Visualizing and Modulating Mitophagy for Therapeutic Studies of Neurodegeneration.

Dysfunctional mitochondria accumulate in many human diseases. Accordingly, mitophagy, which removes these mitochondria through lysosomal degradation, is attracting broad attention. Due to uncertainties in the operational principles of conventional mitophagy probes, however, the specificity and quantitativeness of their readouts are disputable. Thorough investigation of the behaviors and fates of fluorescent proteins inside and outside lysosomes enabled us to develop an indicator for mitophagy, mito-SRAI. Through strict control of its mitochondrial targeting, we were able to monitor mitophagy in fixed biological samples more reproducibly than before. Large-scale image-based high-throughput screening led to the discovery of a hit compound that induces selective mitophagy of damaged mitochondria. In a mouse model of Parkinsons disease, we found that dopaminergic neurons selectively failed to execute mitophagy that promoted their survival within lesions. These results show that mito-SRAI is an essential tool for quantitative studies of mitochondrial quality control.

A bilirubin-inducible fluorescent protein from eel muscle.

The fluorescent protein toolbox has revolutionized experimental biology. Despite this advance, no fluorescent proteins have been identified from vertebrates, nor has chromogenic ligand-inducible activation or clinical utility been demonstrated. Here, we report the cloning and characterization of UnaG, a fluorescent protein from Japanese eel. UnaG belongs to the fatty-acid-binding protein (FABP) family, and expression in eel is restricted to small-diameter muscle fibers. On heterologous expression in cell lines or mouse brain, UnaG produces oxygen-independent green fluorescence. Remarkably, UnaG fluorescence is triggered by an endogenous ligand, bilirubin, a membrane-permeable heme metabolite and clinical health biomarker. The holoUnaG structure at 1.2 Å revealed a biplanar coordination of bilirubin by reversible π-conjugation, and we used this high-affinity and high-specificity interaction to establish a fluorescence-based human bilirubin assay with promising clinical utility. UnaG will be the prototype for a versatile class of ligand-activated fluorescent proteins, with applications in research, medicine, and bioengineering.

StayGold variants for molecular fusion and membrane-targeting applications.

Although StayGold is a bright and highly photostable fluorescent protein, its propensity for obligate dimer formation may hinder applications in molecular fusion and membrane targeting. To attain monovalent as well as bright and photostable labeling, we engineered tandem dimers of StayGold to promote dispersibility. On the basis of the crystal structure of this fluorescent protein, we disrupted the dimerization to generate a monomeric variant that offers improved photostability and brightness compared to StayGold. We applied the new monovalent StayGold tools to live-cell imaging experiments using spinning-disk laser-scanning confocal microscopy or structured illumination microscopy. We achieved cell-wide, high-spatiotemporal resolution and sustained imaging of dynamic subcellular events, including the targeting of endogenous condensin I to mitotic chromosomes, the movement of the Golgi apparatus and its membranous derivatives along microtubule networks, the distribution of cortical filamentous actin and the remolding of cristae membranes within mobile mitochondria.

Contribution of the yeast bi-chaperone system in the restoration of the RNA helicase Ded1 and translational activity under severe ethanol stress.

Preexposure to mild stress often improves cellular tolerance to subsequent severe stress. Severe ethanol stress (10% v/v) causes persistent and pronounced translation repression in Saccharomyces cerevisiae. However, it remains unclear whether preexposure to mild stress can mitigate translation repression in yeast cells under severe ethanol stress. We found that the translational activity of yeast cells pretreated with 6% (v/v) ethanol was initially significantly repressed under subsequent 10% ethanol but was then gradually restored even under severe ethanol stress. We also found that 10% ethanol caused the aggregation of Ded1, which plays a key role in translation initiation as a DEAD-box RNA helicase. Pretreatment with 6% ethanol led to the gradual disaggregation of Ded1 under subsequent 10% ethanol treatment in wild-type cells but not in fes1Δhsp104Δ cells, which are deficient in Hsp104 with significantly reduced capacity for Hsp70. Hsp104 and Hsp70 are key components of the bi-chaperone system that play a role in yeast protein quality control. fes1Δhsp104Δ cells did not restore translational activity under 10% ethanol, even after pretreatment with 6% ethanol. These results indicate that the regeneration of Ded1 through the bi-chaperone system leads to the gradual restoration of translational activity under continuous severe stress. This study provides new insights into the acquired tolerance of yeast cells to severe ethanol stress and the resilience of their translational activity.

Yeast Tor complex 1 phosphorylates eIF4E-binding protein, Caf20.

Tor complex 1 (TORC1), a master regulator of cell growth, is an evolutionarily conserved protein kinase within eukaryotic organisms. To control cell growth, TORC1 governs translational processes by phosphorylating its substrate proteins in response to cellular nutritional cues. Mammalian TORC1 (mTORC1) assumes the responsibility of phosphorylating the eukaryotic translation initiation factor 4E (eIF4E)-binding protein 1 (4E-BP1) to regulate its interaction with eIF4E. The budding yeast Saccharomyces cerevisiae possesses a pair of 4E-BP genes, CAF20 and EAP1. However, the extent to which the TORC1-4E-BP axis regulates translational initiation in yeast remains uncertain. In this study, we demonstrated the influence of TORC1 on the phosphorylation status of Caf20 in vivo, as well as the direct phosphorylation of Caf20 by TORC1 in vitro. Furthermore, we found the TORC1-dependent recruitment of Caf20 to the 80S ribosome. Consequently, our study proposes a plausible involvement of yeast's 4E-BP in the efficacy of translation initiation, an aspect under the control of TORC1.

Severe ethanol stress inhibits yeast proteasome activity at moderate temperatures but not at low temperatures.

Since yeast research under laboratory conditions is usually conducted at 25-30°C (moderate temperature range), most of the findings on yeast physiology are based on analyses in this temperature range. Due to inefficiencies in cultivation and analysis, insufficient information is available on yeast physiology in the low-temperature range, although alcoholic beverage production is often conducted at relatively low temperatures (around 15°C). Recently, we reported that severe ethanol stress (10% v/v) inhibits proteasomal proteolysis in yeast cells under laboratory conditions at 28°C. In this study, proteasomal proteolysis at a low temperature (15°C) was evaluated using cycloheximide chase analysis of a short-lived protein (Gic2-3HA), an auxin-inducible degron system (Paf1-AID*-6FLAG), and Spe1-3HA, which is degraded ubiquitin-independently by the proteasome. At 15°C, proteasomal proteolysis was not inhibited under severe ethanol stress, and sufficient proteasomal activity was maintained. These results provide novel insights into the effects of low temperature and ethanol on yeast physiology.

Two coral fluorescent proteins of distinct colors for sharp visualization of cell-cycle progression.

We cloned and characterized two new coral fluorescent proteins: h2-3 and 1-41. h2-3 formed an obligate dimeric complex and exhibited bright green fluorescence. On the other hand, 1-41 formed a highly multimeric complex and exhibited dim red fluorescence. We engineered 1-41 into AzaleaB5, a practically useful red-emitting fluorescent protein for cellular labeling applications. We fused h2-3 and AzaleaB5 to the ubiquitination domains of human Geminin and Cdt1, respectively, to generate a new color variant of Fucci (Fluorescent Ubiquitination-based Cell-Cycle Indicator): Fucci5. We found Fucci5 provided more reliable nuclear labeling for monitoring cell-cycle progression than the 1st and 2nd generations that used mAG/mKO2 and mVenus/mCherry, respectively.Key words: fluorescent protein, cell cycle, time-lapse imaging, flow cytometry.

StayGold photostability under different illumination modes.

StayGold is a bright fluorescent protein (FP) that is over one order of magnitude more photostable than any of the currently available FPs across the full range of illumination intensities used in widefield microscopy and structured illumination microscopy, the latter of which is a widefield illumination-based technique. To compare the photostability of StayGold under other illumination modes with that of three other green-emitting FPs, namely EGFP, mClover3, and mNeonGreen, we expressed all four FPs as fusions to histone 2B in HeLa cells. Unlike the case of widefield microscopy, the photobleaching behavior of these FPs in laser scanning confocal microscopy (LSCM) is complicated. The outstanding photostability of StayGold observed in multi-beam LSCM was variably attenuated in single-beam LSCM, which produces intermittent and instantaneously strong illumination. We systematically examined the effects of different single-beam LSCM beam-scanning patterns on the photostability of the FPs in living HeLa cells. This study offers relevant guidelines for researchers who aim to achieve sustainable live cell imaging by resolving problems related to FP photostability. We also provide evidence for measurable sensitivity of the photostability of StayGold to chemical fixation.

Acquired resistance to severe ethanol stress-induced inhibition of proteasomal proteolysis in Saccharomyces cerevisiae.

BACKGROUND: Although the budding yeast, Saccharomyces cerevisiae, produces ethanol via alcoholic fermentation, high-concentration ethanol is harmful to yeast cells. Severe ethanol stress (> 9% v/v) inhibits protein synthesis and increases the level of intracellular protein aggregates. However, its effect on proteolysis in yeast cells remains largely unknown. METHODS: We examined the effects of ethanol on proteasomal proteolysis in yeast cells through the cycloheximide-chase analysis of short-lived proteins. We also assayed protein degradation in the auxin-inducible degron system and the ubiquitin-independent degradation of Spe1 under ethanol stress conditions. RESULTS: We demonstrated that severe ethanol stress strongly inhibited the degradation of the short-lived proteins Rim101 and Gic2. Severe ethanol stress also inhibited protein degradation in the auxin-inducible degron system (Paf1-AID*-6FLAG) and the ubiquitin-independent degradation of Spe1. Proteasomal degradation of these proteins, which was inhibited by severe ethanol stress, resumed rapidly once the ethanol was removed. These results suggested that proteasomal proteolysis in yeast cells is reversibly inhibited by severe ethanol stress. Furthermore, yeast cells pretreated with mild ethanol stress (6% v/v) showed proteasomal proteolysis even with 10% (v/v) ethanol, indicating that yeast cells acquired resistance to proteasome inhibition caused by severe ethanol stress. However, yeast cells failed to acquire sufficient resistance to severe ethanol stress-induced proteasome inhibition when new protein synthesis was blocked with cycloheximide during pretreatment, or when Rpn4 was lost. CONCLUSIONS AND GENERAL SIGNIFICANCE: Our results provide novel insights into the adverse effects of severe ethanol stress on proteasomal proteolysis and ethanol adaptability in yeast.

A highly photostable and bright green fluorescent protein.

The low photostability of fluorescent proteins is a limiting factor in many applications of fluorescence microscopy. Here we present StayGold, a green fluorescent protein (GFP) derived from the jellyfish Cytaeis uchidae. StayGold is over one order of magnitude more photostable than any currently available fluorescent protein and has a cellular brightness similar to mNeonGreen. We used StayGold to image the dynamics of the endoplasmic reticulum (ER) with high spatiotemporal resolution over several minutes using structured illumination microscopy (SIM) and observed substantially less photobleaching than with a GFP variant optimized for stability in the ER. Using StayGold fusions and SIM, we also imaged the dynamics of mitochondrial fusion and fission and mapped the viral spike proteins in fixed cells infected with severe acute respiratory syndrome coronavirus 2. As StayGold is a dimer, we created a tandem dimer version that allowed us to observe the dynamics of microtubules and the excitatory post-synaptic density in neurons. StayGold will substantially reduce the limitations imposed by photobleaching, especially in live cell or volumetric imaging.

The three-dimensional morphometry and cell-cell communication of the osteocyte network in chick and mouse embryonic calvaria.

The study of osteocytes has progressed in chicks. We examined whether chick osteocyte data can be applied to other species. We used mice for comparison because they are common clinical tools in biomedical research and useful for future study. We analyzed the three-dimensional (3D) osteocyte network and gap junctional intercellular communication (GJIC) in living embryonic calvaria for the anatomical features. Embryonic parietal bones were stained with fluorescently labeled phalloidin and observed using confocal laser scanning microscopy. GJIC between osteocytes in chick and mouse parietal bone was assessed using fluorescence recovery after photobleaching (FRAP). The values for one chick and mouse osteocyte, respectively, were calculated as follows: cell processes 1,131 ± 139 µm, 2,668 ± 596 µm; surface area 1,128 ± 358 µm(2), 2,654 ± 659 µm(2); and cell volume 455 ± 90 µm(3), 1,328 ± 210 µm(3). The density of 3D osteocyte processes in the bone matrix was not significantly different. FRAP analysis showed dye coupling among osteocytes in chick and mouse bone. The fluorescence intensity recovered to 49.0 ± 2.4% in chicks and 39.9 ± 2.4% in mice after 5 minutes. Fluorescence recovery was similar within 4 minutes. The difference in osteocyte size between the two species might have affected their functions. Osteocyte processes in the two species may sense similarly changes in the exterior environment. We successfully conducted morphological and functional analyses of the osteocyte network in chicks and mice. The size of the osteocytes in bone differed between the two species.

Light-dependent regulation of structural flexibility in a photochromic fluorescent protein.

The structural basis for the photochromism in the fluorescent protein Dronpa is poorly understood, because the crystal structures of the bright state of the protein did not provide an answer to the mechanism of the photochromism, and structural determination of the dark state has been elusive. We performed NMR analyses of Dronpa in solution at ambient temperatures to find structural flexibility of the protein in the dark state. Light-induced changes in interactions between the chromophore and beta-barrel are responsible for switching between the two states. In the bright state, the apex of the chromophore tethers to the barrel by a hydrogen bond, and an imidazole ring protruding from the barrel stabilizes the plane of the chromophore. These interactions are disrupted by strong illumination with blue light, and the chromophore, together with a part of the beta-barrel, becomes flexible, leading to a nonradiative decay process.

How to overcome the difficulty of talking about the experience of a nuclear disaster.

At the ICRP International Conference on Recovery After Nuclear Accidents Session 3.4 Forum on the Transmission of Experience held in December 2020, a panel discussion took place on the topic, 'How to overcome the difficulty to talk about the experience of nuclear accidents?'. The facilitator was Ryoko Ando (NPO Fukushima Dialogue) and the following six people participated as panelists: Atsushi Chiba (teacher at Fukushima Prefectural Asaka High School), Yoshiko Aoki (NPO Group of Storytellers About 311 in Tomioka), Miku Endo (Great East Japan Earthquake and Nuclear Disaster Memorial Museum), Kenji Shiga (former Director of Hiroshima Peace Memorial Museum), Thierry Schneider (Centre d'étude sur l'Evaluation de la Protection dans le domaine Nucléaire), and Noboru Takamura (Director of Great East Japan Earthquake and Nuclear Disaster Memorial Museum, Nagasaki University).

As a resident and a counsellor.

At the time of the accident at Fukushima Daiichi nuclear power plant in 2011, I was living in Iwaki City with my two children and my husband. With our home damaged by the tsunami and the deteriorating status following the situation at the nuclear power plant, we spent 2 years as evacuees before returning to Iwaki City. Subsequently, I decided to work as a radiation counsellor in the Suetsugi district of Iwaki City. I would like to describe my experience of taking measurements and helping to communicate with the residents while respecting the lives of local people.

Higher resolution in localization microscopy by slower switching of a photochromic protein.

Photoswitchable fluorophores play an essential role in super-resolution fluorescence microscopy, including techniques such as photoactivated localization microscopy (PALM). A determining factor in the precision of the images generated by PALM measurements is the photon numbers that can be detected from the fluorophores. Dronpa is a reversibly photoswitchable fluorescent protein that has been successfully used in PALM experiments. The number of photons per switching cycle that can be acquired for Dronpa depends on its off-switching rate, limiting the number of photons that can be recorded. In this study we report our discovery that the tetrameric ancestor of Dronpa, 22G, shows slower switching, and develop a mutant that displays switching kinetics between those of Dronpa and 22G. We show that the kinetics of the photoswitching are strongly related to self-association of the protein, supporting our view of dynamic flexibility as determining in the photoswitching. Similarly we find that higher-resolution PALM images can be acquired with slower-switching proteins due to their higher number of emitted photons per switching cycle.

Relationship between orthodontic expertise and perception of treatment needs for maxillary protrusion: comparison of dental students, residents, and orthodontists.

INTRODUCTION: The aims of this study were to assess the peer assessment rating (PAR) index in relation to perceived treatment needs for maxillary protrusion in Japanese subjects and to investigate how perceived orthodontic treatment needs change with increased experience in dentistry and orthodontics. METHODS: The subjects were 155 persons (73 men, 82 women; mean age, 24.2 years; SD, 4.7 years) including dental students, residents, and orthodontists. We showed them casts from 10 patients with untreated maxillary protrusion and gave them a questionnaire with a 100-point visual analog scale (VAS), concerning their perceptions of orthodontic treatment needs. The PAR index was used for cast evaluation. RESULTS: The PAR index showed significant correlations with the VAS scores. On the casts evaluated with a PAR index below 17, there was no difference in VAS scores between the 3 groups; however, orthodontists perceived significantly greater treatment needs than did students and residents on casts with a PAR index of 18 or 19. The VAS scores were significantly increased when casts with a PAR index of more than 20 and overjet above 5.0 mm were evaluated. Orthodontists and residents perceived greater treatment needs than students on assessing occlusions with severe maxillary protrusion. CONCLUSIONS: The PAR index is clinically useful to evaluate malocclusion, and the perception of treatment needs was significantly greater when the maxillary protrusion cast had a PAR index of more than 20 and overjet above 5.0 mm. Perceived needs for orthodontic treatment for maxillary protrusion changed with increasing experience and skills in dentistry and orthodontics.

A method for observing silver-stained osteocytes in situ in 3-microm sections using ultra-high voltage electron microscopy tomography.

Osteocytes are surrounded by hard bone matrix, and it has not been possible previously to directly observe the in situ architecture of osteocyte morphology in bone. Electron microscope tomography, however, is a technique that has the unique potential to provide three-dimensional (3D) visualization of cellular ultrastructure. This approach is based on reconstruction of 3D volumes from a tilt series of electron micrographs of cells, and resolution at the nanometer level has been achieved. We applied electron microscope tomography to thick sections of silver-stained osteocytes in bone using a Hitachi H-3000 ultra-high voltage electron microscope equipped with a 360 degrees tilt specimen holder, at an accelerating voltage of 2 MeV. Osteocytes with numerous processes and branches were clearly seen in the serial tilt series acquired from 3-microm-thick sections. Reconstruction of young osteocytes showed the 3D topographic morphology of the cell body and processes at high resolution. This morphological data on osteocytes should provide useful information to those who study osteocyte physiology and the several models used to explain their mechanosensory properties.

Influence of anteroposterior mandibular positions on facial attractiveness in Japanese adults.

INTRODUCTION: Our aims in this study were to determine the anteroposterior facial relationship that is regarded as most attractive by Japanese laypersons in a questionnaire survey and to evaluate which analysis of the soft-tissue profile is most suitable for Japanese people. METHODS: We showed 262 Japanese laypersons (121 male, 141 female) 9 morphed profile images with Point B and menton anteriorly or distally moved by software and asked them to number them sequentially according to their attractiveness. To examine which analysis best reflects facial attractiveness as judged by laypersons, we made 5 types of analyses of the facial profile with 11 variables in the 9 images. RESULTS: The normal face was judged favorably; however, an attractive profile might be different for each subject. The 3 highest ranking profiles (normal face and moderate mandibular retrusions) were often favorites, and 2 profiles (severe mandibular protrusions) were liked the least for most subjects. However, the other images showed a wide range of distribution. Mandibular retrusion was generally more favored than mandibular protrusion and bimaxillary protrusion (severe chin retrusion) had a high attractiveness ranking and was well accepted in the Japanese population. CONCLUSIONS: To evaluate the profiles of Japanese subjects, it is important to evaluate not only the esthetic line defined by the nose and chin, but also the balance of the upper and lower lips defined by the posterior reference line--ie, Burstone's Sn-Pog' line.

Moments reconstruction and local dynamic range compression of high order superresolution optical fluctuation imaging.

Super-resolution optical fluctuation imaging (SOFI) offers a simple and affordable alternative to other super-resolution (SR) imaging techniques. The theoretical resolution enhancement of SOFI scales linearly with the order of cumulants, while the imaging conditions exhibit less photo-toxicity to the living samples as compared to other SR methods. High order SOFI could, therefore, be a method of choice for dynamic live cell imaging. However, due to the cusp-artifacts and dynamic range expansion of pixel intensities, this promise has not been materialized as of yet. Here we investigated and compared high order moments vs. high order cumulant SOFI reconstructions. We demonstrate that even-order moments reconstructions are intrinsically free of cusp artifacts, allowing for a subsequent deconvolution operation to be performed, hence enhancing the resolution even further. High order moments reconstruction performance was examined for various (simulated) conditions and applied to (experimental) imaging of QD labeled microtubules in fixed cells, and actin stress fiber dynamics in live cells.

Second-harmonic generation in GFP-like proteins.

The second-order nonlinear optical properties of green fluorescent proteins (GFPs), such as the photoswitchable Dronpa and enhanced GFP (EGFP), have been studied at both the theoretical and experimental levels. In the case of Dronpa, both approaches are consistent in showing the rather counterintuitive result of a larger second-order nonlinear polarizability (or first hyperpolarizability, beta) for the protonated state, which has a higher transition energy, than for the deprotonated, fluorescent state with its absorption at lower energy. Moreover, the value of beta for the protonated form of Dronpa is among the highest reported for proteins. In addition to the pH dependence, we have found a wavelength dependence in the beta values. These properties are essential for the practical use of Dronpa or other GFP-like fluorescent proteins as second-order nonlinear fluorophores for symmetry-sensitive nonlinear microscopy imaging and as nonlinear optical sensors for electrophysiological processes. An accurate value of the first hyperpolarizability is also essential for any qualitative analysis of the nonlinear images.