birth (and miscarriage) rates included treatment allocation (odds ratio: OR 2.167, 95% CI 1.084-4.464, P = 0.031), female age (OR 0.301, 95% CI 0.133-0.761, P = 0.013, per decade) and the AO assay (OR 0.79, 95% CI 0.60-1. 02.761, P = 0.073, per 10 points rise). A model predicting the expected rate of biochemical pregnancy included male age (OR 0.464, 95% CI 0.314-0.674, P < 0.001, per decade) and the SCD assay (OR 1.04, 95% CI 1.007-1.075, P = 0.018, per 10 point rise). A model for conversion from biochemical to clinical pregnancy did not retain any significant patient or assay variables. A model for post-injection fertilization rates included treatment allocation (OR 0.83, 95% CI 0.75-0.91, P < 0.001) and the Comet assay (OR 0.950, 95% CI 0.91-1.00, P = 0.041). LIMITATIONS, REASONS FOR CAUTION: HABSelect was a prospective RCT and the mechanistic study group was drawn from its recruitment cohort for retrospective analysis, without the full benefit of randomization. The clinical and mechanistic aspects of the study were mutually exclusive in that measures of DNAq were obtained from residual samples and not from HA-selected versus unselected sperm. Models for fitting mechanistic with baseline and other clinical data were developed to compensate for variable DNAq data quality. HABSelect used a solid-state version of PICSI and we did not assess the efficacy of any liquid-state alternatives. PICSI reduced fertilization rates and did not improve the outlook for cumulative pregnancy rates. WIDER IMPLICATIONS OF THE FINDINGS: Notwithstanding the interventional effect on fertilization rates and possibly blastocyst formation (neither of which influenced pregnancy rates), poor sperm DNAq, reflected by lower HBS, probably contributed to the depression of all gestational outcomes including live births, in the HABSelect trial. The interventional avoidance of defective sperm is the best explanation for the equalization in live birth rates among older couples randomized to the trial's PICSI arm. As patients going forward for assisted conception cycles globally in future are likely to be dominated by an older demographic, HA-based selection of sperm for ICSI could be considered as part of their treatment plan. STUDY FUNDING/COMPETING INTEREST(S): The study was supported by the National Institute for Health Research (NIHR) EME (Efficacy and Mechanism Evaluation)-11-14-34. National Research Ethics Service approval 11/06/2013: 13/YH/0162. S.L. is CEO of ExamenLab Ltd (company number NI605309). TRIAL REGISTRATION NUMBER: ISRCTN99214271.

Membraneless organelles formed by liquid-liquid phase separation increase bacterial fitness.

Liquid-liquid phase separation is emerging as a crucial phenomenon in several fundamental cell processes. A range of eukaryotic systems exhibit liquid condensates. However, their function in bacteria, which, in general, lack membrane-bound compartments, remains less clear. Here, we used high-resolution optical microscopy to observe single bacterial aggresomes, nanostructured intracellular assemblies of proteins, to undercover their role in cell stress. We find that proteins inside aggresomes are mobile and undergo dynamic turnover, consistent with a liquid state. Our observations are in quantitative agreement with phase-separated liquid droplet formation driven by interacting proteins under thermal equilibrium that nucleate following diffusive collisions in the cytoplasm. We have found aggresomes in multiple species of bacteria and show that these emergent, metastable liquid-structured protein assemblies increase bacterial fitness by enabling cells to tolerate environmental stresses.