Hierarchical Development of Motile Polarity in Durotactic Cells Just Crossing an Elasticity Boundary.

Cellular durotaxis has been extensively studied in the field of mechanobiology. In principle, asymmetric mechanical field of a stiffness gradient generates motile polarity in a cell, which is a driving factor of durotaxis. However, the actual process by which the motile polarity in durotaxis develops is still unclear. In this study, to clarify the details of the kinetics of the development of durotactic polarity, we investigated the dynamics of both cell-shaping and the microscopic turnover of focal adhesions (FAs) for Venus-paxillin-expressing fibroblasts just crossing an elasticity boundary prepared on microelastically patterned gels. The Fourier mode analysis of cell-shaping based on a persistent random deformation model revealed that motile polarity at a cell-body scale was established within the first few hours after the leading edges of a moving cell passed through the boundary from the soft to the stiff regions. A fluorescence recovery after photobleaching (FRAP) analysis showed that the mobile fractions of paxillin at FAs in the anterior part of the cells exhibited an asymmetric increase within several tens of minutes after cells entered the stiff region. The results demonstrated that motile polarity in durotactic cells is established through the hierarchical step-wise development of different types of asymmetricity in the kinetics of FAs activity and cell-shaping with a several-hour time lag.Key words: Microelasticity patterned gel, durotaxis, cell polarity, focal adhesions, paxillin.

Activity-Dependent Dynamics of the Transcription Factor of cAMP-Response Element Binding Protein in Cortical Neurons Revealed by Single-Molecule Imaging.

Transcriptional regulation is crucial for neuronal activity-dependent processes that govern neuronal circuit formation and synaptic plasticity. An intriguing question is how neuronal activity influences the spatiotemporal interactions between transcription factors and their target sites. Here, using a single-molecule imaging technique, we investigated the activity dependence of DNA binding and dissociation events of cAMP-response element binding protein (CREB), a principal factor in activity-dependent transcription, in mouse cortical neurons. To visualize CREB at the single-molecule level, fluorescent-tagged CREB in living dissociated cortical neurons was observed by highly inclined and laminated optical sheet microscopy. We found that a significant fraction of CREB spots resided in the restricted locations in the nucleus for several seconds (dissociation rate constant: 0.42 s-1). In contrast, two mutant CREBs, which cannot bind to the cAMP-response element, scarcely exhibited long-term residence. To test the possibility that CREB dynamics depends on neuronal activity, pharmacological treatments and an optogenetic method involving channelrhodopsin-2 were applied to cultured cortical neurons. Increased neuronal activity did not appear to influence the residence time of CREB spots, but markedly increased the number of restricted locations (hot spots) where CREB spots frequently resided with long residence times (>1 s). These results suggest that neuronal activity promotes CREB-dependent transcription by increasing the frequency of CREB binding to highly localized genome locations. SIGNIFICANCE STATEMENT: The transcription factor, cAMP response element-binding protein (CREB) is known to regulate gene expression in neuronal activity-dependent processes. However, its spatiotemporal interactions with the genome remain unknown. Single-molecule imaging in cortical neurons revealed that fluorescent-tagged CREB spots frequently reside at fixed nuclear locations in the time range of several seconds. Neuronal activity had little effect on the CREB residence time, but increased the rapid and frequent reappearance of long-residence CREB spots at the same nuclear locations. Thus, activity-dependent transcription is attributable to frequent binding of CREB to specific genome loci.

A fast- and positively photoswitchable fluorescent protein for ultralow-laser-power RESOLFT nanoscopy.

Fluorescence nanoscopy has revolutionized our ability to visualize biological structures not resolvable by conventional microscopy. However, photodamage induced by intense light exposure has limited its use in live specimens. Here we describe Kohinoor, a fast-switching, positively photoswitchable fluorescent protein, and show that it has high photostability over many switching repeats. With Kohinoor, we achieved super-resolution imaging of live HeLa cells using biocompatible, ultralow laser intensity (0.004 J/cm(2)) in reversible saturable optical fluorescence transition (RESOLFT) nanoscopy.

Rapid severing and motility of chloroplast-actin filaments are required for the chloroplast avoidance response in Arabidopsis.

Phototropins (phot1 and phot2 in Arabidopsis thaliana) relay blue light intensity information to the chloroplasts, which move toward weak light (the accumulation response) and away from strong light (the avoidance response). Chloroplast-actin (cp-actin) filaments are vital for mediating these chloroplast photorelocation movements. In this report, we examine in detail the cp-actin filament dynamics by which the chloroplast avoidance response is regulated. Although stochastic dynamics of cortical actin fragments are observed on the chloroplasts, the basic mechanisms underlying the disappearance (including severing and turnover) of the cp-actin filaments are regulated differently from those of cortical actin filaments. phot2 plays a pivotal role in the strong blue light-induced severing and random motility of cp-actin filaments, processes that are therefore essential for asymmetric cp-actin formation for the avoidance response. In addition, phot2 functions in the bundling of cp-actin filaments that is induced by dark incubation. By contrast, the function of phot1 is dispensable for these responses. Our findings suggest that phot2 is the primary photoreceptor involved in the rapid reorganization of cp-actin filaments that allows chloroplasts to change direction rapidly and control the velocity of the avoidance movement according to the light's intensity and position.

Determination of Capillary Blood TSH and Free Thyroxine Levels Using Digital Immunoassay.

Background: The remote performance of thyroid function blood tests is complicated because it requires blood collection. Objective: To compare TSH and free thyroxine (FT4) levels between capillary and venous blood and assess the adequacy of measuring each value in capillary blood. Methods: This prospective intervention study was conducted at Ito Hospital and was based on the clinical research method. The participants were 5 healthy female volunteers and 50 patients (41 females and 9 males) between the ages of 23 and 81 years. To measure TSH and FT4 levels in capillary and venous blood, a digital immunoassay (d-IA) method capable of measuring trace samples was used. Chemiluminescence measurements were used as controls. Values obtained for each assay system were compared using Spearman's correlation analysis. Capillary blood was collected using an autologous device (TAP II; not approved in Japan). Results: Capillary plasma volume obtained using TAP II was 125 µL or more in 26 cases, 25 µL to 124 µL in 24 cases, and less than 25 µL in 5 cases. Strong correlations were noted in the TSH and FT4 levels between capillary and venous blood, with correlation coefficients of rs = 0.99 and rs = 0.97, respectively. Conclusion: Capillary TSH and FT4 levels strongly correlate with venous blood values. Trace samples can be used in high-precision d-IA methods. These results may promote telemedicine in assessing thyroid function.

Self-organization of the phosphatidylinositol lipids signaling system for random cell migration.

Phosphatidylinositol (PtdIns) lipids have been identified as key signaling mediators for random cell migration as well as chemoattractant-induced directional migration. However, how the PtdIns lipids are organized spatiotemporally to regulate cellular motility and polarity remains to be clarified. Here, we found that self-organized waves of PtdIns 3,4,5-trisphosphate [PtdIns(3,4,5)P(3)] are generated spontaneously on the membrane of Dictyostelium cells in the absence of a chemoattractant. Characteristic oscillatory dynamics within the PtdIns lipids signaling system were determined experimentally by observing the phenotypic variability of PtdIns lipid waves in single cells, which exhibited characteristics of a relaxation oscillator. The enzymes phosphatase and tensin homolog (PTEN) and phosphoinositide-3-kinase (PI3K), which are regulators for PtdIns lipid concentrations along the membrane, were essential for wave generation whereas functional actin cytoskeleton was not. Defects in these enzymes inhibited wave generation as well as actin-based polarization and cell migration. On the basis of these experimental results, we developed a reaction-diffusion model that reproduced the characteristic relaxation oscillation dynamics of the PtdIns lipid system, illustrating that a self-organization mechanism provides the basis for the PtdIns lipids signaling system to generate spontaneous spatiotemporal signals for random cell migration and that molecular noise derived from stochastic fluctuations within the signaling components gives rise to the variability of these spontaneous signals.

Development of a quantitative thyroid-stimulating hormone assay system for a benchtop digital ELISA desktop analyzer.

Regular checkups for thyroid-stimulating hormone (TSH) levels are essential for the diagnosis of thyroid disease. The enzyme-linked immunosorbent assay (ELISA) technique is a standard method for detecting TSH in the serum or plasma of hospitalized patients. A recently developed next-generation ELISA, the digital immunoassay (d-IA), has facilitated detection of molecules with ultra-high-sensitivity. In this study, we developed a TSH assay system using the d-IA platform. By utilizing the ultrasensitivity of d-IA, we were able to use a sample volume of as little as 5 µL for each assay (the dead volume was 5 µL). The limits of blank, detection, and quantification (i.e., functional sensitivity), were 0.000346, 0.001953, and 0.002280 µIU/mL, respectively, and the precision of the total coefficient of variation did not exceed 10%. The correlation between serum and plasma levels indicated good agreement. Thus, our system successfully measured TSH using d-IA with a small sample volume and equal functional sensitivity to the current third generation like ARCHITECT TSH assay, which has a functional sensitivity of 0.0038 µIU/mL.

Activity-dependent organization of prefrontal hub-networks for associative learning and signal transformation.

Associative learning is crucial for adapting to environmental changes. Interactions among neuronal populations involving the dorso-medial prefrontal cortex (dmPFC) are proposed to regulate associative learning, but how these neuronal populations store and process information about the association remains unclear. Here we developed a pipeline for longitudinal two-photon imaging and computational dissection of neural population activities in male mouse dmPFC during fear-conditioning procedures, enabling us to detect learning-dependent changes in the dmPFC network topology. Using regularized regression methods and graphical modeling, we found that fear conditioning drove dmPFC reorganization to generate a neuronal ensemble encoding conditioned responses (CR) characterized by enhanced internal coactivity, functional connectivity, and association with conditioned stimuli (CS). Importantly, neurons strongly responding to unconditioned stimuli during conditioning subsequently became hubs of this novel associative network for the CS-to-CR transformation. Altogether, we demonstrate learning-dependent dynamic modulation of population coding structured on the activity-dependent formation of the hub network within the dmPFC.

Simultaneous observation of the lever arm and head explains myosin VI dual function.

Myosin VI is an adenosine triphosphate (ATP)-driven dimeric molecular motor that has dual function as a vesicle transporter and a cytoskeletal anchor. Recently, it was reported that myosin VI generates three types of steps by taking either a distant binding or adjacent binding state (noncanonical hand-over-hand step pathway). The adjacent binding state, in which both heads bind to an actin filament near one another, is unique to myosin VI and therefore may help explain its distinct features. However, detailed information of the adjacent binding state remains unclear. Here simultaneous observations of the head and tail domain during stepping are presented. These observations show that the lever arms tilt forward in the adjacent binding state. Furthermore, it is revealed that either head could take the subsequent step with equal probability from this state. Together with previous results, a comprehensive stepping scheme is proposed; it includes the tail domain motion to explain how myosin VI achieves its dual function.

Development of a Fully Automated Desktop Analyzer and Ultrahigh Sensitivity Digital Immunoassay for SARS-CoV-2 Nucleocapsid Antigen Detection.

BACKGROUND: The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) outbreak has had a significant impact on public health and the global economy. Several diagnostic tools are available for the detection of infectious diseases, with reverse transcription-polymerase chain reaction (RT-PCR) testing specifically recommended for viral RNA detection. However, this diagnostic method is costly, complex, and time-consuming. Although it does not have sufficient sensitivity, antigen detection by an immunoassay is an inexpensive and simpler alternative to RT-PCR. Here, we developed an ultrahigh sensitivity digital immunoassay (d-IA) for detecting SARS-CoV-2 nucleocapsid (N) protein as antigens using a fully automated desktop analyzer based on a digital enzyme-linked immunosorbent assay. METHODS: We developed a fully automated d-IA desktop analyzer and measured the viral N protein as an antigen in nasopharyngeal (NP) swabs from patients with coronavirus disease. We studied nasopharyngeal swabs of 159 and 88 patients who were RT-PCR-negative and RT-PCR-positive, respectively. RESULTS: The limit of detection of SARS-CoV-2 d-IA was 0.0043 pg/mL of N protein. The cutoff value was 0.029 pg/mL, with a negative RT-PCR distribution. The sensitivity of RT-PCR-positive specimens was estimated to be 94.3% (83/88). The assay time was 28 min. CONCLUSIONS: Our d-IA system, which includes a novel fully automated desktop analyzer, enabled detection of the SARS-CoV-2 N-protein with a comparable sensitivity to RT-PCR within 30 min. Thus, d-IA shows potential for SARS-CoV-2 detection across multiple diagnostic centers including small clinics, hospitals, airport quarantines, and clinical laboratories.

Conjugation of both on-axis and off-axis light in Nipkow disk confocal microscope to increase availability of incoherent light source.

Laser-scanning confocal microscopy has been employed for exploring structures at subcellular, cellular and tissue level in three dimensions. To acquire the confocal image, a coherent light source, such as laser, is generally required in conventional single-point scanning microscopy. The illuminating beam must be focused onto a small spot with diffraction-limited size, and this determines the spatial resolution of the microscopy system. In contrast, multipoint scanning confocal microscopy using a Nipkow disk enables the use of an incoherent light source. We previously demonstrated successful application of a 100 W mercury arc lamp as a light source for the Yokogawa confocal scanner unit in which a microlens array was coupled with a Nipkow disk to focus the collimated incident light onto a pinhole (Saito et al., Cell Struct. Funct., 33: 133-141, 2008). However, transmission efficiency of incident light through the pinhole array was low because off-axis light, the major component of the incident light, was blocked by the non-aperture area of the disk. To improve transmission efficiency, we propose an optical system in which off-axis light is able to be transmitted through pinholes surrounding the pinhole located on the optical axis of the collimator lens. This optical system facilitates the use of not only the on-axis but also the off-axis light such that the available incident light is considerably improved. As a result, we apply the proposed system to high-speed confocal and multicolor imaging both with a satisfactory signal-to-noise ratio.

Ratiometric Bioluminescent Indicator for Simple and Rapid Diagnosis of Bilirubin.

Bilirubin in human blood is highly important as a general index of one's physical condition because its concentration changes under the influence of several diseases. In particular, in newborns, jaundice is one of the most common diseases involving unconjugated bilirubin (UCBR), causing serious symptoms such as nuclear jaundice and deafness. Therefore, a frequent measurement of the UCBR levels in the blood is important. Here, we report a ratiometric bioluminescent indicator, BABI (bilirubin assessment with a bioluminescent indicator), that changes the emission color from blue to green depending on the UCBR concentration in a sample. Owing to the use of a bioluminescence signal that has a higher signal-to-noise ratio than the absorption and fluorescence signal, BABI enables highly sensitive and quantitative detection of UCBR for small blood samples using a smartphone camera. The establishment of a UCBR measurement assay using BABI provides the possibility of a simple and rapid method for blood-based diagnosis using bioluminescent indicators and a versatile mobile device.

Imaging local brain activity of multiple freely moving mice sharing the same environment.

Electrophysiological field potential dynamics have been widely used to investigate brain functions and related psychiatric disorders. Considering recent demand for its applicability to freely moving subjects, especially for animals in a group and socially interacting with each other, here we propose a new method based on a bioluminescent voltage indicator LOTUS-V. Using our fiber-free recording method based on the LOTUS-V, we succeeded in capturing dynamic change of brain activity in freely moving mice. Because LOTUS-V is the ratiometric indicator, motion and head-angle artifacts were not significantly detected. Taking advantage of our method as a fiber-free system, we further succeeded in simultaneously recording from multiple independently-locomotive mice that were freely interacting with one another. Importantly, this enabled us to find that the primary visual cortex, a center of visual processing, was activated during the interaction of mice. This methodology may further facilitate a wide range of studies in neurobiology and psychiatry.

Visible-wavelength two-photon excitation microscopy with multifocus scanning for volumetric live-cell imaging.

Two-photon excitation microscopy is one of the key techniques used to observe three-dimensional (3-D) structures in biological samples. We utilized a visible-wavelength laser beam for two-photon excitation in a multifocus confocal scanning system to improve the spatial resolution and image contrast in 3-D live-cell imaging. Experimental and numerical analyses revealed that the axial resolution has improved for a wide range of pinhole sizes used for confocal detection. We observed the 3-D movements of the Golgi bodies in living HeLa cells with an imaging speed of 2 s per volume. We also confirmed that the time-lapse observation up to 8 min did not cause significant cell damage in two-photon excitation experiments using wavelengths in the visible light range. These results demonstrate that multifocus, two-photon excitation microscopy with the use of a visible wavelength can constitute a simple technique for 3-D visualization of living cells with high spatial resolution and image contrast.

Biomimetic Chemical Sensing by Fluorescence Signals Using a Virus-like Particle-Based Platform.

The chemical receptors present in living organisms are promising tools for developing biomimetic chemical sensors. However, these receptors require lipid membranes for functioning under physiological conditions, which prevents their utilization in the production of cell-free in vitro chemical sensing systems. Here, we report the development of a cell-free biomimetic sensing platform using virus-like particles (VLPs) with intact ligand-gated Ca2+ channels and genetically encoded Ca2+ indicator (GECI). We observed that targeting GECI to the plasma membrane was essential for efficient loading GECI in the VLPs. Although the physiological Ca2+ concentration [Ca2+] maintained in the cells was low (∼10 nM), the concentration in the VLPs was high. This prevented the detection of the increase in [Ca2+] caused by binding of the ligand to the receptor. To address this problem, we employed Lyn-R-CEPIA1, which had low affinity for Ca2+, and a membrane targeting sequence. Thus, we succeeded in monitoring the activation of cyclic nucleotide-gated channels (CNG) on the VLPs by measuring the increase in fluorescence of Lyn-R-CEPIA1. Our VLP-based sensing system can act as a fundamental platform for all kinds of ligand-gated channels.

Highly biocompatible super-resolution fluorescence imaging using the fast photoswitching fluorescent protein Kohinoor and SPoD-ExPAN with Lp-regularized image reconstruction.

Far-field super-resolution fluorescence microscopy has enabled us to visualize live cells in great detail and with an unprecedented resolution. However, the techniques developed thus far have required high-power illumination (102-106 W/cm2), which leads to considerable phototoxicity to live cells and hampers time-lapse observation of the cells. In this study we show a highly biocompatible super-resolution microscopy technique that requires a very low-power illumination. The present technique combines a fast photoswitchable fluorescent protein, Kohinoor, with SPoD-ExPAN (super-resolution by polarization demodulation/excitation polarization angle narrowing). With this technique, we successfully observed Kohinoor-fusion proteins involving vimentin, paxillin, histone and clathrin expressed in HeLa cells at a spatial resolution of 70-80 nm with illumination power densities as low as ~1 W/cm2 for both excitation and photoswitching. Furthermore, although the previous SPoD-ExPAN technique used L1-regularized maximum-likelihood calculations to reconstruct super-resolved images, we devised an extension to the Lp-regularization to obtain super-resolved images that more accurately describe objects at the specimen plane. Thus, the present technique would significantly extend the applicability of super-resolution fluorescence microscopy for live-cell imaging.

Spontaneously Blinking Fluorescent Protein for Simple Single Laser Super-Resolution Live Cell Imaging.

Super-resolution imaging techniques based on single molecule localization microscopy (SMLM) broke the diffraction limit of optical microscopy in living samples with the aid of photoswitchable fluorescent probes and intricate microscopy systems. Here, we developed a fluorescent protein, SPOON, which can be switched off by excitation light illumination and switched on by thermally induced dehydration, resulting in an apparent spontaneous blinking behavior. This unique property of SPOON provides a simple SMLM-based super-resolution imaging platform which requires only a single 488 nm laser.

Publisher Correction: Alpha-synuclein facilitates to form short unconventional microtubules that have a unique function in the axonal transport.

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Single molecule FRET for the study on structural dynamics of biomolecules.

Single molecule fluorescence resonance energy transfer (FRET) is the technique that has been developed by combining FRET measurement and single molecule fluorescence imaging. This technique allows us to measure the dynamic changes of the interaction and structures of biomolecules. In this study, the validity of the method was tested using fluorescence dyes on double stranded DNA molecules as a rigid spacer. FRET signals from double stranded DNA molecules were stable and their average FRET values provided the distance between the donor and acceptor in agreement with B-DNA type helix model. Next, the single molecule FRET method was applied to the studies on the dynamic structure of Ras, a signaling protein. The data showed that Ras has multiple conformational states and undergoes transition between them. This study on the dynamic conformation of Ras provided a clue for understanding the molecular mechanism of cell signaling switches.