Larval zebrafish swim bouts in three dimensions reveal both new and redundant behaviours.

Two-dimensional swimming of larval zebrafish has been studied extensively. We use a three-dimensional imaging system and neural network for pose estimation to study their three-dimensional behaviour. We answer two questions: (i) are spontaneous or delayed-onset turns from free swim, dark flash and acoustic startle experiments objectively differentiable? and (ii) could larvae use stochastic selection among responses, 'feinting' during an escape? Our analysis identifies two new major modes of dorso-ventral displacement.

Temperature-jump microscopy and interaction of Hsp70 heat shock protein with a client protein in vivo.

Biomolecular processes such as protein-protein interactions can depend strongly on cell type and even vary within a single cell type. Here we develop a microscope with a Peltier-controlled temperature stage, a laser temperature jump to induce heat stress, and an autofocusing feature to mitigate temperature drift during experiments, to study a protein-protein interaction in a selected cell type within a live organism, the zebrafish larva. As an application of the instrument, we show that there is considerable cell-to-cell variation of the heat shock protein Hsp70 binding to one of its clients, phosphoglycerate kinase in vivo.

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.

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.

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.

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.

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 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.

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).

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 .

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.

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.

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.

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.

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.

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.

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.

Blue Light Is a Universal Signal for Chemoreceptors.

Blue light has been shown to elicit a tumbling response in , a nonphototrophic bacterium. The exact mechanism of this phototactic response is still unknown. Here, we quantify phototaxis in by analyzing single-cell trajectories in populations of free-swimming bacteria before and after light exposure.

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.

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 Zn-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.

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 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.

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.

Altering the speed of a DNA packaging motor from bacteriophage T4.

The speed at which a molecular motor operates is critically important for the survival of a virus or an organism but very little is known about the underlying mechanisms. Tailed bacteriophage T4 employs one of the fastest and most powerful packaging motors, a pentamer of gp17 that translocates DNA at a rate of up to ∼2000-bp/s. We hypothesize, guided by structural and genetic analyses, that a unique hydrophobic environment in the catalytic space of gp17-adenosine triphosphatase (ATPase) determines the rate at which the 'lytic water' molecule is activated and OH- nucleophile is generated, in turn determining the speed of the motor.

Mapping cell surface adhesion by rotation tracking and adhesion footprinting.

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.