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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 FUS. Here, we demonstrate that ALS/FTLD-linked FUS mutations in glycine (G) strikingly drive formation of droplets that do not readily interact with wild-type FUS whereas arginine (R) mutants form mixed condensates with wild-type FUS. Remarkably, interactions between wild-type and G mutants are disfavored at the earliest stages of FUS nucleation. In contrast, R mutants physically interact with the wild-type FUS such that wild-type 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.

Helicases utilize the energy of 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 which regulate helicase function. For example, single-stranded DNA binding proteins are known to enhance helicase activity, although the underlying mechanisms remain largely unknown. F. acidarmanus XPD helicase serves as a model for understanding the molecular mechanisms of Superfamily 2B helicases, and previous work has shown that its activity is enhanced by the cognate single-stranded DNA binding protein RPA2. Here, single-molecule 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. These findings provide new insights on mechanisms of helicase regulation by accessory proteins.

Biochemical signaling networks allow living cells to adapt to a changing environment, but these networks must cope with unavoidable number fluctuations (“noise”) in their molecular constituents. Escherichia coli chemotaxis, by which bacteria modulate their random run/tumble swimming pattern to navigate their environment, is a paradigm for the role of noise in cell signaling. The key signaling protein, CheY, when activated by (reversible) phosphorylation, causes a switch in the rotational direction of the flagellar motors propelling the cell, leading to tumbling. CheY-P concentration, [CheY-P], is thus a measure of the chemotaxis network’s output, and temporal fluctuations in [CheY-P] provide a proxy for network noise. However, measuring these fluctuations in the single cell, at the relevant timescale of individual run and tumble events, remains a challenge. Here we quantify the short-timescale (0.5-5 s) fluctuations in [CheY-P] from the switching dynamics of individual flagella, observed using time-resolved fluorescence microscopy of optically trapped E. coli cells. This approach reveals large [CheY-P] fluctuations at steady state, which may play a critical role in driving flagellar switching and cell tumbling. A stochastic theoretical model, inspired by work on gene expression noise, points to CheY activation occurring in bursts, driving the large [CheY-P] fluctuations. When the network is stimulated chemically to higher activity, we observe a dramatic decrease in [CheY-P] fluctuations. Our stochastic model shows that an intrinsic kinetic ceiling on network activity places an upper limit on [CheY-P], which when encountered suppresses its fluctuations. This limit may also prevent cells from tumbling unproductively in steep gradients. Significance Statement Bacteria use intracellular signaling networks to navigate and adapt to their changing environment. These networks must cope with fluctuations in their molecular constituents, but the role this noise plays in cell behavior is not well understood. Here, we present a novel approach to quantify network noise in individual Escherichia coli cells. Our measurements show that the network exhibits larger-than-expected fluctuations when operating at a steady state; these fluctuations decrease dramatically when the network is activated by a chemical stimulus. A model inspired by gene expression noise studies recapitulates our findings and suggests that large fluctuations are driven by ‘bursts’ in signaling, drawing a parallel between the operating principles of gene regulatory and protein signaling networks.

The formation of pathogenic inclusions of RNA-binding proteins in neurons is a hallmark of amyotrophic lateral sclerosis (ALS) and frontotemporal lobar dementia (FTLD). One prominent protein in these inclusions is Fused in sarcoma (FUS), and over 70 mutations in Fus are linked to ALS/FTLD. In patients, all Fus mutations are heterozygous, indicating that the mutant drives pathology despite the presence of wild-type FUS. Here, we demonstrate that ALS FUS mutations in glycine (G) strikingly drive formation of droplets that are immiscible with wild-type FUS whereas arginine (R) mutants form miscible condensates with wild-type FUS. Remarkably, immiscibility between wild-type and G mutants begins at the earliest stages of FUS nucleation. In contrast to G mutants, the miscible R mutants physically interact with the wild-type FUS such that wild-type FUS rescues the mutant defects by reducing droplet size and recovering dynamic interactions with RNA. This result suggests disparate molecular mechanisms underlying pathogenesis and differing rescue potential depending on the type of mutation.

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., eLife digest 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.

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.

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.

Rolling adhesion, in which cells passively roll along surfaces under shear flow, is a critical process involved in inflammatory responses and cancer metastasis. Surface adhesion properties regulated by adhesion receptors and membrane tethers are critical in understanding cell rolling behavior. Locally, adhesion molecules are distributed at the tips of membrane tethers. However, how functional adhesion properties are globally distributed on the individual cell’s surface is unknown. Here, we developed a label-free technique to determine the spatial distribution of adhesive properties on rolling cell surfaces. Using dark-field imaging and particle tracking, we extract the rotational motion of individual rolling cells. The rotational information allows us to construct an adhesion map along the contact circumference of a single cell. To complement this approach, we also developed a fluorescent adhesion footprint assay to record the molecular adhesion events from cell rolling. Applying the combination of the two methods on human promyelocytic leukemia cells, our results surprisingly reveal that adhesion is non-uniformly distributed in patches on the cell surfaces. Our label-free adhesion mapping methods are applicable to the variety of cell types that undergo rolling adhesion and provide a quantitative picture of cell surface adhesion at the functional and molecular level.

Rolling adhesion is the behaviour that leukocytes and circulating tumour cells exhibit as they passively roll along blood vessel walls under flow. It plays a critical role in capturing cells in the blood, guiding them toward inflammation sites, and activating cell signalling pathways to enable their subsequent transmigration. Rolling adhesion is mediated by catch-bond-like interactions between selectins expressed on endothelial cells lining blood vessels and P-selectin glycoprotein ligand-1 (PSGL-1) found at microvilli tips of leukocytes. Despite our understanding of individual components of this process, how the molecular details of adhesion bonds scale to cell-surface adhesion and rolling behaviour remains poorly understood. Here, we developed a method that maps the functional adhesion sites and their strength on a leukocyte surface. The method relies on tracking the rotational angle of a single rolling cell, which confers advantages over standard methods that track the centre-of-mass alone. Constructing the adhesion map from the instantaneous angular velocity reveals that the adhesion profile along the rolling circumference is inhomogeneous. We corroborated these findings by obtaining a footprint of molecular adhesion events using DNA-based molecular force probes. Our results reveal that adhesion at the functional level is not uniformly distributed over the leukocyte surface as previously assumed, but is instead patchy.

Escherichia coli chemotaxis serves as a paradigm for the way living cells respond and adapt to changes in their environment. The chemotactic response has been characterized at the level of individual flagellar motors and in populations of swimming cells. However, it has not been previously possible to quantify accurately the adaptive response of a single, multiflagellated cell. Here, we use our recently developed optical trapping technique to characterize the swimming behavior of individual bacteria as they respond to sudden changes in the chemical environment. We follow the adaptation kinetics of E. coli to varying magnitudes of step-up and step-down changes in concentration of chemoattractant. We quantify two features of adaptation and how they vary with stimulus strength: abruptness (the degree to which return to prestimulus behavior occurs within a small number of run/tumble events) and overshoot (the degree of excessive response before the return to prestimulus behavior). We also characterize the asymmetry between step-up and step-down responses, observed at the single-cell level. Our findings provide clues to an improved understanding of chemotactic adaptation.

Escherichia coli single-stranded (ss)DNA binding (SSB) protein mediates genome maintenance processes by regulating access to ssDNA. This homotetrameric protein wraps ssDNA in multiple distinct binding modes that may be used selectively in different DNA processes, and whose detailed wrapping topologies remain speculative. Here, we used single-molecule force and fluorescence spectroscopy to investigate E. coli SSB binding to ssDNA. Stretching a single ssDNA-SSB complex reveals discrete states that correlate with known binding modes, the likely ssDNA conformations and diffusion dynamics in each, and the kinetic pathways by which the protein wraps ssDNA and is dissociated. The data allow us to construct an energy landscape for the ssDNA-SSB complex, revealing that unwrapping energy costs increase the more ssDNA is unraveled. Our findings provide insights into the mechanism by which proteins gain access to ssDNA bound by SSB, as demonstrated by experiments in which SSB is displaced by the E. coli recombinase RecA. DOI: http://dx.doi.org/10.7554/eLife.08193.001, eLife digest The DNA double helix consists of two strands coiled around each other. However, there are many instances when DNA must be separated into its individual strands—for example, when the DNA sequence needs to be copied. These single-stranded structures are highly prone to damage. For protection, the single-stranded DNA can wrap around single-stranded DNA binding (SSB) proteins, which also control how other maintenance proteins interact with the DNA. SSB proteins from the bacteria species Escherichia coli wrap single-stranded DNA into a variety of topologies known as binding modes. By using a technique that uses a laser to exert forces on an individual DNA molecule, Suksombat et al. unraveled DNA from a single SSB protein. This revealed that the unraveling occurs in a series of steps that correspond well to the known binding modes. These steps also provide the energies required to unravel the single-stranded DNA. Further experiments showed that SSBs can slide along DNA without having to change their binding mode. The unraveling and sliding mechanisms are likely to be used by other proteins to gain access to DNA coated with SSBs. The next step is to understand how SSBs interact with these other proteins, and how their various wrapping configurations affect this interaction. DOI: http://dx.doi.org/10.7554/eLife.08193.002

Bacterial chemotaxis is a paradigm for how environmental signals modulate cellular behavior. Although the network underlying this process has been studied extensively, we do not yet have an end-to-end understanding of chemotaxis. Specifically, how the rotational states of a cell’s flagella cooperatively determine whether the cell ‘runs’ or ‘tumbles’ remains poorly characterized. Here, we measure the swimming behavior of individual E. coli cells while simultaneously detecting the rotational states of each flagellum. We find that a simple mathematical expression relates the cell’s run/tumble bias to the number and average rotational state of its flagella. However, due to inter-flagellar correlations, an ‘effective number’ of flagella—smaller than the actual number—enters into this relation. Data from a chemotaxis mutant and stochastic modeling suggest that fluctuations of the regulator CheY-P are the source of flagellar correlations. A consequence of inter-flagellar correlations is that run/tumble behavior is only weakly dependent on number of flagella. DOI: http://dx.doi.org/10.7554/eLife.01916.001, eLife digest Escherichia coli is a rod-shaped bacterium commonly found in the lower intestines of humans and other warm-blooded animals. While most strains of E. coli are harmless, including most of those found in the human gut, some can cause diseases such as food poisoning. Due to its close association with humans and the fact that it is easy to grow and work with in the laboratory, E. coli has been intensively studied for over 60 years. Many bacteria are capable of ‘swimming’ by using one or more flagella. These rotating whip-like structures are each driven by a reversible motor, and they act a bit like a propeller on a boat. While some bacteria have only a single flagellum, others, such as E. coli, have multiple flagella distributed over the cell surface. Rotating all their flagella in a counterclockwise direction allows the bacterium to swim—and it has been proposed that the clockwise movement of at least one flagellum will cause the bacterium cell to stop swimming and start tumbling. E. coli is able to control the time it spends swimming or tumbling to move towards a nutrient, such as glucose, or away from certain harmful chemicals. However, the details of how the number of flagella and the direction of rotation (clockwise or counterclockwise) influence the motion of the bacterium are not fully understood. Now, Mears et al. have used ‘optical tweezers’ to immobilize individual E. coli cells under a microscope, and then track both their swimming behavior and the movements of their flagella. This revealed that the individual flagella on the same cell tend to move in a coordinated way. Therefore, whilst tumbling could be caused by a single flagellum stopping swimming behavior, it often involved a concerted effort by many of the cell’s flagella. After observing that E. coli cells with more flagella spent less time tumbling than would be predicted if a single flagella always ‘vetoed’ swimming, Mears et al. propose a new mathematical relationship between the number of flagella on the cell, the direction of rotation, and the resulting probability that the cell will tumble. This work shows that swimming behavior in bacteria is less affected by variations in the number of flagella than previously thought—and this phenomenon may provide evolutionary advantages to E. coli. The next step is to explore the mechanism by which bacteria coordinate their flagella. DOI: http://dx.doi.org/10.7554/eLife.01916.002

Helicases couple the chemical energy of ATP hydrolysis to directional translocation along nucleic acids and transient duplex separation. Understanding helicase mechanism requires that the basic physicochemical process of base pair separation be understood. This necessitates monitoring helicase activity directly, at high spatio-temporal resolution. Using optical tweezers with single base pair (bp) resolution, we analyzed DNA unwinding by XPD helicase, a Superfamily 2 (SF2) DNA helicase involved in DNA repair and transcription initiation. We show that monomeric XPD unwinds duplex DNA in 1-bp steps, yet exhibits frequent backsteps and undergoes conformational transitions manifested in 5-bp backward and forward steps. Quantifying the sequence dependence of XPD stepping dynamics with near base pair resolution, we provide the strongest and most direct evidence thus far that forward, single-base pair stepping of a helicase utilizes the spontaneous opening of the duplex. The proposed unwinding mechanism may be a universal feature of DNA helicases that move along DNA phosphodiester backbones. DOI: http://dx.doi.org/10.7554/eLife.00334.001, eLife digest During many cellular processes, the double helix must be transiently unwound so that the enzymes responsible for maintaining the genome can access the two strands. During DNA synthesis, for instance, the two strands of DNA are first separated and then used as templates for the production of new strands. The role of destabilizing, separating and unwinding the double helix falls to enzymes known as DNA helicases. Helicases are also involved in separating strands of nucleic acids during myriad other cellular processes, including DNA repair, transcription and translation. While the functions of helicases are clear, the precise mechanisms by which they unwind DNA are not. Here, Qi et al. have investigated the mechanism of a helicase called XPD, which is involved in DNA repair and the initiation of transcription of DNA into RNA. Using optical tweezers—in which a laser beam is used to exert extremely small forces on a single DNA molecule—they followed the activity of individual molecules of XPD as they unwound DNA with base pair resolution. Qi et al. observed that the helicase unwinds DNA strands 1 base pair at a time, but that it sometimes moves backwards by 1 base pair and at other times makes 5 base pair backward and forward steps. The frequency of these backwards steps depends on the availability of ATP, and the sequence of the DNA. Due to the high resolution of the data, Qi et al. were able to correlate these stepping dynamics with the DNA sequence with base pair level accuracy. While some helicases actively separate the strands, using energy derived from ATP to break the hydrogen bonds between pairs of bases, Qi et al. showed that XPD appears to take advantage of momentary separations that arise spontaneously between base pairs. As well as providing insights into the role of XPD in DNA repair and transcription, the work of Qi et al. presents a method that could be used to explore the mechanisms of other helicases. Given that the unwinding mechanism described here is likely to be a universal feature of enzymes related to XPD, the current work could shed light on a number of other cellular processes involving XPD-like helicases, such as homologous DNA recombination, inter-strand cross-link repair, and accurate chromosome segregation. DOI: http://dx.doi.org/10.7554/eLife.00334.002

FacXPD is the archaeal homolog of yeast Rad3 and human xeroderma pigmentosum group D protein (XPD) helicase from Ferroplasma acidarmanus. This enzyme serves as a model for understanding the molecular mechanism of human Superfamily 2 helicase XPD involved in both transcription initiation and nucleotide excision repair, and for the related 5’-3’ helicases FancJ, Rtel and ChlR1 important for maintaining genomic integrity and DNA repair. We developed a single-molecule, high-resolution optical tweezers assay to decipher the mechanism by which a single XPD helicase unwinds dsDNA while translocating in the 5’-3’ direction. This assay monitors the unwinding of an 89-bp DNA hairpin substrate with single base pair resolution. Our substrate design allows us to control the length of a poly-dT ssDNA “translocation” strand (the strand to which XPD binds and along which it translocates), and a “displaced” strand (the strand displaced upon unwinding the duplex), located at the 5’ and 3’ tails of the hairpin, respectively. We found that the displaced strand interacts with XPD and that this interaction controls the helicase activity. When the 3’ tail of the substrate hairpin is substituted for dsDNA, a single XPD molecule displays repetitive “non-processive” bursts of substrate unwinding in which only ∼10-bp of the hairpin is unwound at a time. However, in the presence of a ssDNA displaced strand (of length ranging from 3 to 10 nt), we observe two types of activity: the same 10-bp non-processive mode as above, and also a “processive” mode in which the entire 89-bp hairpin is unwound. These data suggest two different binding modes for XPD resulting in non-processive or processive unwinding which are regulated by its interaction with the displaced strand. We propose a model for how the domains of XPD bind to its DNA substrate in these two modes.