Coinfecting phages impede each other's entry into the cell.

The developmental choice made by temperate phages, between cell death (lysis) and viral dormancy (lysogeny), is influenced by the relative abundance of viruses and hosts in the environment. The paradigm for this abundance-driven decision is phage lambda of E. coli, whose propensity to lysogenize increases with the number of viruses coinfecting the same bacterium. It is believed that lambda uses this number to infer whether phages or bacteria outnumber each other. However, this interpretation is premised on an accurate mapping between the extracellular phage-to-bacteria ratio and the intracellular multiplicity of infection (MOI). Here, we show this premise to be faulty. By simultaneously labeling phage capsids and genomes, we find that, while the number of phages landing on each cell reliably samples the population ratio, the number of phages entering the cell does not. Single-cell infections, performed in a microfluidic device and interpreted using a stochastic model, reveal that the probability and rate of phage entry decrease with the number of adsorbed phages. This decrease reflects an MOI-dependent perturbation to host physiology caused by phage attachment, as evidenced by compromised membrane integrity and loss of membrane potential. The dependence of entry dynamics on the surrounding medium results in a strong impact on the infection outcome, while the protracted entry of coinfecting phages increases the heterogeneity in infection outcome at a given MOI. Our findings in lambda, and similar results we obtained for phages T5 and P1, demonstrate the previously unappreciated role played by entry dynamics in determining the outcome of bacteriophage infection.

Flagellar dynamics reveal fluctuations and kinetic limit in the Escherichia coli chemotaxis network.

The Escherichia coli chemotaxis network, by which bacteria modulate their random run/tumble swimming pattern to navigate their environment, must cope with unavoidable number fluctuations ("noise") in its molecular constituents like other signaling networks. The probability of clockwise (CW) flagellar rotation, or CW bias, is a measure of the chemotaxis network's output, and its temporal fluctuations provide a proxy for network noise. Here we quantify fluctuations in the chemotaxis signaling network from the switching statistics of flagella, observed using time-resolved fluorescence microscopy of individual optically trapped E. coli cells. This approach allows noise to be quantified across the dynamic range of the network. Large CW bias fluctuations are revealed at steady state, which may play a critical role in driving flagellar switching and cell tumbling. When the network is stimulated chemically to higher activity, fluctuations dramatically decrease. A stochastic theoretical model, inspired by work on gene expression noise, points to CheY activation occurring in bursts, driving CW bias fluctuations. This model also shows that an intrinsic kinetic ceiling on network activity places an upper limit on activated CheY and CW bias, which when encountered suppresses network fluctuations. This limit may also prevent cells from tumbling unproductively in steep gradients.

Rapid automated 3-D pose estimation of larval zebrafish using a physical model-trained neural network.

Quantitative ethology requires an accurate estimation of an organism's postural dynamics in three dimensions plus time. Technological progress over the last decade has made animal pose estimation in challenging scenarios possible with unprecedented detail. Here, we present (i) a fast automated method to record and track the pose of individual larval zebrafish in a 3-D environment, applicable when accurate human labeling is not possible; (ii) a rich annotated dataset of 3-D larval poses for ethologists and the general zebrafish and machine learning community; and (iii) a technique to generate realistic, annotated larval images in different behavioral contexts. Using a three-camera system calibrated with refraction correction, we record diverse larval swims under free swimming conditions and in response to acoustic and optical stimuli. We then employ a convolutional neural network to estimate 3-D larval poses from video images. The network is trained against a set of synthetic larval images rendered using a 3-D physical model of larvae. This 3-D model samples from a distribution of realistic larval poses that we estimate a priori using a template-based pose estimation of a small number of swim bouts. Our network model, trained without any human annotation, performs larval pose estimation three orders of magnitude faster and with accuracy comparable to the template-based approach, capturing detailed kinematics of 3-D larval swims. It also applies accurately to other datasets collected under different imaging conditions and containing behavioral contexts not included in our training.

Optical traps induce fluorophore photobleaching by two-photon excitation.

Techniques combining optical tweezers with fluorescence microscopy have become increasingly popular. Unfortunately, the high-power, infrared lasers used to create optical traps can have a deleterious effect on dye stability. Previous studies have shown that dye photobleaching is enhanced by absorption of visible fluorescence excitation plus infrared trap photons, a process that can be significantly reduced by minimizing simultaneous exposure to both light sources. Here, we report another photobleaching pathway that results from direct excitation by the trapping laser alone. Our results show that this trap-induced fluorescence loss is a two-photon absorption process, as demonstrated by a quadratic dependence on the intensity of the trapping laser. We further show that, under conditions typical of many trap-based experiments, fluorescence emission of certain fluorophores near the trap focus can drop by 90% within 1 min. We investigate how photostability is affected by the choice of dye molecule, excitation and emission wavelength, and labeled molecule. Finally, we discuss the different photobleaching pathways in combined trap-fluorescence measurements, which guide the selection of optimal dyes and conditions for more robust experimental protocols.

Helicase Activity Modulation with On-Demand Light-Based Conformational Control.

Engineering a protein variant with a desired role relies on deep knowledge of the relationship between a protein's native structure and function. Using our structural understanding of a regulatory subdomain found in a family of DNA helicases, we engineered novel helicases for which the subdomain orientation is designed to switch between unwinding-inactive and -active conformations upon trans-cis isomerization of an azobenzene-based crosslinker. This on-demand light-based conformational control directly alters helicase activity as demonstrated by both bulk phase experiments and single-molecule optical tweezers analysis of one of the engineered helicases. The "opto-helicase" may be useful in future applications that require spatiotemporal control of DNA hybridization states.

CO-INFECTING PHAGES IMPEDE EACH OTHER'S ENTRY INTO THE CELL.

Bacteriophage lambda tunes its propensity to lysogenize based on the number of viral genome copies inside the infected cell. Viral self-counting is believed to serve as a way of inferring the abundance of available hosts in the environment. This interpretation is premised on an accurate mapping between the extracellular phage-to-bacteria ratio and the intracellular multiplicity of infection (MOI). However, here we show this premise to be untrue. By simultaneously labeling phage capsids and genomes, we find that, while the number of phages landing on each cell reliably samples the population ratio, the number of phages entering the cell does not. Single-cell infections, followed in a microfluidic device and interpreted using a stochastic model, reveal that the probability and rate of individual phage entries decrease with MOI. This decrease reflects an MOI-dependent perturbation to host physiology caused by phage landing, evidenced by compromised membrane integrity and loss of membrane potential. The dependence of phage entry dynamics on the surrounding medium is found to result in a strong impact of environmental conditions on the infection outcome, while the protracted entry of co-infecting phages increases the cell-to-cell variability in infection outcome at a given MOI. Our findings demonstrate the previously unappreciated role played by entry dynamics in determining the outcome of bacteriophage infection.

High-throughput force measurement of individual kinesin-1 motors during multi-motor transport.

Molecular motors often work in teams to move a cellular cargo. Yet measuring the forces exerted by each motor is challenging. Using a sensor made with denatured ssDNA and multi-color fluorescence, we measured picoNewtons of forces and nanometer distances exerted by individual constrained kinesin-1 motors acting together while driving a common microtubule in vitro. We find that kinesins primarily exerted less than 1 pN force, even while the microtubule is bypassing artificial obstacles of 20-100 nanometer size. Occasionally, individual forces increase upon encountering obstacles, although at other times they do not, with the cargo continuing in a directional manner. Our high-throughput technique, which can measure forces by many motors simultaneously, is expected to be useful for many different types of molecular motors.

Optical tweezers in single-molecule biophysics.

Optical tweezers have become the method of choice in single-molecule manipulation studies. In this Primer, we first review the physical principles of optical tweezers and the characteristics that make them a powerful tool to investigate single molecules. We then introduce the modifications of the method to extend the measurement of forces and displacements to torques and angles, and to develop optical tweezers with single-molecule fluorescence detection capabilities. We discuss force and torque calibration of these instruments, their various modes of operation and most common experimental geometries. We describe the type of data obtained in each experimental design and their analyses. This description is followed by a survey of applications of these methods to the studies of protein-nucleic acid interactions, protein/RNA folding and molecular motors. We also discuss data reproducibility, the factors that lead to the data variability among different laboratories and the need to develop field standards. We cover the current limitations of the methods and possible ways to optimize instrument operation, data extraction and analysis, before suggesting likely areas of future growth.

Kinetic and structural mechanism for DNA unwinding by a non-hexameric helicase.

UvrD, a model for non-hexameric Superfamily 1 helicases, utilizes ATP hydrolysis to translocate stepwise along single-stranded DNA and unwind the duplex. Previous estimates of its step size have been indirect, and a consensus on its stepping mechanism is lacking. To dissect the mechanism underlying DNA unwinding, we use optical tweezers to measure directly the stepping behavior of UvrD as it processes a DNA hairpin and show that UvrD exhibits a variable step size averaging ~3 base pairs. Analyzing stepping kinetics across ATP reveals the type and number of catalytic events that occur with different step sizes. These single-molecule data reveal a mechanism in which UvrD moves one base pair at a time but sequesters the nascent single strands, releasing them non-uniformly after a variable number of catalytic cycles. Molecular dynamics simulations point to a structural basis for this behavior, identifying the protein-DNA interactions responsible for strand sequestration. Based on structural and sequence alignment data, we propose that this stepping mechanism may be conserved among other non-hexameric helicases.

A viral genome packaging ring-ATPase is a flexibly coordinated pentamer.

Multi-subunit ring-ATPases carry out a myriad of biological functions, including genome packaging in viruses. Though the basic structures and functions of these motors have been well-established, the mechanisms of ATPase firing and motor coordination are poorly understood. Here, using single-molecule fluorescence, we determine that the active bacteriophage T4 DNA packaging motor consists of five subunits of gp17. By systematically doping motors with an ATPase-defective subunit and selecting single motors containing a precise number of active or inactive subunits, we find that the packaging motor can tolerate an inactive subunit. However, motors containing one or more inactive subunits exhibit fewer DNA engagements, a higher failure rate in encapsidation, reduced packaging velocity, and increased pausing. These findings suggest a DNA packaging model in which the motor, by re-adjusting its grip on DNA, can skip an inactive subunit and resume DNA translocation, suggesting that strict coordination amongst motor subunits of packaging motors is not crucial for function.

Switch-like control of helicase processivity by single-stranded DNA binding protein.

Helicases utilize nucleotide triphosphate (NTP) hydrolysis to translocate along single-stranded nucleic acids (NA) and unwind the duplex. In the cell, helicases function in the context of other NA-associated proteins such as single-stranded DNA binding proteins. Such encounters regulate helicase function, although the underlying mechanisms remain largely unknown. Ferroplasma acidarmanus xeroderma pigmentosum group D (XPD) helicase serves as a model for understanding the molecular mechanisms of superfamily 2B helicases, and its activity is enhanced by the cognate single-stranded DNA binding protein replication protein A 2 (RPA2). Here, optical trap measurements of the unwinding activity of a single XPD helicase in the presence of RPA2 reveal a mechanism in which XPD interconverts between two states with different processivities and transient RPA2 interactions stabilize the more processive state, activating a latent 'processivity switch' in XPD. A point mutation at a regulatory DNA binding site on XPD similarly activates this switch. These findings provide new insights on mechanisms of helicase regulation by accessory proteins.

ALS/FTLD-Linked Mutations in FUS Glycine Residues Cause Accelerated Gelation and Reduced Interactions with Wild-Type FUS.

The RNA-binding protein fused in sarcoma (FUS) can form pathogenic inclusions in neurodegenerative diseases like amyotrophic lateral sclerosis (ALS) and frontotemporal lobar dementia (FTLD). Over 70 mutations in Fus are linked to ALS/FTLD. In patients, all Fus mutations are heterozygous, indicating that the mutant drives disease progression despite the presence of wild-type (WT) FUS. Here, we demonstrate that ALS/FTLD-linked FUS mutations in glycine (G) strikingly drive formation of droplets that do not readily interact with WT FUS, whereas arginine (R) mutants form mixed condensates with WT FUS. Remarkably, interactions between WT and G mutants are disfavored at the earliest stages of FUS nucleation. In contrast, R mutants physically interact with the WT FUS such that WT FUS recovers the mutant defects by reducing droplet size and increasing dynamic interactions with RNA. This result suggests disparate molecular mechanisms underlying ALS/FTLD pathogenesis and differing recovery potential depending on the type of mutation.

Multiple kinesins induce tension for smooth cargo transport.

How cargoes move within a crowded cell-over long distances and at speeds nearly the same as when moving on unimpeded pathway-has long been mysterious. Through an in vitro force-gliding assay, which involves measuring nanometer displacement and piconewtons of force, we show that multiple mammalian kinesin-1 (from 2 to 8) communicate in a team by inducing tension (up to 4 pN) on the cargo. Kinesins adopt two distinct states, with one-third slowing down the microtubule and two-thirds speeding it up. Resisting kinesins tend to come off more rapidly than, and speed up when pulled by driving kinesins, implying an asymmetric tug-of-war. Furthermore, kinesins dynamically interact to overcome roadblocks, occasionally combining their forces. Consequently, multiple kinesins acting as a team may play a significant role in facilitating smooth cargo motion in a dense environment. This is one of few cases in which single molecule behavior can be connected to ensemble behavior of multiple motors.

The inside of a cell is a crowded space, full of proteins and other molecules. Yet, the molecular motors that transport some of those molecules within the cell move at the same speed as they would in pure water ­ about one micrometer per second. How the molecular motors could achieve such speeds in crowded cells was unclear. Nevertheless, Tjioe et al. suspected that the answer might be related to how multiple motors work together. Molecular motors move by walking along filaments inside the cell and pulling their cargo from one location to another. Other molecules that bind to the filaments should, in theory, act like "roadblocks" and impede the movement of the cargo. Tjioe et al. studied a motor protein called kinesin, which walks on filaments called microtubules. But instead of looking at these motors moving along microtubules inside a cell, Tjioe et al. used a simpler system where the cell was eliminated, and all parts were purified. Specifically, Tjioe et al. tethered purified motors to a piece of glass and then observed them under an extremely accurate microscope as they moved free-floating, fluorescently labelled microtubules. The microtubules, in this scenario, were acting like cargoes, where many kinesins could bind. Each kinesin motor also had a small chemical tag that could emit light. By following the movement of the lights, it was possible to calculate what each kinesin was doing and how the cargo moved. When more than one kinesin molecule was acting, the tension and speed of one kinesin affected the movement of the others. In any group of kinesins, about two-thirds of kinesin pulled the cargo, and unexpectedly, about one-third tended to resist and slow the cargo. These latter kinesins were moved along with the group without actually driving the cargo. These resisting kinesins did come off more rapidly than the driving kinesins, meaning the cargo should be able to quickly bypass roadblocks. This would help to keep the whole group travelling in the right direction at a steady pace.

Extreme mechanical diversity of human telomeric DNA revealed by fluorescence-force spectroscopy.

G-quadruplexes (GQs) can adopt diverse structures and are functionally implicated in transcription, replication, translation, and maintenance of telomere. Their conformational diversity under physiological levels of mechanical stress, however, is poorly understood. We used single-molecule fluorescence-force spectroscopy that combines fluorescence resonance energy transfer with optical tweezers to measure human telomeric sequences under tension. Abrupt GQ unfolding with K+ in solution occurred at as many as four discrete levels of force. Added to an ultrastable state and a gradually unfolding state, there were six mechanically distinct structures. Extreme mechanical diversity was also observed with Na+, although GQs were mechanically weaker. Our ability to detect small conformational changes at low forces enabled the determination of refolding forces of about 2 pN. Refolding was rapid and stochastically redistributed molecules to mechanically distinct states. A single guanine-to-thymine substitution mutant required much higher ion concentrations to display GQ-like unfolding and refolded via intermediates, contrary to the wild type. Contradicting an earlier proposal, truncation to three hexanucleotide repeats resulted in a single-stranded DNA-like mechanical behavior under all conditions, indicating that at least four repeats are required to form mechanically stable structures.

Blue Light Is a Universal Signal for Escherichia coli Chemoreceptors.

Blue light has been shown to elicit a tumbling response in Escherichia coli, a nonphototrophic bacterium. The exact mechanism of this phototactic response is still unknown. Here, we quantify phototaxis in E. coli by analyzing single-cell trajectories in populations of free-swimming bacteria before and after light exposure. Bacterial strains expressing only one type of chemoreceptor reveal that all five E. coli receptors (Aer, Tar, Tsr, Tap, and Trg) are capable of mediating responses to light. In particular, light exposure elicits a running response in the Tap-only strain, the opposite of the tumbling responses observed for all other strains. Therefore, light emerges as a universal stimulus for all E. coli chemoreceptors. We also show that blue light exposure causes a reversible decrease in swimming velocity, a proxy for proton motive force. This result is consistent with a previously proposed hypothesis that, rather than sensing light directly, chemoreceptors sense light-induced perturbations in proton motive force, although other factors are also likely to contribute.IMPORTANCE Our findings provide new insights into the mechanism of E. coli phototaxis, showing that all five chemoreceptor types respond to light and their interactions play an important role in cell behavior. Our results also open up new avenues for examining and manipulating E. coli taxis. Since light is a universal stimulus, it may provide a way to quantify interactions among different types of receptors. Because light is easier to control spatially and temporally than chemicals, it may be used to study swimming behavior in complex environments. Since phototaxis can cause migration of E. coli bacteria in light gradients, light may be used to control bacterial density for studying density-dependent processes in bacteria.

Regulation of Rep helicase unwinding by an auto-inhibitory subdomain.

Helicases are biomolecular motors that unwind nucleic acids, and their regulation is essential for proper maintenance of genomic integrity. Escherichia coli Rep helicase, whose primary role is to help restart stalled replication, serves as a model for Superfamily I helicases. The activity of Rep-like helicases is regulated by two factors: their oligomeric state, and the conformation of the flexible subdomain 2B. However, the mechanism of control is not well understood. To understand the factors that regulate the active state of Rep, here we investigate the behavior of a 2B-deficient variant (RepΔ2B) in relation to wild-type Rep (wtRep). Using a single-molecule optical tweezers assay, we explore the effects of oligomeric state, DNA geometry, and duplex stability on wtRep and RepΔ2B unwinding activity. We find that monomeric RepΔ2B unwinds more processively and at a higher speed than the activated, dimeric form of wtRep. The unwinding processivity of RepΔ2B and wtRep is primarily limited by 'strand-switching'-during which the helicases alternate between strands of the duplex-which does not require the 2B subdomain, contrary to a previous proposal. We provide a quantitative model of the factors that enhance unwinding processivity. Our work sheds light on the mechanisms of regulation of unwinding by Rep-like helicases.

Optical Control of Metal Ion Probes in Cells and Zebrafish Using Highly Selective DNAzymes Conjugated to Upconversion Nanoparticles.

Spatial and temporal distributions of metal ions in vitro and in vivo are crucial in our understanding of the roles of metal ions in biological systems, and yet there is a very limited number of methods to probe metal ions with high space and time resolution, especially in vivo. To overcome this limitation, we report a Zn2+-specific near-infrared (NIR) DNAzyme nanoprobe for real-time metal ion tracking with spatiotemporal control in early embryos and larvae of zebrafish. By conjugating photocaged DNAzymes onto lanthanide-doped upconversion nanoparticles (UCNPs), we have achieved upconversion of a deep tissue penetrating NIR 980 nm light into 365 nm emission. The UV photon then efficiently photodecages a substrate strand containing a nitrobenzyl group at the 2'-OH of adenosine ribonucleotide, allowing enzymatic cleavage by a complementary DNA strand containing a Zn2+-selective DNAzyme. The product containing a visible FAM fluorophore that is initially quenched by BHQ1 and Dabcyl quenchers is released after cleavage, resulting in higher fluorescent signals. The DNAzyme-UCNP probe enables Zn2+ sensing by exciting in the NIR biological imaging window in both living cells and zebrafish embryos and detecting in the visible region. In this study, we introduce a platform that can be used to understand the Zn2+ distribution with spatiotemporal control, thereby giving insights into the dynamical Zn2+ ion distribution in intracellular and in vivo models.

Free-energy simulations reveal molecular mechanism for functional switch of a DNA helicase.

Helicases play key roles in genome maintenance, yet it remains elusive how these enzymes change conformations and how transitions between different conformational states regulate nucleic acid reshaping. Here, we developed a computational technique combining structural bioinformatics approaches and atomic-level free-energy simulations to characterize how the Escherichia coli DNA repair enzyme UvrD changes its conformation at the fork junction to switch its function from unwinding to rezipping DNA. The lowest free-energy path shows that UvrD opens the interface between two domains, allowing the bound ssDNA to escape. The simulation results predict a key metastable 'tilted' state during ssDNA strand switching. By simulating FRET distributions with fluorophores attached to UvrD, we show that the new state is supported quantitatively by single-molecule measurements. The present study deciphers key elements for the 'hyper-helicase' behavior of a mutant and provides an effective framework to characterize directly structure-function relationships in molecular machines.

Ultrashort Nucleic Acid Duplexes Exhibit Long Wormlike Chain Behavior with Force-Dependent Edge Effects.

Despite their importance in biology and use in nanotechnology, the elastic behavior of nucleic acids on "ultrashort" (<15 nt) length scales remains poorly understood. Here, we use optical tweezers combined with fluorescence imaging to observe directly the hybridization of oligonucleotides (7-12 nt) to a complementary strand under tension and to measure the difference in end-to-end extension between the single-stranded and duplex states. Data are consistent with long-polymer models at low forces (<8 pN) but smaller than predicted at higher forces (>8 pN), the result of the sequence-dependent duplex edge effects.