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Live cell imaging using fluorescent DNA markers are an indispensable molecular tool in various biological and biomedical fields. It is a challenge to develop DNA probes that avoid UV light photo-excitation, have high specificity for DNA, are cell-permeable and are compatible with cutting-edge imaging techniques such as super-resolution microscopy. Herein, we present N-aryl pyrido cyanine (N-aryl-PC) derivatives as a class of long absorption DNA markers with absorption in the wide range of visible light. The high DNA specificity and membrane permeability allow the staining of both organelle DNA as well as nuclear DNA, in various cell types, including plant tissues, without the need for washing post-staining. N-aryl-PC dyes are also highly compatible with a separation of photon by lifetime tuning method in stimulated emission depletion microscopy (SPLIT-STED) for super-resolution imaging as well as two-photon microscopy for deep tissue imaging, making it a powerful tool in the life sciences. It is challenging to develop DNA probes that allow staining of both organelle and nuclear DNA, are compatible with super resolution imaging and avoid UV-light photo-excitation. The authors overcome these issues with N-aryl pyrido cyanine derivatives showing high DNA specificity and membrane permeability.

Stimulation emission depletion (STED) microscopy enables ultrastructural imaging of organelle dynamics with a high spatiotemporal resolution in living cells. For the visualization of the mitochondrial membrane dynamics in STED microscopy, rationally designed mitochondrial fluorescent markers with enhanced photostability are required. Herein, we report the development of a superphotostable fluorescent labeling reagent with long fluorescence lifetime, whose design is based on a structurally reinforced naphthophosphole fluorophore that is conjugated with an electron-donating diphenylamino group. The combination of long-lived fluorescence and superphotostable features of the fluorophore allowed us to selectively capture the ultrastructures of the mitochondrial cristae with a resolution of ∼60 nm when depleted at 660 nm. This chemical tool provides morphological information of the cristae, which has so far only been observed in fixed cells using electron microscopy. Moreover, this method gives information about the dynamic ultrastructures such as the intermembrane fusion in different mitochondria as well as the intercristae mergence in a single mitochondrion during the apoptosis-like mitochondrial swelling process.

Metabolic distribution of fatty acid to organelles is an essential biological process for energy homeostasis as well as for the maintenance of membrane integrity, and the metabolic pathways are strictly regulated in response to environmental stimuli. Herein, we report a fluorescent fatty acid probe, which bears an azapyrene dye that changes its absorption and emission features depending on the microenvironment polarity of the organelle into which it is transported. Owing to the environmental sensitivity of this dye, the distribution of the metabolically incorporated probe in non-polar lipid droplets, medium-polarity membranes, and the polar aqueous regions, can be visualized in different colors. Based on density scatter plots of the fluorophore, we demonstrate that the degradation of triacylglycerols in lipid droplets occurs predominantly via lipolysis rather than lipophagy in nutrition-starved hepatocytes. This tool can thus be expected to significantly advance our understanding of the lipid metabolism in living organisms. Metabolic distribution of fatty acids to organelles is an essential biological process for energy homeostasis. Here the authors report a fluorescent probe that allows multicolour visualisation of the intracellular distribution of exogenous fatty acids, metabolically incorporated as lipid components.

The nuclear lamina plays an important role in the regulation of chromatin organization and gene positioning in animals. CROWDED NUCLEI (CRWN) is a strong candidate for the plant nuclear lamina protein in Arabidopsis thaliana but its biological function was largely unknown. Here, we show that CRWNs localize at the nuclear lamina and build the meshwork structure. Fluorescence in situ hybridization and RNA-seq analyses revealed that CRWNs regulate chromatin distribution and gene expression. More than 2000 differentially expressed genes were identified in the crwn1crwn4 double mutant. Copper-associated ( CA ) genes that form a gene cluster on chromosome 5 were among the downregulated genes in the double mutant exhibiting low tolerance to excess copper. Our analyses showed this low tolerance to copper was associated with the suppression of CA gene expression and that CRWN1 interacts with the CA gene locus, enabling the locus to localize at the nuclear lamina under excess copper conditions. The nuclear lamina regulates chromatin organization and gene positioning. Here the authors show that CROWDED NUCLEI proteins contribute to the meshwork lamina structure in Arabidopsis nuclei and regulate copper tolerance by promoting lamina association and expression of copper response genes.

C 4 plants have C 4 photosynthetic cycle, a CO 2 -concentrating pump that functions across mesophyll (M) and bundle sheath (BS) cells. M chloroplasts aggregate toward BS cells in response to environmental stress, which would contribute to adjustment in C 4 photosynthetic cycle. However, it remains unclear whether M chloroplast movement is an intercellular response mediated by BS cells. One major challenge to resolve this is the difficulty in observing chloroplast movement due to scattering and absorption of observation light in live-leaf tissues. We established a live leaf-section imaging technique that enables the long-term observation of sections of chemically unfixed leaf blades, with which we quantitatively analyzed M chloroplast movements. Another challenge in clarifying the contribution of BS cells to M chloroplast movement is the selective ablation of BS cells without impairing M cell function. To investigate the necessity of BS cells for M chloroplast movement, we developed a method to remove BS cells only based on differences in shape between M and BS cells. We also found that chloroplasts in M cells without adjacent BS cell contents did not show typical aggregative movement. This indicates that the M chloroplast aggregative movement occurs during communication with BS cells.

Mitochondrial DNA (mtDNA) is organized in nucleoprotein complexes called mitochondrial nucleoids (mt-nucleoids), which are critical units of mtDNA replication and transmission. In humans, several hundreds of mt-nucleoids exist in a cell. However, how numerous mt-nucleoids are maintained during the cell cycle remains elusive, because cell cycle synchronization procedures affect mtDNA replication. Here, we analyzed regulation of the maintenance of mt-nucleoids in the cell cycle, using a fluorescent cell cycle indicator, Fucci2. Live imaging of mt-nucleoids with higher temporal resolution showed frequent attachment and detachment of mt-nucleoids throughout the cell cycle. TFAM, an mtDNA packaging protein, was involved in the regulation of this dynamic process, which was important for maintaining proper mt-nucleoid number. Both an increase in mt-nucleoid number and activation of mtDNA replication occurred during S phase. To increase mt-nucleoid number, mtDNA replication, but not nuclear DNA replication, was necessary. We propose that these dynamic and regulatory processes in the cell cycle maintain several hundred mt-nucleoids in proliferating cells.

Tip-growing fungal cells maintain cell polarity at the apical regions and elongate by de novo synthesis of the cell wall. Cell polarity and tip growth rate affect mycelial morphology. Tip-growing fungal cells maintain cell polarity at the apical regions and elongate by de novo synthesis of the cell wall. Cell polarity and tip growth rate affect mycelial morphology. However, it remains unclear how both features act cooperatively to determine cell shape. Here, we investigated this relationship by analyzing hyphal tip growth of filamentous fungi growing inside extremely narrow 1 μm-width channels of microfluidic devices. Since the channels are much narrower than the diameter of hyphae, any hypha growing through the channel must adapt its morphology. Live-cell imaging analyses revealed that hyphae of some species continued growing through the channels, whereas hyphae of other species often ceased growing when passing through the channels, or had lost apical polarity after emerging from the other end of the channel. Fluorescence live-cell imaging analyses of the Spitzenkörper, a collection of secretory vesicles and polarity-related proteins at the hyphal tip, in Neurospora crassa indicates that hyphal tip growth requires a very delicate balance of ordered exocytosis to maintain polarity in spatially confined environments. We analyzed the mycelial growth of seven fungal species from different lineages, including phytopathogenic fungi. This comparative approach revealed that the growth defects induced by the channels were not correlated with their taxonomic classification or with the width of hyphae, but, rather, correlated with the hyphal elongation rate. This report indicates a trade-off between morphological plasticity and velocity in mycelial growth and serves to help understand fungal invasive growth into substrates or plant/animal cells, with direct impact on fungal biotechnology, ecology, and pathogenicity. IMPORTANCE Cell morphology, which is controlled by polarity and growth, is fundamental for all cellular functions. However how polarity and growth act cooperatively to control cell shape remains unclear. Here we investigated their relationship by analyzing hyphal tip growth of filamentous fungi growing inside extremely narrow 1 μm-width channels of microfluidic devices. We found that most fast growing hyphae often lost the cell polarity after emerging from the channels, whereas slow growing hyphae retained polarity and continued growing, indicating a trade-off between plasticity and velocity in mycelial growth. These results serve to understand fungal invasive growth into substrates or plant/animal cells, with direct impact on fungal biotechnology, ecology and pathogenicity.

This study highlights the critical role of hyphal plasticity and cell wall remodeling in the pathogenicity of Fusarium oxysporum , a major plant pathogen affecting over a hundred crops. The ability of fungal hyphae to traverse narrow plant tissue spaces, such as apoplastic gaps and plasmodesmata, is essential for successful host colonization. The research demonstrates that the Mpk1 mitogen-activated protein kinase (MAPK) pathway, responsible for cell wall integrity, is crucial for hyphal growth through microspaces, while other MAPK pathways are not. Staining with specific probes showed a reduction in both chitin and glucan content in the cell wall of hyphae growing within 1 µm width channels. Both the cell wall integrity mutants impaired in microchannel passage, as well as the invasive growth mutants unable to penetrate cellophane membranes, caused significantly reduced mortality in tomato plants. Our study reveals that two morphogenetic processes essential for plant infection, which are governed by distinct cellular pathways, contribute independently to fungal phytopathogenicity.

The precise onset of flowering is crucial for successful reproduction. In longer days, the florigen gene FLOWERING LOCUS T ( FT ) is induced in specific leaf phloem companion cells in Arabidopsis . However, the molecular nature of these cells remains elusive. Here, we utilized bulk nuclei RNA-seq and single nuclei RNA (snRNA)-seq to investigate transcription in FT -expressing cells and other companion cells. Our bulk nuclei RNA-seq demonstrated that FT -expressing cells in cotyledons and true leaves showed differences in FT repressor gene expression. Within true leaves, our snRNA-seq analysis revealed that companion cells with high FT expression form a unique cluster. The cluster expresses other genes encoding small proteins, including the flowering and stem growth inducer FPF1-LIKE PROTEIN 1 (FLP1) and the anti-florigen BROTHER OF FT AND TFL1 (BFT). We also found that the promoters of FT and the genes co-expressed with FT in the cluster were enriched for the binding motif of NITRATE-INDUCIBLE GARP-TYPE TRANSCRIPTIONAL REPRESSOR 1 (NIGT1). Overexpression of NIGT1.2 and NIGT1.4 repressed FT and delayed flowering under nitrogen-rich conditions, implying the roles of NIGT1s as nitrogen-dependent FT repressors. Taken together, our results indicate that unique FT -expressing phloem cells may produce multiple systemic signals to regulate plant growth and development.

Key message Mitochondria change their distribution from nuclear peripheral to uniformly distributed in cytoplasm during zygotic development of rice, and the mitochondria re-distribute around nucleus for even segregation into daughter cells. Mitochondria are highly dynamic organelles that actively move and change their localization along with actin filaments during the cell cycle. Studies of mitochondrial dynamics and distribution in plant cells have mainly been conducted on somatic cells, and our understanding about these aspects during the formation and development of zygotes remains limited. In this study, mitochondrial nucleoids of rice egg cells and zygotes were successfully stained by using N-aryl pyrido cyanine 3 (PC3), and their intracellular localization and distribution were demonstrated. Mitochondria in rice egg cells were small and coccoid in shape and were primarily distributed around the nucleus. Upon gamete fusion, the resulting zygotes showed mitochondrial dispersion and accumulation equivalent to those in rice egg cells until 8 h after fusion (HAF). Around 12 HAF, the mitochondria started to disperse throughout the cytoplasm of the zygotes, and this dispersive distribution pattern continued until the zygotes entered the mitotic phase. At early prophase, the mitochondria redistributed from dispersive to densely accumulated around the nucleus, and during the metaphase and anaphase, the mitochondria were depleted from possible mitotic spindle region. Thereafter, during cell plate formation between daughter nuclei, the mitochondria distributed along the phragmoplast, where the new cell wall was formed. Finally, relatively equivalent amounts of mitochondria were detected in the apical and basal cells which were produced through asymmetric division of the zygotes. Further observation by treating the egg cell with latrunculin B revealed that the accumulation of mitochondria around the nuclear periphery in egg cells and early zygotes depended on the actin meshwork converging toward the egg or zygote nucleus.