Myosin-5 varies its step length to carry cargo straight along the irregular F-actin track.

Molecular motors employ chemical energy to generate unidirectional mechanical output against a track while navigating a chaotic cellular environment, potential disorder on the track, and against Brownian motion. Nevertheless, decades of nanometer-precise optical studies suggest that myosin-5a, one of the prototypical molecular motors, takes uniform steps spanning 13 subunits (36 nm) along its F-actin track. Here, we use high-resolution interferometric scattering microscopy to reveal that myosin takes strides spanning 22 to 34 actin subunits, despite walking straight along the helical actin filament. We show that cumulative angular disorder in F-actin accounts for the observed proportion of each stride length, akin to crossing a river on variably spaced stepping stones. Electron microscopy revealed the structure of the stepping molecule. Our results indicate that both motor and track are soft materials that can adapt to function in complex cellular conditions.

Modelling 3D supramolecular structure from sparse single-molecule localisation microscopy data.

Single-molecule localisation microscopy (SMLM) has the potential to reveal the underlying organisation of specific molecules within supramolecular complexes and their conformations, which is not possible with conventional microscope resolution. However, the detection efficiency for fluorescent molecules in cells can be limited in SMLM, even to below 1% in thick and dense samples. Segmentation of individual complexes can also be challenging. To overcome these problems, we have developed a software package termed PERPL: Pattern Extraction from Relative Positions of Localisations. This software assesses the relative likelihoods of models for underlying patterns behind incomplete SMLM data, based on the relative positions of pairs of localisations. We review its principles and demonstrate its use on the 3D lattice of Z-disk proteins in mammalian cardiomyocytes. We find known and novel features at ~20 nm with localisations of less than 1% of the target proteins, using mEos fluorescent protein constructs.

Myosin-5 varies its steps along the irregular F-actin track.

Molecular motors employ chemical energy to generate unidirectional mechanical output against a track. By contrast to the majority of macroscopic machines, they need to navigate a chaotic cellular environment, potential disorder in the track and Brownian motion. Nevertheless, decades of nanometer-precise optical studies suggest that myosin-5a, one of the prototypical molecular motors, takes uniform steps spanning 13 subunits (36 nm) along its F-actin track. Here, we use high-resolution interferometric scattering (iSCAT) microscopy to reveal that myosin takes strides spanning 22 to 34 actin subunits, despite walking straight along the helical actin filament. We show that cumulative angular disorder in F-actin accounts for the observed proportion of each stride length, akin to crossing a river on variably-spaced stepping stones. Electron microscopy revealed the structure of the stepping molecule. Our results indicate that both motor and track are soft materials that can adapt to function in complex cellular conditions.

PIEZO1 and PECAM1 interact at cell-cell junctions and partner in endothelial force sensing.

Two prominent concepts for the sensing of shear stress by endothelium are the PIEZO1 channel as a mediator of mechanically activated calcium ion entry and the PECAM1 cell adhesion molecule as the apex of a triad with CDH5 and VGFR2. Here, we investigated if there is a relationship. By inserting a non-disruptive tag in native PIEZO1 of mice, we reveal in situ overlap of PIEZO1 with PECAM1. Through reconstitution and high resolution microscopy studies we show that PECAM1 interacts with PIEZO1 and directs it to cell-cell junctions. PECAM1 extracellular N-terminus is critical in this, but a C-terminal intracellular domain linked to shear stress also contributes. CDH5 similarly drives PIEZO1 to junctions but unlike PECAM1 its interaction with PIEZO1 is dynamic, increasing with shear stress. PIEZO1 does not interact with VGFR2. PIEZO1 is required in Ca2+-dependent formation of adherens junctions and associated cytoskeleton, consistent with it conferring force-dependent Ca2+ entry for junctional remodelling. The data suggest a pool of PIEZO1 at cell junctions, the coming together of PIEZO1 and PECAM1 mechanisms and intimate cooperation of PIEZO1 and adhesion molecules in tailoring junctional structure to mechanical requirement.

Affimers and nanobodies as molecular probes and their applications in imaging.

Antibodies are the most widely used, traditional tool for labelling molecules in cells. In the past five to ten years, many new labelling tools have been developed with significant advantages over the traditional antibody. Here, we focus on nanobodies and the non-antibody binding scaffold proteins called Affimers. We explain how they are generated, selected and produced, and we describe how their small size, high binding affinity and specificity provides them with many advantages compared to antibodies. Of particular importance, their small size enables them to better penetrate dense cytoskeletal regions within cells, as well as tissues, providing them with specific advantage for super-resolution imaging, as they place the fluorophore with a few nanometres of the target protein being imaged. We expect these novel tools to be of broad interest to many cell biologists and anticipate them becoming the tools of choice for super-resolution imaging.

Receptor tyrosine kinases regulate signal transduction through a liquid-liquid phase separated state.

The recruitment of signaling proteins into activated receptor tyrosine kinases (RTKs) to produce rapid, high-fidelity downstream response is exposed to the ambiguity of random diffusion to the target site. Liquid-liquid phase separation (LLPS) overcomes this by providing elevated, localized concentrations of the required proteins while impeding competitor ligands. Here, we show a subset of phosphorylation-dependent RTK-mediated LLPS states. We then investigate the formation of phase-separated droplets comprising a ternary complex including the RTK, (FGFR2); the phosphatase, SHP2; and the phospholipase, PLCγ1, which assembles in response to receptor phosphorylation. SHP2 and activated PLCγ1 interact through their tandem SH2 domains via a previously undescribed interface. The complex of FGFR2 and SHP2 combines kinase and phosphatase activities to control the phosphorylation state of the assembly while providing a scaffold for active PLCγ1 to facilitate access to its plasma membrane substrate. Thus, LLPS modulates RTK signaling, with potential consequences for therapeutic intervention.

Nanoscale Pattern Extraction from Relative Positions of Sparse 3D Localizations.

Inferring the organization of fluorescently labeled nanosized structures from single molecule localization microscopy (SMLM) data, typically obscured by stochastic noise and background, remains challenging. To overcome this, we developed a method to extract high-resolution ordered features from SMLM data that requires only a low fraction of targets to be localized with high precision. First, experimentally measured localizations are analyzed to produce relative position distributions (RPDs). Next, model RPDs are constructed using hypotheses of how the molecule is organized. Finally, a statistical comparison is used to select the most likely model. This approach allows pattern recognition at sub-1% detection efficiencies for target molecules, in large and heterogeneous samples and in 2D and 3D data sets. As a proof-of-concept, we infer ultrastructure of Nup107 within the nuclear pore, DNA origami structures, and α-actinin-2 within the cardiomyocyte Z-disc and assess the quality of images of centrioles to improve the averaged single-particle reconstruction.

A novel workflow for three-dimensional analysis of tumour cell migration.

The limitations of two-dimensional analysis in three-dimensional (3D) cellular imaging impair the accuracy of research findings in biological studies. Here, we report a novel 3D approach to acquisition, analysis and interpretation of tumour spheroid images. Our research interest in mesenchymal-amoeboid transition led to the development of a workflow incorporating the generation and analysis of 3D data with instant structured illumination microscopy and a new ImageJ plugin.

Visualisation and analysis of hepatitis C virus non-structural proteins using super-resolution microscopy.

Hepatitis C virus (HCV) RNA replication occurs in the cytosol of infected cells within a specialised membranous compartment. How the viral non-structural (NS) proteins are associated and organised within these structures remains poorly defined. We employed a super-resolution microscopy approach to visualise NS3 and NS5A in HCV infected cells. Using single molecule localisation microscopy, both NS proteins were resolved as clusters of localisations smaller than the diffraction-limited volume observed by wide-field. Analysis of the protein clusters identified a significant difference in size between the NS proteins. We also observed a reduction in NS5A cluster size following inhibition of RNA replication using daclatasvir, a phenotype which was maintained in the presence of the Y93H resistance associated substitution and not observed for NS3 clusters. These results provide insight into the NS protein organisation within hepatitis C virus RNA replication complexes and the mode of action of NS5A inhibitors.

Site-Specific Labeling of Affimers for DNA-PAINT Microscopy.

Optical super-resolution techniques allow fluorescence imaging below the classical diffraction limit of light. From a technology standpoint, recent methods are approaching molecular-scale spatial resolution. However, this remarkable achievement is not easily translated to imaging of cellular components, since current labeling approaches are limited by either large label sizes (antibodies) or the sparse availability of small and efficient binders (nanobodies, aptamers, genetically-encoded tags). In this work, we combined recently developed Affimer reagents with site-specific DNA modification for high-efficiency labeling and imaging using DNA-PAINT. We assayed our approach using an actin Affimer. The small DNA-conjugated affinity binders could provide a solution for efficient multitarget super-resolution imaging in the future.

Persistent Replication of a Chikungunya Virus Replicon in Human Cells Is Associated with Presence of Stable Cytoplasmic Granules Containing Nonstructural Protein 3.

Chikungunya virus (CHIKV), a mosquito-borne human pathogen, causes a disabling disease characterized by severe joint pain that can persist for weeks, months, or even years in patients. The nonstructural protein 3 (nsP3) plays essential roles during acute infection, but little is known about the function of nsP3 during chronic disease. Here, we used subdiffraction multicolor microscopy for spatial and temporal analysis of CHIKV nsP3 within human cells that persistently replicate replicon RNA. Round cytoplasmic granules of various sizes (i) contained nsP3 and stress granule assembly factors 1 and 2 (G3BP1/2), (ii) were next to double-stranded RNA foci and nsP1-positive structures, and (iii) were close to the nuclear membrane and the nuclear pore complex protein Nup98. Analysis of protein turnover and mobility by live-cell microscopy revealed that the granules could persist for hours to days, accumulated newly synthesized protein, and moved through the cytoplasm at various speeds. The granules also had a static internal architecture and were stable in cell lysates. Refractory cells that had cleared the noncytotoxic replicon regained the ability to respond to arsenite-induced stress. In summary, nsP3 can form uniquely stable granular structures that persist long-term within the host cell. This continued presence of viral and cellular protein complexes has implications for the study of the pathogenic consequences of lingering CHIKV infection and the development of strategies to mitigate the burden of chronic musculoskeletal disease brought about by a medically important arthropod-borne virus (arbovirus).IMPORTANCE Chikungunya virus (CHIKV) is a reemerging alphavirus transmitted by mosquitos and causes transient sickness but also chronic disease affecting muscles and joints. No approved vaccines or antivirals are available. Thus, a better understanding of the viral life cycle and the role of viral proteins can aid in identifying new therapeutic targets. Advances in microscopy and development of noncytotoxic replicons (A. Utt, P. K. Das, M. Varjak, V. Lulla, A. Lulla, A. Merits, J Virol 89:3145-3162, 2015, https://doi.org/10.1128/JVI.03213-14) have allowed researchers to study viral proteins within controlled laboratory environments over extended durations. Here we established human cells that stably replicate replicon RNA and express tagged nonstructural protein 3 (nsP3). The ability to track nsP3 within the host cell and during persistent replication can benefit fundamental research efforts to better understand long-term consequences of the persistence of viral protein complexes and thereby provide the foundation for new therapeutic targets to control CHIKV infection and treat chronic disease symptoms.

Affimer proteins are versatile and renewable affinity reagents.

Molecular recognition reagents are key tools for understanding biological processes and are used universally by scientists to study protein expression, localisation and interactions. Antibodies remain the most widely used of such reagents and many show excellent performance, although some are poorly characterised or have stability or batch variability issues, supporting the use of alternative binding proteins as complementary reagents for many applications. Here we report on the use of Affimer proteins as research reagents. We selected 12 diverse molecular targets for Affimer selection to exemplify their use in common molecular and cellular applications including the (a) selection against various target molecules; (b) modulation of protein function in vitro and in vivo; (c) labelling of tumour antigens in mouse models; and (d) use in affinity fluorescence and super-resolution microscopy. This work shows that Affimer proteins, as is the case for other alternative binding scaffolds, represent complementary affinity reagents to antibodies for various molecular and cell biology applications.

TMEM107 recruits ciliopathy proteins to subdomains of the ciliary transition zone and causes Joubert syndrome.

The transition zone (TZ) ciliary subcompartment is thought to control cilium composition and signalling by facilitating a protein diffusion barrier at the ciliary base. TZ defects cause ciliopathies such as Meckel-Gruber syndrome (MKS), nephronophthisis (NPHP) and Joubert syndrome (JBTS). However, the molecular composition and mechanisms underpinning TZ organization and barrier regulation are poorly understood. To uncover candidate TZ genes, we employed bioinformatics (coexpression and co-evolution) and identified TMEM107 as a TZ protein mutated in oral-facial-digital syndrome and JBTS patients. Mechanistic studies in Caenorhabditis elegans showed that TMEM-107 controls ciliary composition and functions redundantly with NPHP-4 to regulate cilium integrity, TZ docking and assembly of membrane to microtubule Y-link connectors. Furthermore, nematode TMEM-107 occupies an intermediate layer of the TZ-localized MKS module by organizing recruitment of the ciliopathy proteins MKS-1, TMEM-231 (JBTS20) and JBTS-14 (TMEM237). Finally, MKS module membrane proteins are immobile and super-resolution microscopy in worms and mammalian cells reveals periodic localizations within the TZ. This work expands the MKS module of ciliopathy-causing TZ proteins associated with diffusion barrier formation and provides insight into TZ subdomain architecture.

Construction of an instant structured illumination microscope.

A challenge in biological imaging is to capture high-resolution images at fast frame rates in live cells. The "instant structured illumination microscope" (iSIM) is a system designed for this purpose. Similarly to standard structured illumination microscopy (SIM), an iSIM provides a twofold improvement over widefield microscopy, in x, y and z, but also allows much faster image acquisition, with real-time display of super-resolution images. The assembly of an iSIM is reasonably complex, involving the combination and alignment of many optical components, including three micro-optics arrays (two lenslet arrays and an array of pinholes, all with a pitch of 222 µm) and a double-sided scanning mirror. In addition, a number of electronic components must be correctly controlled. Construction of the system is therefore not trivial, but is highly desirable, particularly for live-cell imaging. We report, and provide instructions for, the construction of an iSIM, including minor modifications to a previous design in both hardware and software. The final instrument allows us to rapidly acquire fluorescence images at rates faster than 100 fps, with approximately twofold improvement in resolution in both x-y and z; sub-diffractive biological features have an apparent size (full width at half maximum) of 145 nm (lateral) and 320 nm (axial), using a 1.49 NA objective and 488 nm excitation.

Processing blur of conflicting stimuli during the latency and onset of accommodation.

The accommodative response (AR) to changes in dioptric accommodative stimulus (AS) during the latency period and onset of accommodation was investigated. Participants monocularly observed one period of a square wave in AS, with a 2-D baseline and mean, and amplitude 1 D or 2 D; the period of the square wave ranged from 0.10 s to 1.00 s; both increases and decreases were used for the first step in AS. At periods of 0.30s and longer, accommodation was found to respond to both levels of the stimulus. Rapid retinal monitoring appeared to be taking place for such stimuli. The amplitudes of peaks in AR did not usually depend on whether a particular level of AS occurred first or second, but for 8/40 conditions, a significant difference was found, with a stronger response when the level of AS occurred second. Null or incorrect responses were also observed in many trials, possibly linked with the natural microfluctuations of accommodation. Minimum response times to the changes in AS were observed, which increased with decreasing period of the AS. The time interval between peaks in the AR decreased with decreasing period of the AS. The findings were consistent with a parallel processing model previously proposed for saccades, where input from a later change in stimulus may enter an element of the control system when that element has finished processing an earlier change. More than one change in stimulus may therefore be passing through the multi-element control system at a time.