Deep learning-based segmentation of subcellular organelles in high-resolution phase-contrast images.

Although quantitative analysis of biological images demands precise extraction of specific organelles or cells, it remains challenging in broad-field grayscale images, where traditional thresholding methods have been hampered due to complex image features. Nevertheless, rapidly growing artificial intelligence technology is overcoming obstacles. We previously reported the fine-tuned apodized phase-contrast microscopy system to capture high-resolution, label-free images of organelle dynamics in unstained living cells (Shimasaki, K. et al. (2024). Cell Struct. Funct., 49: 21-29). We here showed machine learning-based segmentation models for subcellular targeted objects in phase-contrast images using fluorescent markers as origins of ground truth masks. This method enables accurate segmentation of organelles in high-resolution phase-contrast images, providing a practical framework for studying cellular dynamics in unstained living cells.Key words: label-free imaging, organelle dynamics, apodized phase contrast, deep learning-based segmentation.

A high-resolution phase-contrast microscopy system for label-free imaging in living cells.

Cell biologists have long sought the ability to observe intracellular structures in living cells without labels. This study presents procedures to adjust a commercially available apodized phase-contrast (APC) microscopy system for better visualizing the dynamic behaviors of various subcellular organelles in living cells. By harnessing the versatility of this technique to capture sequential images, we could observe morphological changes in cellular geometry after virus infection in real time without probes or invasive staining. The tune-up APC microscopy system is a highly efficient platform for simultaneously observing the dynamic behaviors of diverse subcellular structures with exceptional resolution.

Chloroplast-actin filaments decide the direction of chloroplast avoidance movement under strong light in Arabidopsis thaliana.

Chloroplast-actin (cp-actin) filaments are crucial for light-induced chloroplast movement, and appear in the front region of moving chloroplasts when visualized using GFP-mouse Talin. They are short and thick, exist between a chloroplast and the plasma membrane, and move actively and rapidly compared to cytoplasmic long actin filaments that run through a cell. The average period during which a cp-actin filament was observed at the same position was less than 0.5 s. The average lengths of the cp-actin filaments calculated from those at the front region of the moving chloroplast and those around the chloroplast periphery after stopping the movement were almost the same, approximately 0.8 µm. Each cp-actin filament is shown as a dotted line consisting of 4-5 dots. The vector sum of cp-actin filaments in a moving chloroplast is parallel to the moving direction of the chloroplast, suggesting that the direction of chloroplast movement is regulated by the vector sum of cp-actin filaments. However, once the chloroplasts stopped moving, the vector sum of the cp-actin filaments around the chloroplast periphery was close to zero, indicating that the direction of movement was undecided. To determine the precise structure of cp-actin filaments under electron microscopy, Arabidopsis leaves and fern Adiantum capillus-veneris gametophytes were frozen using a high-pressure freezer, and observed under electron microscopy. However, no bundled microfilaments were found, suggesting that the cp-actin filaments were unstable even under high-pressure freezing.

CHLOROPLAST UNUSUAL POSITIONING 1 is a plant-specific actin polymerization factor regulating chloroplast movement.

Plants have unique responses to fluctuating light conditions. One such response involves chloroplast photorelocation movement, which optimizes photosynthesis under weak light by the accumulation of chloroplasts along the periclinal side of the cell, which prevents photodamage under strong light by avoiding chloroplast positioning toward the anticlinal side of the cell. This light-responsive chloroplast movement relies on the reorganization of chloroplast actin (cp-actin) filaments. Previous studies have suggested that CHLOROPLAST UNUSUAL POSITIONING 1 (CHUP1) is essential for chloroplast photorelocation movement as a regulator of cp-actin filaments. In this study, we conducted comprehensive analyses to understand CHUP1 function. Functional, fluorescently tagged CHUP1 colocalized with and was coordinately reorganized with cp-actin filaments on the chloroplast outer envelope during chloroplast movement in Arabidopsis thaliana. CHUP1 distribution was reversibly regulated in a blue light- and phototropin-dependent manner. X-ray crystallography revealed that the CHUP1-C-terminal domain shares structural homology with the formin homology 2 (FH2) domain, despite lacking sequence similarity. Furthermore, the CHUP1-C-terminal domain promoted actin polymerization in the presence of profilin in vitro. Taken together, our findings indicate that CHUP1 is a plant-specific actin polymerization factor that has convergently evolved to assemble cp-actin filaments and enables chloroplast photorelocation movement.

The C10orf76-PI4KB axis orchestrates CERT-mediated ceramide trafficking to the distal Golgi.

Phosphatidylinositol 4-monophosphate [PtdIns(4)P] is a precursor for various phosphoinositides but also a membrane-embedded component crucial for membrane contact sites (MCSs). Several lipid transfer proteins are recruited to MCSs by recognizing PtdIns(4)P; however, it remains poorly elucidated how the production of PtdIns(4)P for lipid transport at MCSs is regulated. Following human genome-wide screening, we discovered that the PtdIns(4)P-related genes PI4KB, ACBD3, and C10orf76 are involved in endoplasmic reticulum-to-Golgi trafficking of ceramide by the ceramide transport protein CERT. CERT preferentially utilizes PtdIns(4)P generated by PI4KB recruited to the Golgi by C10orf76 rather than by ACBD3. Super-resolution microscopy observation revealed that C10orf76 predominantly localizes at distal Golgi regions, where sphingomyelin (SM) synthesis primarily occurs, while the majority of ACBD3 localizes at more proximal regions. This study provides a proof-of-concept that distinct pools of PtdIns(4)P are generated in different subregions, even within the same organelle, to facilitate interorganelle metabolic channeling for the ceramide-to-SM conversion.

RNaseH2A downregulation drives inflammatory gene expression via genomic DNA fragmentation in senescent and cancer cells.

Cellular senescence caused by oncogenic stimuli is associated with the development of various age-related pathologies through the senescence-associated secretory phenotype (SASP). SASP is mediated by the activation of cytoplasmic nucleic acid sensors. However, the molecular mechanism underlying the accumulation of nucleotide ligands in senescent cells is unclear. In this study, we revealed that the expression of RNaseH2A, which removes ribonucleoside monophosphates (rNMPs) from the genome, is regulated by E2F transcription factors, and it decreases during cellular senescence. Residual rNMPs cause genomic DNA fragmentation and aberrant activation of cytoplasmic nucleic acid sensors, thereby provoking subsequent SASP factor gene expression in senescent cells. In addition, RNaseH2A expression was significantly decreased in aged mouse tissues and cells from individuals with Werner syndrome. Furthermore, RNaseH2A degradation using the auxin-inducible degron system induced the accumulation of nucleotide ligands and induction of certain tumourigenic SASP-like factors, promoting the metastatic properties of colorectal cancer cells. Our results indicate that RNaseH2A downregulation provokes SASP through nucleotide ligand accumulation, which likely contributes to the pathological features of senescent, progeroid, and cancer cells.

Establishment of Vero cell lines persistently harboring a yellow fever virus 17D subgenomic replicon.

Yellow fever virus (YFV), a member of the genus Flavivirus, family Flaviviridae, is the etiological agent for an acute viral hemorrhagic disease, yellow fever. Although effective live attenuated vaccines based on the strain YFV 17D are currently available, no specific antiviral drug is available, and the disease remains a major public health concern. Hence, the discovery and development of antiviral drugs should lead to great benefits in controlling the disease. To provide a screening platform for antiviral agents targeting YFV RNA translation/replication, we have established and characterized two Vero cell lines that persistently harbor a subgenomic replicon derived from YFV 17D-204 (referred to as replicon cells). The replicon carries YFV nucleotides (1 - 176 and 2382-10,862) and a green fluorescent protein (GFP)-Zeocin resistance fusion gene as a selection marker and indicator of persistent replication. Immunofluorescence analysis revealed that both replicon cells and YFV 17D-infected cells showed similar distribution patterns of viral NS4B protein and replication intermediate, double-stranded RNA. Sequencing analysis of persistent replicons from the two replicon cell lines suggested that their nucleotide sequences did not vary greatly following multiple passages. We examined the effect of five agents, the antiviral cytokines interferon-ß and -γ, the nucleoside analog ribavirin, the squalene synthase inhibitor zaragozic acid A, and the antibiotic rifapentine, a recently reported entry and replication inhibitor against YFV, on the persistent replication in the two replicon cell lines. These agents were selected because they inhibited both production of YFV 17D and transient replication of a luciferase-expressing replicon in Vero cells, without greatly affecting cell viability. We found that each of the agents decreased GFP fluorescence in the replicon cells, albeit to varying degrees. The agents other than rifapentine also showed a decrease in viral RNA levels in the replicon cells comparable to that seen for GFP fluorescence. These results indicate that persistent replication is susceptible to each of these five agents, although their mechanisms of action may differ. Taken together, these results provide evidence that translation/replication of the replicon in the replicon cells mimics that of the viral genome upon YFV 17D infection, indicating that the replicon cell lines can serve as a useful tool for high-throughput antiviral drug screening.

Characterization of phalloidin-negative nuclear actin filaments in U2OS cells expressing cytoplasmic actin-EGFP.

Actin is a major structural component of the cytoskeleton in eukaryotic cells, including fungi, plants, and animals, and exists not only in the cytoplasm as cytoskeleton but also in the nucleus. Recently, we developed a novel actin probe, ß-actin-EGFP fusion protein, which exhibited similar monomeric to filamentous ratio as that of endogenous actin, in contrast to the widely used EGFP-ß-actin fusion protein that over-assembles in cells. Unexpectedly, this novel probe visualized an interconnected meshwork of slightly curved beam-like bundles of actin filaments in the nucleus of U2OS cells. These structures were not labeled with rhodamine phalloidin, Lifeact-EGFP or anti-actin antibodies. In addition, immunofluorescence staining and expression of cofilin-EGFP revealed that this nuclear actin structures contained cofilin. We named these actin filaments as phalloidin-negative intranuclear (PHANIN) actin filaments. Since PHANIN actin filaments could not be detected by general detection methods for actin filaments, we propose that PHANIN actin filaments are different from previously reported nuclear actin structures.

Protein lactylation induced by neural excitation.

Lactate has diverse roles in the brain at the molecular and behavioral levels under physiological and pathophysiological conditions. This study investigates whether lysine lactylation (Kla), a lactate-derived post-translational modification in macrophages, occurs in brain cells and if it does, whether Kla is induced by the stimuli that accompany changes in lactate levels. Here, we show that Kla in brain cells is regulated by neural excitation and social stress, with parallel changes in lactate levels. These stimuli increase Kla, which is associated with the expression of the neuronal activity marker c-Fos, as well as with decreased social behavior and increased anxiety-like behavior in the stress model. In addition, we identify 63 candidate lysine-lactylated proteins and find that stress preferentially increases histone H1 Kla. This study may open an avenue for the exploration of a role of neuronal activity-induced lactate mediated by protein lactylation in the brain.

Internal constraints and arrested relaxation in main-chain nematic elastomers.

Nematic liquid crystal elastomers (N-LCE) exhibit intriguing mechanical properties, such as reversible actuation and soft elasticity, which manifests as a wide plateau of low nearly-constant stress upon stretching. N-LCE also have a characteristically slow stress relaxation, which sometimes prevents their shape recovery. To understand how the inherent nematic order retards and arrests the equilibration, here we examine hysteretic stress-strain characteristics in a series of specifically designed main-chain N-LCE, investigating both macroscopic mechanical properties and the microscopic nematic director distribution under applied strains. The hysteretic features are attributed to the dynamics of thermodynamically unfavoured hairpins, the sharp folds on anisotropic polymer strands, the creation and transition of which are restricted by the nematic order. These findings provide a new avenue for tuning the hysteretic nature of N-LCE at both macro- and microscopic levels via different designs of polymer networks, toward materials with highly nonlinear mechanical properties and shape-memory applications.

Coefficient of variation as an image-intensity metric for cytoskeleton bundling.

The evaluation of cytoskeletal bundling is a fundamental experimental method in the field of cell biology. Although the skewness of the pixel intensity distribution derived from fluorescently-labeled cytoskeletons has been widely used as a metric to evaluate the degree of bundling in digital microscopy images, its versatility has not been fully validated. Here, we applied the coefficient of variation (CV) of intensity values as an alternative metric, and compared its performance with skewness. In synthetic images representing extremely bundled conditions, the CV successfully detected degrees of bundling that could not be distinguished by skewness. On actual microscopy images, CV was better than skewness, especially on variable-angle epifluorescence microscopic images or stimulated emission depletion and confocal microscopy images of very small areas of around 1 µm2. When blur or noise was added to synthetic images, CV was found to be robust to blur but deleteriously affected by noise, whereas skewness was robust to noise but deleteriously affected by blur. For confocal images, CV and skewness showed similar sensitivity to noise, possibly because optical blurring is often present in microscopy images. Therefore, in practical use with actual microscopy images, CV may be more appropriate than skewness, unless the image is extremely noisy.

Wipi3 is essential for alternative autophagy and its loss causes neurodegeneration.

Alternative autophagy is an Atg5/Atg7-independent type of autophagy that contributes to various physiological events. We here identify Wipi3 as a molecule essential for alternative autophagy, but which plays minor roles in canonical autophagy. Wipi3 binds to Golgi membranes and is required for the generation of isolation membranes. We establish neuron-specific Wipi3-deficient mice, which show behavioral defects, mainly as a result of cerebellar neuronal loss. The accumulation of iron and ceruloplasmin is also found in the neuronal cells. These abnormalities are suppressed by the expression of Dram1, which is another crucial molecule for alternative autophagy. Although Atg7-deficient mice show similar phenotypes to Wipi3-deficient mice, electron microscopic analysis shows that they have completely different subcellular morphologies, including the morphology of organelles. Furthermore, most Atg7/Wipi3 double-deficient mice are embryonic lethal, indicating that Wipi3 functions to maintain neuronal cells via mechanisms different from those of canonical autophagy.

Modeling a Microtubule Filaments Mesh Structure from Confocal Microscopy Imaging.

This study introduces a modeling method for a supermolecular structure of microtubules for the development of a force generation material using motor proteins. 3D imaging by confocal laser scanning microscopy (CLSM) was used to obtain 3D volume density data. The density data were then interpreted by a set of cylinders with the general-purpose 3D modeling software Blender, and a 3D network structure of microtubules was constructed. Although motor proteins were not visualized experimentally, they were introduced into the model to simulate pulling of the microtubules toward each other to yield shrinking of the network, resulting in contraction of the artificial muscle. From the successful force generation simulation of the obtained model structure of artificial muscle, the modeling method introduced here could be useful in various studies for potential improvements of this contractile molecular system.

Functional Correlation between Subcellular Localizations of Japanese Encephalitis Virus Capsid Protein and Virus Production.

The flavivirus capsid protein is considered to be essential for the formation of nucleocapsid complexes with viral genomic RNA at the viral replication organelle that appears on the endoplasmic reticulum (ER), as well as for incorporation into virus particles. However, this protein is also detected at the lipid droplet (LD) and nucleolus, and physiological roles of these off-site localizations are still unclear. In this study, we made a series of alanine substitution mutants of Japanese encephalitis virus (JEV) capsid protein that cover all polar and hydrophobic amino acid residues to identify the molecular surfaces required for virus particle formation and for localization at the LD and nucleolus. Five mutants exhibited a defect in the formation of infectious particles, and two of these mutants failed to be incorporated into the subviral particles (SVP). Three mutants lost the ability to localize to the nucleolus, and only a single mutant, with mutations at α2, was unable to localize to the LD. Unlike the cytoplasmic capsid protein, the nucleolar capsid protein was resistant to detergent treatment, and the α2 mutant was hypersensitive to detergent treatment. To scrutinize the relationship between these localizations and viral particle formation, we made eight additional alanine substitution mutants and found that all the mutants that did not localize at the LD or nucleolus failed to form normal viral particles. These results support the functional correlation between LD or nucleolus localization of the flaviviral capsid protein and the formation of infectious viral particles.IMPORTANCE This study is the first to report the comprehensive mutagenesis of a flavivirus capsid protein. We assessed the requirement of each molecular surface for infectious viral particle formation as well as for LD and nucleolar localization and found functional relationships between the subcellular localization of the virus capsid protein and infectious virus particle formation. We developed a system to independently assess the packaging of viral RNA and that of the capsid protein and found a molecular surface of the capsid protein that is crucial for packaging of viral RNA but not for packaging of the capsid protein itself. We also characterized the biochemical properties of capsid protein mutants and found that the capsid protein localizes at the nucleolus in a different manner than for its localization to the LD. Our comprehensive alanine-scanning mutagenesis study will aid in the development of antiflavivirus small molecules that can target the flavivirus capsid protein.

Bacterial Cell Culture at the Single-cell Level Inside Giant Vesicles.

We developed a method for culturing bacterial cells at the single-cell level inside giant vesicles (GVs). Bacterial cell culture is important for understanding the function of bacterial cells in the natural environment. Because of technological advances, various bacterial cell functions can be revealed at the single-cell level inside a confined space. GVs are spherical micro-sized compartments composed of amphiphilic lipid molecules and can hold various materials, including cells. In this study, a single bacterial cell was encapsulated into 10-30 µm GVs by the droplet transfer method and the GVs containing bacterial cells were immobilized on a supported membrane on a glass substrate. Our method is useful for observing the real-time growth of single bacteria inside GVs. We cultured Escherichia coli (E. coli) cells as a model inside GVs, but this method can be adapted to other cell types. Our method can be used in the science and industrial fields of microbiology, biology, biotechnology, and synthetic biology.

Fascin in lamellipodia contributes to cell elasticity by controlling the orientation of filamentous actin.

Fascin, an actin-bundling protein, is present in the filopodia and lamellipodia of growth cones. However, few studies have examined lamellipodial fascin because it is difficult to observe. In this study, we evaluated lamellipodial fascin. We visualized the actin meshwork of lamellipodia in live growth cones by super-resolution microscopy. Fascin was colocalized with the actin meshwork in lamellipodia. Ser39 of fascin is a well-known phosphorylation site that controls the binding of fascin to actin filaments. Fluorescence recovery after photobleaching experiments with confocal microscopy showed that binding of fascin was controlled by phosphorylation of Ser39 in lamellipodia. Moreover, TPA, an agonist of protein kinase C, induced phosphorylation of fascin and dissociation from actin filaments in lamellipodia. Time series images showed that dissociation of fascin from the actin meshwork was induced by TPA. As fascin dissociated from actin filaments, the orientation of the actin filaments became parallel to the leading edge. The angle of actin filaments against the leading edge was changed from 73° to 15°. This decreased the elasticity of the lamellipodia by 40%, as measured by atomic force microscopy. These data suggest that actin bundles made by fascin contribute to elasticity of the growth cone.

Cdk1-dependent phosphorylation of KIF4A at S1186 triggers lateral chromosome compaction during early mitosis.

Chromosome organization during cell division is achieved through the timely association of proteins with chromatin and is regulated by protein phosphorylation. Kinesin family member 4A (KIF4A) plays an important role in the chromosome organization through the formation of the chromosome scaffold structure. However, the relationship between the function of KIF4A and its phosphorylation remains unclear. Here, we demonstrate that Cdk1-dependent phosphorylation of KIF4A at S1186 is required for chromosome binding and chromosome scaffold formation. The KIF4A mutant, which is not phosphorylated at S1186, was found to localize to the nucleus during interphase but did not accumulate in the chromosome scaffold after nuclear envelope breakdown. In addition, defects in KIF4A phosphorylation were found to disrupt the interaction of KIF4A with the condensin I complex. As a result, the morphology of the chromosomes was observed to be laterally decondensed, without condensin I in the chromosome scaffold. Additionally, a defect in chromosome segregation, chromosome bridge formation, was often observed. Although both KIF4A and condensin I disappeared from the chromosomes, the chromosomal localization of condensin II was not affected. Collectively, our novel results revealed that Cdk1-dependent KIF4A phosphorylation at S1186 is a trigger for chromosomal organization during early mitosis.

Direct Observation of Bacterial Growth in Giant Unilamellar Vesicles: A Novel Tool for Bacterial Cultures.

Invited for this month's cover picture are Dr. Masamune Morita, Dr. Kaoru Katoh and Dr. Naohiro Noda from the Biomedical Research Institute at the National Institute of Advanced Industrial Science and Technology (AIST, Japan). The cover picture shows direct monitoring of real-time activity of bacterial growth at the single-cell level inside giant unilamellar vesicles (GUVs); entrapped single bacterial cells are actively increasing to a great number of cells inside GUVs. This study shows new applications for GUVs, and can offer a novel tool for culturing bacteria in bacterial studies. Read the full text of their Communication at https://doi.org/10.1002/open.201800126.

Direct Observation of Bacterial Growth in Giant Unilamellar Vesicles: A Novel Tool for Bacterial Cultures.

Bacterial cultivation techniques are classic, basic, and common processes used to characterize the physiological activity of bacteria in their environment. Owing to recent advances in bacterial cultivation techniques, the physiological activity of bacteria can be elucidated at the single-cell culture level. Here, we report a novel method to monitor the real-time activity of bacterial growth at the single-cell level inside giant unilamellar vesicles (GUVs). This method consists of two steps: 1) encapsulation of single bacteria in 1-33 pL scale GUVs and 2) immobilization of the GUVs on a planar lipid bilayer membrane on a glass surface. We directly observed single E. coli cells actively growing to a great number of cells inside GUVs. GUVs also protected the bacteria from external antibiotic compounds during prolonged cultivation for more than 24 h. This approach can be applied widely in the fields of biochemistry, biotechnology, microbiology, and synthetic biology.

Arabidopsis vegetative actin isoforms, AtACT2 and AtACT7, generate distinct filament arrays in living plant cells.

Flowering plants express multiple actin isoforms. Previous studies suggest that individual actin isoforms have specific functions; however, the subcellular localization of actin isoforms in plant cells remains obscure. Here, we transiently expressed and observed major Arabidopsis vegetative actin isoforms, AtACT2 and AtACT7, as fluorescent-fusion proteins. By optimizing the linker sequence between fluorescent protein and actin, we succeeded in observing filaments that contained these expressed actin isoforms fused with green fluorescent protein (GFP) in Arabidopsis protoplasts. Different colored fluorescent proteins fused with AtACT2 and AtACT7 and co-expressed in Nicotiana benthamiana mesophyll cells co-polymerized in a segregated manner along filaments. In epidermal cells, surprisingly, AtACT2 and AtACT7 tended to polymerize into different types of filaments. AtACT2 was incorporated into thinner filaments, whereas AtACT7 was incorporated into thick bundles. We conclude that different actin isoforms are capable of constructing unique filament arrays, depending on the cell type or tissue. Interestingly, staining patterns induced by two indirect actin filament probes, Lifeact and mTalin1, were different between filaments containing AtACT2 and those containing AtACT7. We suggest that filaments containing different actin isoforms bind specific actin-binding proteins in vivo, since the two probes comprise actin-binding domains from different actin-binding proteins.