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.

Atypical Protein Kinase C Promotes its own Asymmetric Localisation by Phosphorylating Cdc42 in Polarising Cells.

Atypical protein kinase C (aPKC) is a major regulator of cell polarity. Acting in conjunction with Par6, Par3 and the small GTPase Cdc42, aPKC becomes asymmetrically localised and drives the polarisation of cells. aPKC activity is crucial for its own asymmetric localisation, suggesting a hitherto unknown feedback mechanism contributing to polarisation. Here we show in C. elegans zygotes that the feedback relies on CDC-42 phosphorylation at serine 71 by aPKC, which in turn results in aPKC dissociation from CDC-42. The dissociated aPKC then associates with PAR-3 clusters, which are transported anteriorly by actomyosin-based cortical flow. Moreover, the turnover of aPKC-mediated CDC-42 phosphorylation regulates the organisation of the actomyosin cortex that drives aPKC asymmetry. Given the widespread role of aPKC and Cdc42 in cell polarity, this form of self-regulation of aPKC may be vital for the robust polarisation of many cell types.

The case for complement component 5 as a target in neurodegenerative disease.

INTRODUCTION: Complement-based drug discovery is undergoing a renaissance, empowered by new advances in structural biology, complement biology and drug development. Certain components of the complement pathway, particularly C1q and C3, have been extensively studied in the context of neurodegenerative disease, and established as key therapeutic targets. C5 also has huge therapeutic potential in this arena, with its druggability clearly demonstrated by the success of C5-inhibitor eculizumab. AREAS COVERED: We will discuss the evidence supporting C5 as a target in neurodegenerative disease, along with the current progress in developing different classes of C5 inhibitors and the gaps in knowledge that will help progress in the field. EXPERT OPINION: Validation of C5 as a therapeutic target for neurodegenerative disease would represent a major step forward for complement therapeutics research and has the potential to furnish disease-modifying drugs for millions of patients suffering worldwide. Key hurdles that need to be overcome for this to be achieved are understanding how C5a and C5b should be targeted to bring therapeutic benefit and demonstrating the ability to target C5 without creating vulnerability to infection in patients. This requires greater biological elucidation of its precise role in disease pathogenesis, supported by better chemical/biological tools.

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-Molecule Narrow-Field Microscopy of Protein-DNA Binding Dynamics in Glucose Signal Transduction of Live Yeast Cells.

Single-molecule narrow-field microscopy is a versatile tool to investigate a diverse range of protein dynamics in live cells and has been extensively used in bacteria. Here, we describe how these methods can be extended to larger eukaryotic, yeast cells, which contain subcellular compartments. We describe how to obtain single-molecule microscopy data but also how to analyze these data to track and obtain the stoichiometry of molecular complexes diffusing in the cell. We chose glucose-mediated signal transduction of live yeast cells as the system to demonstrate these single-molecule techniques as transcriptional regulation is fundamentally a single-molecule problem-a single repressor protein binding a single binding site in the genome can dramatically alter behavior at the whole cell and population levels.

Large scale, single-cell FRET-based glucose uptake measurements within heterogeneous populations.

Fluorescent biosensors are powerful tools allowing the concentration of metabolites and small molecules, and other properties such as pH and molecular crowding to be measured inside live single cells. The technology has been hampered by lack of simple software to identify cells and quantify biosensor signals in single cells. We have developed a new software package, FRETzel, to address this gap and demonstrate its use by measuring insulin-stimulated glucose uptake in individual fat cells of varying sizes for the first time. Our results support the long-standing hypothesis that larger fat cells are less sensitive to insulin than smaller ones, a finding that has important implications for the battle against type 2 diabetes. FRETzel has been optimized using the messy and crowded environment of cultured adipocytes, demonstrating its utility for quantification of FRET biosensors in a wide range of other cell types, including fibroblasts and yeast via a simple user-friendly quantitative interface.

Global impacts of climate change on avian functional diversity.

Climate change is predicted to drive geographical range shifts, leading to fluctuations in species richness (SR) worldwide. However, the effect of these changes on functional diversity (FD) remains unclear, in part because comprehensive species-level trait data are generally lacking at global scales. Here, we use morphometric and ecological traits for 8268 bird species to estimate the impact of climate change on avian FD. We show that future bird assemblages are likely to undergo substantial shifts in trait structure, with a magnitude of change greater than predicted from SR alone, and a direction of change varying according to geographical location and trophic guild. For example, our models predict that FD of insect predators will increase at higher latitudes with concurrent losses at mid-latitudes, whereas FD of seed dispersing birds will fluctuate across the tropics. Our findings highlight the potential for climate change to drive continental-scale shifts in avian FD with implications for ecosystem function and resilience.

AVONET: morphological, ecological and geographical data for all birds.

Functional traits offer a rich quantitative framework for developing and testing theories in evolutionary biology, ecology and ecosystem science. However, the potential of functional traits to drive theoretical advances and refine models of global change can only be fully realised when species-level information is complete. Here we present the AVONET dataset containing comprehensive functional trait data for all birds, including six ecological variables, 11 continuous morphological traits, and information on range size and location. Raw morphological measurements are presented from 90,020 individuals of 11,009 extant bird species sampled from 181 countries. These data are also summarised as species averages in three taxonomic formats, allowing integration with a global phylogeny, geographical range maps, IUCN Red List data and the eBird citizen science database. The AVONET dataset provides the most detailed picture of continuous trait variation for any major radiation of organisms, offering a global template for testing hypotheses and exploring the evolutionary origins, structure and functioning of biodiversity.

Cost comparison of intra-detrusor injection of botulinum toxin versus augmentation cystoplasty for refractory neurogenic detrusor overactivity in children.

INTRODUCTION: Treatment options for refractory neurogenic detrusor overactivity (NDO) in children include botulinum toxin type A (BTX-A) and augmentation cystoplasty (AC). Although BTX-A is accepted in contemporary pediatric urologic practice, cost and long-term outcomes data for BTX-A are limited relative to the gold standard, AC. The purpose of this study was to compare the projected 10-year costs of AC versus BTX-A. METHODS: We performed a cost analysis from the payer perspective by computationally modeling treatment sequences by a Markov model. In the model, we used probabilities derived from published sources, and costs obtained at a tertiary medical center. The base case was a pediatric patient with refractory NDO. In the model, we assumed biannual BTX-A treatments. Treatment costs over 10 years were compared between immediate AC versus bridging therapy with BTX-A. Using the computational model, we simulated 100,000 instances of 10-year treatment cost for each of the two treatment modalities. The costs for the two treatment approaches were then compared using t-test and Wilcoxon test. RESULTS: The projected median and mean 10-year cost of immediately AC were $51,798.72 (95% CI [$51,798.72, $327,483.80]) and $123,473.4 (SD: $98,085.23) respectfully, while the projected median and mean 10-year cost of bridging therapy with BTX-A prior to proceeding to AC as needed were $74,552.46 (95% CI [$53,188.56, $309,913.07]) and $124,858.80 (SD: $84,495.35) (p < 0.001). CONCLUSIONS: For a typical index pediatric patient with NDO, bridging therapy with intravesical BTX-A is associated with an increased cost compared to immediate AC over a ten-year period.

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.

PySTACHIO: Python Single-molecule TrAcking stoiCHiometry Intensity and simulatiOn, a flexible, extensible, beginner-friendly and optimized program for analysis of single-molecule microscopy data.

As camera pixel arrays have grown larger and faster, and optical microscopy techniques ever more refined, there has been an explosion in the quantity of data acquired during routine light microscopy. At the single-molecule level, analysis involves multiple steps and can rapidly become computationally expensive, in some cases intractable on office workstations. Complex bespoke software can present high activation barriers to entry for new users. Here, we redevelop our quantitative single-molecule analysis routines into an optimized and extensible Python program, with GUI and command-line implementations to facilitate use on local machines and remote clusters, by beginners and advanced users alike. We demonstrate that its performance is on par with previous MATLAB implementations but runs an order of magnitude faster. We tested it against challenge data and demonstrate its performance is comparable to state-of-the-art analysis platforms. We show the code can extract fluorescence intensity values for single reporter dye molecules and, using these, estimate molecular stoichiometries and cellular copy numbers of fluorescently-labeled biomolecules. It can evaluate 2D diffusion coefficients for the characteristically short single-particle tracking data. To facilitate benchmarking we include data simulation routines to compare different analysis programs. Finally, we show that it works with 2-color data and enables colocalization analysis based on overlap integration, to infer interactions between differently labelled biomolecules. By making this freely available we aim to make complex light microscopy single-molecule analysis more democratized.

A CLK1-KKT2 Signaling Pathway Regulating Kinetochore Assembly in Trypanosoma brucei.

During mitosis, eukaryotic cells must duplicate and separate their chromosomes in a precise and timely manner. The apparatus responsible for this is the kinetochore, which is a large protein structure that links chromosomal DNA and spindle microtubules to facilitate chromosome alignment and segregation. The proteins that comprise the kinetochore in the protozoan parasite Trypanosoma brucei are divergent from yeast and mammals and comprise an inner kinetochore complex composed of 24 distinct proteins (KKT1 to KKT23, KKT25) that include four protein kinases, CLK1 (KKT10), CLK2 (KKT19), KKT2, and KKT3. We recently reported the identification of a specific trypanocidal inhibitor of T. brucei CLK1, an amidobenzimidazole, AB1. We now show that chemical inhibition of CLK1 with AB1 impairs inner kinetochore recruitment and compromises cell cycle progression, leading to cell death. Here, we show that KKT2 is a substrate for CLK1 and identify phosphorylation of S508 by CLK1 to be essential for KKT2 function and for kinetochore assembly. Additionally, KKT2 protein kinase activity is required for parasite proliferation but not for assembly of the inner kinetochore complex. We also show that chemical inhibition of the aurora kinase AUK1 does not affect CLK1 phosphorylation of KKT2, indicating that AUK1 and CLK1 are in separate regulatory pathways. We propose that CLK1 is part of a divergent signaling cascade that controls kinetochore function via phosphorylation of the inner kinetochore protein kinase KKT2. IMPORTANCE In eukaryotic cells, kinetochores are large protein complexes that link chromosomes to dynamic microtubule tips, ensuring proper segregation and genomic stability during cell division. Several proteins tightly coordinate kinetochore functions, including the protein kinase aurora kinase B. The kinetochore has diverse evolutionary roots. For example, trypanosomatids, single-cell parasitic protozoa that cause several neglected tropical diseases, possess a unique repertoire of kinetochore components whose regulation during the cell cycle remains unclear. Here, we shed light on trypanosomatid kinetochore biology by showing that the protein kinase CLK1 coordinates the assembly of the inner kinetochore by phosphorylating one of its components, KKT2, allowing the timely spatial recruitment of the rest of the kinetochore proteins and posterior attachment to microtubules in a process that is aurora kinase B independent.

Comparison of endogenously expressed fluorescent protein fusions behaviour for protein quality control and cellular ageing research.

The yeast Hsp104 protein disaggregase is often used as a reporter for misfolded or damaged protein aggregates and protein quality control and ageing research. Observing Hsp104 fusions with fluorescent proteins is a popular approach to follow post stress protein aggregation, inclusion formation and disaggregation. While concerns that bigger protein tags, such as genetically encoded fluorescent tags, may affect protein behaviour and function have been around for quite some time, experimental evidence of how exactly the physiology of the protein of interest is altered within fluorescent protein fusions remains limited. To address this issue, we performed a comparative assessment of endogenously expressed Hsp104 fluorescent fusions function and behaviour. We provide experimental evidence that molecular behaviour may not only be altered by introducing a fluorescent protein tag but also varies depending on such a tag within the fusion. Although our findings are especially applicable to protein quality control and ageing research in yeast, similar effects may play a role in other eukaryotic systems.

Correlative single-molecule fluorescence barcoding of gene regulation in Saccharomyces cerevisiae.

Most cells adapt to their environment by switching combinations of genes on and off through a complex interplay of transcription factor proteins (TFs). The mechanisms by which TFs respond to signals, move into the nucleus and find specific binding sites in target genes is still largely unknown. Single-molecule fluorescence microscopes, which can image single TFs in live cells, have begun to elucidate the problem. Here, we show that different environmental signals, in this case carbon sources, yield a unique single-molecule fluorescence pattern of foci of a key metabolic regulating transcription factor, Mig1, in the nucleus of the budding yeast, Saccharomyces cerevisiae. This pattern serves as a 'barcode' of the gene regulatory state of the cells which can be correlated with cell growth characteristics and other biological function.

Single-molecule optical microscopy of protein dynamics and computational analysis of images to determine cell structure development in differentiating Bacillus subtilis.

Here we use singe-molecule optical proteomics and computational analysis of live cell bacterial images, using millisecond super-resolved tracking and quantification of fluorescently labelled protein SpoIIE in single live Bacillus subtilis bacteria to understand its crucial role in cell development. Asymmetric cell division during sporulation in Bacillus subtilis presents a model system for studying cell development. SpoIIE is a key integral membrane protein phosphatase that couples morphological development to differential gene expression. However, the basic mechanisms behind its operation remain unclear due to limitations of traditional tools and technologies. We instead used advanced single-molecule imaging of fluorescently tagged SpoIIE in real time on living cells to reveal vital changes to the patterns of expression, localization, mobility and stoichiometry as cells undergo asymmetric cell division then engulfment of the smaller forespore by the larger mother cell. We find, unexpectedly, that SpoIIE forms tetramers capable of cell- and stage-dependent clustering, its copy number rising to ~ 700 molecules as sporulation progresses. We observed that slow moving SpoIIE clusters initially located at septa are released as mobile clusters at the forespore pole as phosphatase activity is manifested and compartment-specific RNA polymerase sigma factor, σF, becomes active. Our findings reveal that information captured in its quaternary organization enables one protein to perform multiple functions, extending an important paradigm for regulatory proteins in cells. Our findings more generally demonstrate the utility of rapid live cell single-molecule optical proteomics for enabling mechanistic insight into the complex processes of cell development during the cell cycle.

Biophysical characterisation of DNA origami nanostructures reveals inaccessibility to intercalation binding sites.

Intercalation of drug molecules into synthetic DNA nanostructures formed through self-assembled origami has been postulated as a valuable future method for targeted drug delivery. This is due to the excellent biocompatibility of synthetic DNA nanostructures, and high potential for flexible programmability including facile drug release into or near to target cells. Such favourable properties may enable high initial loading and efficient release for a predictable number of drug molecules per nanostructure carrier, important for efficient delivery of safe and effective drug doses to minimise non-specific release away from target cells. However, basic questions remain as to how intercalation-mediated loading depends on the DNA carrier structure. Here we use the interaction of dyes YOYO-1 and acridine orange with a tightly-packed 2D DNA origami tile as a simple model system to investigate intercalation-mediated loading. We employed multiple biophysical techniques including single-molecule fluorescence microscopy, atomic force microscopy, gel electrophoresis and controllable damage using low temperature plasma on synthetic DNA origami samples. Our results indicate that not all potential DNA binding sites are accessible for dye intercalation, which has implications for future DNA nanostructures designed for targeted drug delivery.

Design and validation of a low-cost, high-fidelity model for robotic pyeloplasty simulation training.

INTRODUCTION/BACKGROUND: Owing to restrictions in operative experiences, urology residents can no longer solely rely on 'hands-on' operative time to master their surgical skills by the end of residency. Simulation training could help residents master basic surgical skills and steps of a procedure to maximize time in the operative room. However, simulators can be expensive or tedious to set up, limiting the availability to residents and training programs. OBJECTIVE: The authors sought to develop and validate an inexpensive, high-fidelity training model for robotic pyeloplasty. STUDY DESIGN: Pyeloplasty models were created using Dragon Skin® FX-Pro tissue-mimicking silicone cast over 3-dimensional molds. Urology faculty and trainees completed a demographic questionnaire. The participants viewed a brief instructional video and then independently performed robotic dismembered pyeloplasty on the model. Acceptability and content validity were evaluated via post-task evaluation of the model. Construct validity was evaluated by comparing procedure completion time, the Global Evaluative Assessment of Robotic Skills (GEARS) score, blinded subjective physical evaluation of repair quality (1-10 scale), and flow rate between experts and novices. RESULTS: In total, 5 urology faculty, 6 fellows, and 14 residents participated. The median robotic console experience among faculty, fellows, and residents was 8 years (interquartile range [IQR] = 6-11), 3.5 years (IQR = 2-4 years), and 0 years (IQR = 0-0.5 years), respectively. The median procedure completion time was 29 min (IQR = 26-40 min), and the median flow rate was 1.11 mL/s (IQR = 0-1.34 mL/s). All faculty had flow rates >1.25 mL/s and procedure times <30 min compared with 2 of 6 fellows and none of the residents (P < 0.001). All faculty, half of the fellows, and none of the residents achieved a GEARS score ≥20, with a median resident score of 12.5 (IQR = 8-13) (P < 0.001). For repair quality, all faculty scored ≥9 (out of 10), all fellows scored ≥8, and the median score among residents was 6 (IQR = 2-6) (P < 0.001). The material cost was $1.32/model, and the average production time was 0.12 person-hours/model. DISCUSSION AND CONCLUSION: This low-cost pyeloplasty model exhibits acceptability and content validity. Construct validity is supported by significant correlation between participant expertise and simulator performance across multiple assessment domains. The model has excellent potential to be used as a training tool in urology and allows for repetitive practice of pyeloplasty skills before live cases.

Towards mapping the 3D genome through high speed single-molecule tracking of functional transcription factors in single living cells.

How genomic DNA is organized in the nucleus is a long-standing question. We describe a single-molecule bioimaging method utilizing super-localization precision coupled to fully quantitative image analysis tools, towards determining snapshots of parts of the 3D genome architecture of model eukaryote budding yeast Saccharomyces cerevisiae with exceptional millisecond time resolution. We employ astigmatism imaging to enable robust extraction of 3D position data on genomically encoded fluorescent protein reporters that bind to DNA. Our relatively straightforward method enables snippets of 3D architectures of likely single genome conformations to be resolved captured via DNA-sequence specific binding proteins in single functional living cells.

Single-Organelle Quantification Reveals Stoichiometric and Structural Variability of Carboxysomes Dependent on the Environment.

The carboxysome is a complex, proteinaceous organelle that plays essential roles in carbon assimilation in cyanobacteria and chemoautotrophs. It comprises hundreds of protein homologs that self-assemble in space to form an icosahedral structure. Despite its significance in enhancing CO2 fixation and potentials in bioengineering applications, the formation of carboxysomes and their structural composition, stoichiometry, and adaptation to cope with environmental changes remain unclear. Here we use live-cell single-molecule fluorescence microscopy, coupled with confocal and electron microscopy, to decipher the absolute protein stoichiometry and organizational variability of single ß-carboxysomes in the model cyanobacterium Synechococcus elongatus PCC7942. We determine the physiological abundance of individual building blocks within the icosahedral carboxysome. We further find that the protein stoichiometry, diameter, localization, and mobility patterns of carboxysomes in cells depend sensitively on the microenvironmental levels of CO2 and light intensity during cell growth, revealing cellular strategies of dynamic regulation. These findings, also applicable to other bacterial microcompartments and macromolecular self-assembling systems, advance our knowledge of the principles that mediate carboxysome formation and structural modulation. It will empower rational design and construction of entire functional metabolic factories in heterologous organisms, for example crop plants, to boost photosynthesis and agricultural productivity.