Selective-plane-activation structured illumination microscopy.

The background light from out-of-focus planes hinders resolution enhancement in structured illumination microscopy when observing volumetric samples. Here we used selective plane illumination and reversibly photoswitchable fluorescent proteins to realize structured illumination within the focal plane and eliminate the out-of-focus background. Theoretical investigation of the imaging properties and experimental demonstrations show that selective plane activation is beneficial for imaging dense microstructures in cells and cell spheroids.

Visualization of trans homophilic interaction of clustered protocadherin in neurons.

Clustered protocadherin (Pcdh) functions as a cell recognition molecule through the homophilic interaction in the central nervous system. However, its interactions have not yet been visualized in neurons. We previously reported PcdhγB2-Förster resonance energy transfer (FRET) probes to be applicable only to cell lines. Herein, we designed γB2-FRET probes by fusing FRET donor and acceptor fluorescent proteins to a single γB2 molecule and succeeded in visualizing γB2 homophilic interaction in cultured hippocampal neurons. The γB2-FRET probe localized in the soma and neurites, and FRET signals, which were observed at contact sites between neurites, eliminated by ethylene glycol tetraacetic acid (EGTA) addition. Live imaging revealed that the FRET-negative γB2 signals rapidly moved along neurites and soma, whereas the FRET-positive signals remained in place. We observed that the γB2 proteins at synapses rarely interact homophilically. The γB2-FRET probe might allow us to elucidate the function of the homophilic interaction and the cell recognition mechanism.

Dynamic Organization of Chromatin Domains Revealed by Super-Resolution Live-Cell Imaging.

The eukaryotic genome is organized within cells as chromatin. For proper information output, higher-order chromatin structures can be regulated dynamically. How such structures form and behave in various cellular processes remains unclear. Here, by combining super-resolution imaging (photoactivated localization microscopy [PALM]) and single-nucleosome tracking, we developed a nuclear imaging system to visualize the higher-order structures along with their dynamics in live mammalian cells. We demonstrated that nucleosomes form compact domains with a peak diameter of ∼160 nm and move coherently in live cells. The heterochromatin-rich regions showed more domains and less movement. With cell differentiation, the domains became more apparent, with reduced dynamics. Furthermore, various perturbation experiments indicated that they are organized by a combination of factors, including cohesin and nucleosome-nucleosome interactions. Notably, we observed the domains during mitosis, suggesting that they act as building blocks of chromosomes and may serve as information units throughout the cell cycle.

Calcium-induced upregulation of energy metabolism heats neurons during neural activity.

Cellular temperature affects every biochemical reaction, underscoring its critical role in cellular functions. In neurons, temperature not only modulates neurotransmission but is also a key determinant of neurodegenerative diseases. Considering that the brain consumes a disproportionately high amount of energy relative to its weight, neural circuits likely generate a lot of heat, which can increase cytosolic temperature. However, the changes in temperature within neurons and the mechanisms of heat generation during neural excitation remain unclear. In this study, we achieved simultaneous imaging of Ca2+ and temperature using the genetically encoded indicators, B-GECO and B-gTEMP. We then compared the spatiotemporal distributions of Ca2+ responses and temperature. Following neural excitation induced by veratridine, an activator of the voltage-gated Na+ channel, we observed an approximately 2 °C increase in cytosolic temperature occurring 30 s after the Ca2+ response. The temperature elevation was observed in the non-nuclear region, while Ca2+ increased throughout the cell body. Moreover, this temperature increase was suppressed under Ca2+-free conditions and by inhibitors of ATP synthesis. These results indicate that Ca2+-induced upregulation of energy metabolism serves as the heat source during neural excitation.

Intracellular Heat Transfer and Thermal Property Revealed by Kilohertz Temperature Imaging with a Genetically Encoded Nanothermometer.

Despite improved sensitivity of nanothermometers, direct observation of heat transport inside single cells has remained challenging for the lack of high-speed temperature imaging techniques. Here, we identified insufficient temperature resolution under short signal integration time and slow sensor kinetics as two major bottlenecks. To overcome the limitations, we developed B-gTEMP, a nanothermometer based on the tandem fusion of mNeonGreen and tdTomato fluorescent proteins. We visualized the propagation of heat inside intracellular space by tracking the temporal variation of local temperature at a time resolution of 155 µs and a temperature resolution 0.042 °C. By comparing the fast in situ temperature dynamics with computer-simulated heat diffusion, we estimated the thermal diffusivity of live HeLa cells. The present thermal diffusivity in cells was about 1/5.3 of that of water and much smaller than the values reported for bulk tissues, which may account for observations of heterogeneous intracellular temperature distributions.

Palmitoylated CKAP4 regulates mitochondrial functions through an interaction with VDAC2 at ER-mitochondria contact sites.

Cytoskeleton-associated protein 4 (CKAP4) is a palmitoylated type II transmembrane protein localized to the endoplasmic reticulum (ER). Here, we found that knockout (KO) of CKAP4 in HeLaS3 cells induces the alteration of mitochondrial structures and increases the number of ER-mitochondria contact sites. To understand the involvement of CKAP4 in mitochondrial functions, the binding proteins of CKAP4 were explored, enabling identification of the mitochondrial porin voltage-dependent anion-selective channel protein 2 (VDAC2), which is localized to the outer mitochondrial membrane. Palmitoylation at Cys100 of CKAP4 was required for the binding between CKAP4 and VDAC2. In CKAP4 KO cells, the binding of inositol trisphosphate receptor (IP3R) and VDAC2 was enhanced, the intramitochondrial Ca2+ concentration increased and the mitochondrial membrane potential decreased. In addition, CKAP4 KO decreased the oxidative consumption rate, in vitro cancer cell proliferation under low-glucose conditions and in vivo xenograft tumor formation. The phenotypes were not rescued by expression of a palmitoylation-deficient CKAP4 mutant. These results suggest that CKAP4 plays a role in maintaining mitochondrial functions through the binding to VDAC2 at ER-mitochondria contact sites and that palmitoylation is required for this novel function of CKAP4.This article has an associated First Person interview with the first author of the paper.

Application of Green-enhanced Nano-lantern as a bioluminescent ratiometric indicator for measurement of Arabidopsis thaliana root apoplastic fluid pH.

Plant root absorbs water and nutrients from the soil, and the root apoplastic fluid (AF) is an important intermediate between cells and the surrounding environment. The acid growth theory suggests that an acidic AF is needed for cell wall expansion during root growth. However, technical limitations have precluded the quantification of root apoplastic fluid pH (AF-pH). Here, we used Green-enhanced Nano-lantern (GeNL), a chimeric protein of the luciferase NanoLuc (Nluc) and the green fluorescent protein mNeonGreen (mNG), as a ratiometric pH indicator based on the pH dependency of bioluminescence resonance energy transfer efficiency from Nluc to mNG. Luminescence spectrum of GeNL changed reciprocally from pH 4.5 to 7.5, with a pKa of 5.5. By fusing GeNL to a novel signal peptide from Arabidopsis thaliana Cellulase 1, we localised GeNL in A. thaliana AF. We visualised AF dynamics at subcellular resolution over 30 min and determined flow velocity in the maturation zone to be 0.97± 0.06 µm/s. We confirmed that the developing root AF is acidic in the pH range of 5.1-5.7, suggesting that the AF-pH is tightly regulated during root elongation. These results support the acid growth theory and provide evidence for AF-pH maintenance despite changes in ambient pH.

Ratiometric Bioluminescent Indicator for a Simple and Rapid Measurement of Thrombin Activity Using a Smartphone.

Hemostasis is an essential function that repairs tissues and maintains the survival of living organisms. To prevent diseases caused by the abnormality of the blood coagulation mechanism, it is important to carry out a blood test periodically by a method that is convenient and less burdensome for examiners. Thrombin is a protease that catalyzes the conversion of fibrinogen, and its cleavage activity can be an index of coagulation activity. Here, we developed a ratiometric bioluminescent indicator, Thrombastor (thrombin activity sensing indicator), which reflects the thrombin cleavage activity in blood by changing the emission color from green to blue. Compared to the current thrombin activity indicator, the rapid color change of the emission indicated a 2.5-fold decrease in the Km for thrombin, and the cleavage rate was more than two times faster. By improving the absolute bioluminescence intensity, detection from a small amount of plasma could be achieved with a smartphone camera. Using Thrombastor and a versatile device, an effective diagnosis for preventing coagulation disorders can be provided.

Hyperspectral two-photon excitation microscopy using visible wavelength.

We demonstrate hyperspectral imaging by visible-wavelength two-photon excitation microscopy using line illumination and slit-confocal detection. A femtosecond pulsed laser light at 530 nm was used for the simultaneous excitation of fluorescent proteins with different emission wavelengths. The use of line illumination enabled efficient detection of hyperspectral images and achieved simultaneous detection of three fluorescence spectra in the observation of living HeLa cells with an exposure time of 1 ms per line, which is equivalent to about 2 µs per pixel in point scanning, with 160 data points per spectrum. On combining linear spectral unmixing techniques, localization of fluorescent probes in the cells was achieved. A theoretical investigation of the imaging property revealed high-depth discrimination property attained through the combination of nonlinear excitation and slit detection.

Genetically Encoded Photosensitizer for Destruction of Protein or Cell Function.

There are several paths when excited molecules return to the ground state. In the case of fluorescent molecules, the dominant path is fluorescence emission that is greatly contributing to bioimaging. Meanwhile, photosensitizers transfer electron or energy from chromophore to the surrounding molecules, including molecular oxygen. Generated reactive oxygen species has potency to attack other molecules by oxidation. In this chapter, we introduce the chromophore-assisted light inactivation (CALI) method using a photosensitizer to inactivate proteins in a spatiotemporal manner and development of CALI tools, which is useful for investigation of protein functions and dynamics, by inactivation of the target molecules. Moreover, photosensitizers with high efficiency make it possible optogenetic control of cell ablation in living organisms and photodynamic therapy. Further development of photosensitizers with different excitation wavelengths will contribute to the investigation of multiple proteins or cell functions through inactivation in the different positions and timings.

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.

Distinct intracellular Ca2+ dynamics regulate apical constriction and differentially contribute to neural tube closure.

Early in the development of the central nervous system, progenitor cells undergo a shape change, called apical constriction, that triggers the neural plate to form a tubular structure. How apical constriction in the neural plate is controlled and how it contributes to tissue morphogenesis are not fully understood. In this study, we show that intracellular calcium ions (Ca2+) are required for Xenopus neural tube formation and that there are two types of Ca2+-concentration changes, a single-cell and a multicellular wave-like fluctuation, in the developing neural plate. Quantitative imaging analyses revealed that transient increases in Ca2+ concentration induced cortical F-actin remodeling, apical constriction and accelerations of the closing movement of the neural plate. We also show that extracellular ATP and N-cadherin (cdh2) participate in the Ca2+-induced apical constriction. Furthermore, our mathematical model suggests that the effect of Ca2+ fluctuations on tissue morphogenesis is independent of fluctuation frequency and that fluctuations affecting individual cells are more efficient than those at the multicellular level. We propose that distinct Ca2+ signaling patterns differentially modulate apical constriction for efficient epithelial folding and that this mechanism has a broad range of physiological outcomes.

A simple microfluidic device for live-imaging of the vertical section of epithelial cells.

We investigated the capability of simple microfluidic devices with trenches having vertical sidewalls for live-cell fluorescence imaging of adherent cells. An epithelial cell line that forms a two-dimensional (2D) sheet was cultured to adhere to the vertical sidewall so that its vertical section can be imaged directly using ordinal inverted-type laser-scanning microscopy. The material and the structure of the device were characterized. We show that the detailed distribution of intracellular organelles, such as microtubules and mitochondria, and of intercellular apparatus, such as claudin and zonula occludens, can be imaged with high spatio-temporal resolution with a single scan.

Smartphone-Based Portable Bioluminescence Imaging System Enabling Observation at Various Scales from Whole Mouse Body to Organelle.

Current smartphones equipped with high-sensitivity and high-resolution sensors in the camera can respond to the needs of low-light imaging, streaming acquisition, targets of various scales, etc. Therefore, a smartphone has great potential as an imaging device even in the scientific field and has already been introduced into biomolecular imaging using fluorescence tags. However, owing to the necessity of an excitation light source, fluorescence methods impair its mobility. Bioluminescence does not require illumination; therefore, imaging with a smartphone camera is compact and requires minimal devices, thus making it suitable for personal and portable imaging devices. Here, we report smartphone-based methods to observe biological targets in various scales using bioluminescence. In particular, we demonstrate, for the first time, that bioluminescence can be observed in an organelle in a single living cell using a smartphone camera by attaching a detachable objective lens. Through capturing color changes with the camera, changes in the amount of target molecules was detected using bioluminescent indicators. The combination of bioluminescence and a mobile phone makes possible a compact imaging system without an external light source and expands the potential of portable devices.

Bioluminescent Ratiometric Indicator for Analysis of Water Hardness in Household Water.

Water hardness (WH) is a useful parameter for testing household water, such as drinking, cooking, and washing water. Many countries around the world use pipeline water in their houses, but there is a need to monitor the WH because hard water has a negative impact on appliances. Currently, WH is often measured using chemical dye-based WH indicators, and these techniques require expensive equipment, and trained personnel. Therefore, a low-cost and simple measurement method has been desired. Here, we report LOTUS-W, which consists of a luciferase, Nanoluc, a yellow fluorescent protein Venus, and a Ca2+/Mg2+ detection domain of human centrin 3. The binding of Ca2+/Mg2+ to this indicator changes the conformation of human centrin 3, and induces bioluminescence resonance energy transfer (BRET) from Nanoluc to Venus, which changes its emission spectrum about 140%. The dissociation constants of LOTUS-W for Ca2+/Mg2+ are approximately several mM, making it suitable for measuring WH in the household water. With this indicator in combination with a smartphone, we have demonstrated that it is possible to evaluate WH easily and quickly. This novel indicator has the potential to be used for measuring not only household water but also water used in the food industry, etc.

Significance of PGR5-dependent cyclic electron flow for optimizing the rate of ATP synthesis and consumption in Arabidopsis chloroplasts.

The proton motive force (PMF) across the chloroplast thylakoid membrane that is generated by electron transport during photosynthesis is the driving force for ATP synthesis in plants. The PMF mainly arises from the oxidation of water in photosystem II and from electron transfer within the cytochrome b6f complex. There are two electron transfer pathways related to PMF formation: linear electron flow and cyclic electron flow. Proton gradient regulation 5 (PGR5) is a major component of the cyclic electron flow pathway, and the Arabidopsis pgr5 mutant shows a substantial reduction in the PMF. How the PGR5-dependent cyclic electron flow contributes to ATP synthesis has not, however, been fully delineated. In this study, we monitored in vivo ATP levels in Arabidopsis chloroplasts in real time using a genetically encoded bioluminescence-based ATP indicator, Nano-lantern(ATP1). The increase in ATP in the chloroplast stroma of pgr5 leaves upon illumination with actinic light was significantly slower than in wild type, and the decrease in ATP levels when this illumination stopped was significantly faster in pgr5 leaves than in wild type. These results indicated that PGR5-dependent cyclic electron flow around photosystem I helps to sustain the rate of ATP synthesis, which is important for growth under fluctuating light conditions.

Simultaneous monitoring of Ca2+ responses and salivary secretion in live animals reveals a threshold intracellular Ca2+ concentration for salivation.

NEW FINDINGS: What is the central question of this study? The effects of Ca2+ responses on salivary fluid secretion have been studied indirectly by monitoring ion channel activities and other indices. Therefore, Ca2+ responses during salivary secretion remain poorly understood. What is the main finding and its importance? Herein, we developed a simultaneous monitoring system for Ca2+ responses and salivary secretion in live animals using a YC-Nano50-expressing submandibular gland and a fibre-optic pressure sensor. This new approach revealed a clear time lag between the onset of Ca2+ responses and salivary secretion. We also estimated the [Ca2+ ]i and provided direct evidence for the regulation of salivary secretion by small increases in [Ca2+ ]i in submandibular gland acinar cells. ABSTRACT: We monitored changes in [Ca2+ ]i during salivary secretion in the rat submandibular gland in live animals using a combination of intravital Ca2+ imaging with the ultrasensitive Ca2+ indicator YC-Nano50 and a fibre-optic pressure sensor. Intravenous infusion of ACh (10-720 nmol min-1 ) increased [Ca2+ ]i and salivary flow rate in a dose-dependent manner. Repetitive stimulation with ACh induced equivalent Ca2+ responses and salivary secretion in the same individual animals. The accurate ACh stimulation experiments revealed a clear time lag between the onset of the increase in [Ca2+ ]i and salivary secretion. The time lag with the lowest dose of ACh (30 nmol min-1 ) was 106 s, which shortened to 19 s with the dose used for maximal salivary secretion (360 nmol min-1 ). This time lag might reflect the time required for [Ca2+ ]i to reach the level required to activate molecules for fluid secretion. The resting [Ca2+ ]i in submandibular gland was 37 nm, and [Ca2+ ]i at the onset of salivary secretion was 45-57 nm, irrespective of ACh dose. These results indicate that low [Ca2+ ]i is sufficient to trigger fluid secretion in the rat submandibular gland in vivo.

Green monomeric photosensitizing fluorescent protein for photo-inducible protein inactivation and cell ablation.

BACKGROUND: Photosensitizing fluorescent proteins, which generate reactive oxygen species (ROS) upon light irradiation, are useful for spatiotemporal protein inactivation and cell ablation. They give us clues about protein function, intracellular signaling pathways and intercellular interactions. Since ROS generation of a photosensitizer is specifically controlled by certain excitation wavelengths, utilizing colour variants of photosensitizing protein would allow multi-spatiotemporal control of inactivation. To expand the colour palette of photosensitizing protein, here we developed SuperNova Green from its red predecessor, SuperNova. RESULTS: SuperNova Green is able to produce ROS spatiotemporally upon blue light irradiation. Based on protein characterization, SuperNova Green produces insignificant amounts of singlet oxygen and predominantly produces superoxide and its derivatives. We utilized SuperNova Green to specifically inactivate the pleckstrin homology domain of phospholipase C-δ1 and to ablate cancer cells in vitro. As a proof of concept for multi-spatiotemporal control of inactivation, we demonstrate that SuperNova Green can be used with its red variant, SuperNova, to perform independent protein inactivation or cell ablation studies in a spatiotemporal manner by selective light irradiation. CONCLUSION: Development of SuperNova Green has expanded the photosensitizing protein toolbox to optogenetically control protein inactivation and cell ablation.

Fluorescent Protein-Based Indicators for Functional Super-Resolution Imaging of Biomolecular Activities in Living Cells.

Super-resolution light microscopy (SRM) offers a unique opportunity for diffraction-unlimited imaging of biomolecular activities in living cells. To realize such potential, genetically encoded indicators were developed recently from fluorescent proteins (FPs) that exhibit phototransformation behaviors including photoactivation, photoconversion, and photoswitching, etc. Super-resolution observations of biomolecule interactions and biochemical activities have been demonstrated by exploiting the principles of bimolecular fluorescence complementation (BiFC), points accumulation for imaging nanoscale topography (PAINT), and fluorescence fluctuation increase by contact (FLINC), etc. To improve functional nanoscopy with the technology of genetically encoded indicators, it is essential to fully decipher the photo-induced chemistry of FPs and opt for innovative indicator designs that utilize not only fluorescence intensity but also multi-parametric readouts such as phototransformation kinetics. In parallel, technical improvements to both the microscopy optics and image analysis pipeline are promising avenues to increase the sensitivity and versatility of functional SRM.

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.