Predicting Rubisco-Linker Condensation from Titration in the Dilute Phase.

The condensation of Rubisco holoenzymes and linker proteins into "pyrenoids," a crucial supercharger of photosynthesis in algae, is qualitatively understood in terms of "sticker-and-spacer" theory. We derive semianalytical partition sums for small Rubisco-linker aggregates, which enable the calculation of both dilute-phase titration curves and dimerization diagrams. By fitting the titration curves to surface plasmon resonance and single-molecule fluorescence microscopy data, we extract the molecular properties needed to predict dimerization diagrams. We use these to estimate typical concentrations for condensation, and successfully compare these to microscopy observations.

Correlating fluorescence microscopy, optical and magnetic tweezers to study single chiral biopolymers such as DNA.

Biopolymer topology is critical for determining interactions inside cell environments, exemplified by DNA where its response to mechanical perturbation is as important as biochemical properties to its cellular roles. The dynamic structures of chiral biopolymers exhibit complex dependence with extension and torsion, however the physical mechanisms underpinning the emergence of structural motifs upon physiological twisting and stretching are poorly understood due to technological limitations in correlating force, torque and spatial localization information. We present COMBI-Tweez (Combined Optical and Magnetic BIomolecule TWEEZers), a transformative tool that overcomes these challenges by integrating optical trapping, time-resolved electromagnetic tweezers, and fluorescence microscopy, demonstrated on single DNA molecules, that can controllably form and visualise higher order structural motifs including plectonemes. This technology combined with cutting-edge MD simulations provides quantitative insight into complex dynamic structures relevant to DNA cellular processes and can be adapted to study a range of filamentous biopolymers.

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.

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.

GFP fusions of Sec-routed extracellular proteins in Staphylococcus aureus reveal surface-associated coagulase in biofilms.

Staphylococcus aureus is a major human pathogen that utilises many surface-associated and secreted proteins to form biofilms and cause disease. However, our understanding of these processes is limited by challenges of using fluorescent protein reporters in their native environment, because they must be exported and fold correctly to become fluorescent. Here, we demonstrate the feasibility of using the monomeric superfolder GFP (msfGFP) exported from S. aureus. By fusing msfGFP to signal peptides for the Secretory (Sec) and Twin Arginine Translocation (Tat) pathways, the two major secretion pathways in S. aureus, we quantified msfGFP fluorescence in bacterial cultures and cell-free supernatant from the cultures. When fused to a Tat signal peptide, we detected msfGFP fluorescence inside but not outside bacterial cells, indicating a failure to export msfGFP. However, when fused to a Sec signal peptide, msfGFP fluorescence was present outside cells, indicating successful export of the msfGFP in the unfolded state, followed by extracellular folding and maturation to the photoactive state. We applied this strategy to study coagulase (Coa), a secreted protein and a major contributor to the formation of a fibrin network in S. aureus biofilms that protects bacteria from the host immune system and increases attachment to host surfaces. We confirmed that a genomically integrated C-terminal fusion of Coa to msfGFP does not impair the activity of Coa or its localisation within the biofilm matrix. Our findings demonstrate that msfGFP is a good candidate fluorescent reporter to consider when studying proteins secreted by the Sec pathway in S. aureus.

Fluidification of Entanglements by a DNA Bending Protein.

In spite of the nanoscale and single-molecule insights into nucleoid associated proteins (NAPs), their role in modulating the mesoscale viscoelasticity of entangled DNA has been overlooked so far. By combining microrheology and molecular dynamics simulation, we find that the abundant NAP "integration host factor" (IHF) lowers the viscosity of entangled λDNA 20-fold at physiological concentrations and stoichiometries. Our results suggest that IHF may play a previously unappreciated role in resolving DNA entanglements and in turn may be acting as a "genomic fluidizer" for bacterial genomes.

Sensitive bacterial Vm sensors revealed the excitability of bacterial Vm and its role in antibiotic tolerance.

As an important free energy source, the membrane voltage (Vm) regulates many essential physiological processes in bacteria. However, in comparison with eukaryotic cells, knowledge of bacterial electrophysiology is very limited. Here, we developed a set of novel genetically encoded bacterial Vm sensors which allow single-cell recording of bacterial Vm dynamics in live cells with high temporal resolution. Using these new sensors, we reveal the electrically "excitable" and "resting" states of bacterial cells dependent on their metabolic status. In the electrically excitable state, frequent hyperpolarization spikes in bacterial Vm are observed, which are regulated by Na+/K+ ratio of the medium and facilitate increased antibiotic tolerance. In the electrically resting state, bacterial Vm displays significant cell-to-cell heterogeneity and is linked to the cell fate after antibiotic treatment. Our findings demonstrate the potential of our newly developed voltage sensors to reveal the underpinning connections between bacterial Vm and antibiotic tolerance.

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.

Correlating viscosity and molecular crowding with fluorescent nanobeads and molecular probes: in vitro and in vivo.

In eukaryotes, intracellular physico-chemical properties like macromolecular crowding and cytoplasmic viscoelasticity influence key processes such as metabolic activities, molecular diffusion and protein folding. However, mapping crowding and viscoelasticity in living cells remains challenging. One approach uses passive rheology in which diffusion of exogenous fluorescent particles internalized in cells is tracked and physico-chemical properties inferred from derived mean square displacement relations. Recently, the crGE2.3 Förster resonance energy transfer biosensor was developed to quantify crowding in cells, though it is unclear how this readout depends on viscoelasticity and the molecular weight of the crowder. Here, we present correlative, multi-dimensional data to explore diffusion and molecular crowding characteristics of molecular crowding agents using super-resolved fluorescence microscopy and ensemble time-resolved spectroscopy. We firstly characterize in vitro and then apply these insights to live cells of budding yeast Saccharomyces cerevisiae. It is to our knowledge the first time this has been attempted. We demonstrate that these are usable both in vitro and in the case of endogenously expressed sensors in live cells. Finally, we present a method to internalize fluorescent beads as in situ viscoelasticity markers in the cytoplasm of live yeast cells and discuss limitations of this approach including impairment of cellular function.

Crowding-induced morphological changes in synthetic lipid vesicles determined using smFRET.

Lipid vesicles are valuable mesoscale molecular confinement vessels for studying membrane mechanics and lipid-protein interactions, and they have found utility among bio-inspired technologies, including drug delivery vehicles. While vesicle morphology can be modified by changing the lipid composition and introducing fusion or pore-forming proteins and detergents, the influence of extramembrane crowding on vesicle morphology has remained under-explored owing to a lack of experimental tools capable of capturing morphological changes on the nanoscale. Here, we use biocompatible polymers to simulate molecular crowding in vitro, and through combinations of FRET spectroscopy, lifetime analysis, dynamic light scattering, and single-vesicle imaging, we characterize how crowding regulates vesicle morphology. We show that both freely diffusing and surface-tethered vesicles fluorescently tagged with the DiI and DiD FRET pair undergo compaction in response to modest concentrations of sorbitol, polyethylene glycol, and Ficoll. A striking observation is that sorbitol results in irreversible compaction, whereas the influence of high molecular weight PEG-based crowders was found to be reversible. Regulation of molecular crowding allows for precise control of the vesicle architecture in vitro, with vast implications for drug delivery and vesicle trafficking systems. Furthermore, our observations of vesicle compaction may also serve to act as a mechanosensitive readout of extramembrane crowding.

Structural interplay between DNA-shape protein recognition and supercoiling: The case of IHF.

The integration host factor (IHF) is a prominent example of indirect readout as it imposes one of the strongest bends on relaxed linear DNA. However, the relation between IHF and torsionally constrained DNA, as occurs physiologically, remains unclear. By using atomistic molecular dynamics simulations on DNA minicircles, we reveal, for the first time, the reciprocal influence between a DNA-bending protein and supercoiling. On one hand, the increased curvature of supercoiled DNA enhances wrapping around IHF making the final complex topologically dependent. On the other hand, IHF acts as a 'supercoiling relief' factor by compacting relaxed DNA loops and, when supercoiled, it pins the position of plectonemes in a unique and specific manner. In addition, IHF restrains under- or overtwisted DNA depending on whether the complex is formed in negatively or positively supercoiled DNA, becoming effectively a 'supercoiling buffer'. We finally provide evidence of DNA bridging by IHF and reveal that these bridges divide DNA into independent topological domains. We anticipate that the crosstalk detected here between the 'active' DNA and the multifaceted IHF could be common to other DNA-protein complexes relying on the deformation of DNA.

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.

Tween-20 Induces the Structural Remodeling of Single Lipid Vesicles.

The solubilization of lipid membranes by Tween-20 is crucial for a number of biotechnological applications, but the mechanistic details remain elusive. Evidence from ensemble assays supports a solubilization model that encompasses surfactant association with the membrane and the release of mixed micelles to solution, but whether this process also involves intermediate transitions between regimes is unanswered. In search of mechanistic origins, increasing focus is placed on identifying Tween-20 interactions with controllable membrane mimetics. Here, we employed ultrasensitive biosensing approaches, including single-vesicle spectroscopy based on fluorescence and energy transfer from membrane-encapsulated molecules, to interrogate interactions between Tween-20 and submicrometer-sized vesicles below the optical diffraction limit. We discovered that Tween-20, even at concentrations below the critical micellar concentration, triggers stepwise and phase-dependent structural remodeling events, including permeabilization and swelling, in both freely diffusing and surface-tethered vesicles, highlighting the substantial impact the surfactant has on vesicle conformation and stability prior to lysis.

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.

A Next Generation of Advances in Chromosome Architecture.

New insight into the architecture of chromosomes, their molecular composition, structure and spatial location, and time-resolved features, has grown enormously through developments of a range of pioneering interdisciplinary approaches that lie at the interface of the life and physical sciences. These involve several state-of-the-art "physics of life" tools that are both experimental and theoretical, used in conjunction with molecular biology methods which enable investigation of chromosome structure and function in vitro, in vivo, and even in silico. In particular, a move towards far greater quantitation has enabled transformative leaps in our understanding. These have involved valuable improvements to the spatial and temporal resolution of quantitative measurements, such as in vivo super-resolved light microscopy and single-molecule biophysics methods, which facilitate probing of dynamic chromosome processes hitherto impossible. Similarly, there have been important advances in the theoretical biophysics approaches which have enabled advances in predictive modeling to generate new understanding of the modes of operation of chromosomes across all domains of life. Here, I discuss these advances, and review the current state of our knowledge of chromosome architecture and speculation where future advances may lead.

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.

Fluorescence Recovery After Photobleaching (FRAP) to Study Dynamics of the Structural Maintenance of Chromosome (SMC) Complex in Live Escherichia coli Bacteria.

MukBEF, a structural maintenance of chromosome (SMC) complex, is an important molecular machine for chromosome organization and segregation in Escherichia coli. Fluorescently tagged MukBEF forms distinct spots (or "foci") composed of molecular assemblies in the cell, where it is thought to carry out most of its chromosome-associated activities. Here, we outline the technique of fluorescence recovery after photobleaching (FRAP) as a method to study the properties of YFP-tagged MukB in fluorescent foci. This method can provide important insight into the dynamics of MukB on DNA and be used to study its biochemical properties in vivo.

Copy Number Analysis of the Yeast Histone Deacetylase Complex Component Cti6 Directly in Living Cells.

Proteins are one of the key components of cellular life that play a crucial role in most biological processes. Therefore, quantification of protein copy numbers is essential for revealing and better understanding of cellular behavior and functions. Here we describe a single-molecule fluorescence-based method of protein copy number quantification directly in living cells. This enables quick and reliable estimations and comparison of the protein of interest abundance without implementing large-scale studies.

The End Restraint Method for Mechanically Perturbing Nucleic Acids In Silico.

Far from being a passive information store, the genome is a mechanically dynamic and diverse system in which torsion and tension fluctuate and combine to determine structure and help regulate gene expression. Much of this mechanical perturbation is due to molecular machines such as topoisomerases which must stretch and twist DNA as part of various functions including DNA repair and replication. While the broad-scale mechanical response of nucleic acids to tension and torsion is well characterized, detail at the single base pair level is beyond the limits of even super-resolution imaging. Here, we present a straightforward, flexible, and extensible umbrella-sampling protocol to twist and stretch nucleic acids in silico using the popular biomolecular simulation package Amber-though the principles we describe are applicable also to other packages such as GROMACS. We discuss how to set up the simulation system, decide force fields and solvation models, and equilibrate. We then introduce the torsionally constrained stretching protocol, and finally we present some analysis techniques we have used to characterize structural motif formation. Rather than defining forces or fictional pseudoatoms, we instead define a fixed translation of specified atoms between each umbrella-sampling step, which allows comparison with experiment without needing to estimate applied forces by simply using the fractional end-to-end displacement as a comparison metric. We hope that this easy-to-implement solution will be valuable for interrogating optical and magnetic tweezers data on nucleic acids at base pair resolution.

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.