Biology subjects

Sato, Y.

Publications and source records attributed to Sato, Y..

At least 73 records · Page 4Linked to original sources

bioRxiv2024

Development of bright fluorescent auxin

Polar transport of the phytohormone auxin plays a crucial role in plant growth and response to environmental stimuli. Small-molecule tools that visualize auxin distribution in intact plants enable us to understand how plants dynamically regulate auxin transport to modulate growth. In this study, we developed a new fluorescent auxin probe, BODIPY-IAA2, which effectively visualizes auxin distribution in various plant tissues. We designed this probe to be transported by auxin transporters while lacking the ability to elicit auxin signaling. Using BODIPY as the fluorophore provides bright and stable fluorescence signals, making it suitable for live-imaging under standard fluorescent microscopy. We tested the probe with auxin reporter lines in Arabidopsis and performed yeast two-hybrid assays. The results showed that BODIPY-IAA2 did not activate auxin signaling through the auxin receptor TIR1. However, BODIPY-IAA2 did mildly compete with both exogenous and endogenous auxins for transport, indicating that the probe is transported by auxin transporters in vivo. The probe not only enables visualization of its tissue distribution but also allows sub-cellular staining, including the endoplasmic reticulum and tip regions in elongating cells in moss. We also observed unusual staining patterns in the main root of non-model parasitic plants where genetic transformation is not feasible. Our new fluorescent auxin probe demonstrates significant potential for detailed studies on auxin transport and distribution across diverse plant species.

plant biology↗

bioRxiv2024

Apusomonad photophobic behavior highlights cytoskeletal responses to blue light in early eukaryotes

Light response is a fundamental characteristic of eukaryotic organisms, both unicellular and multicellular. However, no photoresponse has previously been reported in Apusomonadida, a group of small, free-living biflagellates phylogenetically positioned as a sister group to Opisthokonta (animals, fungi, and their unicellular relatives). Apusomonads are thus crucial for understanding the evolution of opisthokonts. Here, we report for the first time an avoidance response to blue light in the apusomonad Podomonas kaiyoae. This avoidance response is accompanied by an increase in gliding velocity, transient changes in flagellar waveforms, and alterations in cell shape. Dynamic cell contraction is induced by an increase in intracellular free Ca2+ and is inhibited by either a dynein inhibitor or an actin-disrupting drug. These findings suggest that the photophobic behavior of Podomonas kaiyoae relies on cytoskeletal responses mediated by both the dynein/tubulin and myosin/actin systems, which were acquired early in eukaryotic evolution. The dominance of the posterior flagellum in cilia-driven directional changes further supports the phylogenetic placement of apusomonads as a sister group to opisthokonts rather than other eukaryotic lineages.

evolutionary biology↗

bioRxiv2024

Automated non-invasive laser speckle imaging of the chick heart rate and extraembryonic blood vessels and their response to nifedipine and amlodipine drugs

Using our recently developed laser speckle contrast imaging (LSCI) to visualize blood vessels and monitor blood flow, here we test the utility of the chick embryo for drug screening. To this end, we examined the effects of antihypertensive agents Nifedipine and Amlodipine, belonging to the L-type calcium channel antagonist family, on blood flow visualized noninvasively through the intact shell. Guided by the live view mode, the drugs were injected through the shell and ventral to HH16-19 chick embryos. Our results show a significant reduction in the chick heart rate, blood flow, and vascular size within 5-20 minutes after Nifedipine or Amlodipine injection. For moderate Nifedipine concentrations, these parameters returned to initial values within 2-3 hours. In contrast, Amlodipine showed a rapid reduction in heart rate and blood flow dynamics at a more than ten times higher concentration than Nifedipine. These findings show that our LSCI system can monitor and distinguish the chick hearts response to injected drugs from the same family. This serves as proof-of-concept, paving the way for a rapid, cost effective, and quantitative test system for screening drugs that affect the cardiovascular system of live chick embryos. Live noninvasive imaging may also provide insights into the development and functioning of the vertebrate heart. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=136 SRC="FIGDIR/small/609812v1_ufig1.gif" ALT="Figure 1"> View larger version (43K): org.highwire.dtl.DTLVardef@c22831org.highwire.dtl.DTLVardef@dcab67org.highwire.dtl.DTLVardef@1792bf3org.highwire.dtl.DTLVardef@346fb0_HPS_FORMAT_FIGEXP M_FIG C_FIG HighlightsO_LINon-invasive Laser Speckle Contrast Imaging (LSCI) of the chick chorioallantoic membrane (CAM) in whole incubated eggs C_LIO_LISimultaneous recording images of the CAM, dynamics of blood flow, and heart rate C_LIO_LILive view mode to identify size, heart position, and location of the embryo in the egg C_LIO_LIAutomated system for data acquisition and analysis C_LIO_LILongitudinal quantification of the impact of a calcium channel antagonists, nifedipidine and amlodipine on the embryonic heart rate, CAMs blood flow, size and number of vessels C_LI

bioengineering↗

bioRxiv2024

A florigen-expressing subpopulation of companion cells expresses other small proteins and reveals a nitrogen-sensitive FT repressor

The precise onset of flowering is crucial to ensure successful plant reproduction. The gene FLOWERING LOCUS T (FT) encodes florigen, a mobile signal produced in leaves that initiates flowering at the shoot apical meristem. In response to seasonal changes, FT is induced in phloem companion cells located in distal leaf regions. Thus far, a detailed molecular characterization of the FT-expressing cells has been lacking. Here, we used bulk nuclei RNA-seq and single nuclei RNA (snRNA)-seq to investigate gene expression in FT-expressing cells and other phloem companion cells. Our bulk nuclei RNA-seq demonstrated that FT-expressing cells in cotyledons and true leaves showed differences especially in FT repressor genes. Within the true leaves, our snRNA-seq analysis revealed that companion cells with high FT expression form a unique cluster in which many genes involved in ATP biosynthesis are highly upregulated. The cluster also 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). In addition, we found that the promoters of FT and the genes co-expressed with FT in the cluster were enriched for the consensus binding motifs of NITRATE-INDUCIBLE GARP-TYPE TRANSCRIPTIONAL REPRESSOR 1 (NIGT1). Overexpression of the paralogous NIGT1.2 and NIGT1.4 repressed FT expression and significantly delayed flowering under nitrogen-rich conditions, consistent with NIGT1s acting as nitrogen-dependent FT repressors. Taken together, our results demonstrate that major FT-expressing cells show a distinct expression profile that suggests that these cells may produce multiple systemic signals to regulate plant growth and development.

plant biology↗

bioRxiv2024

Decoding the Centromeric Region with a Near Complete Genome Assembly of the Oshima Cherry Cerasus speciosa

The Oshima cherry (Cerasus speciosa), which is endemic to Japan, has significant cultural and horticultural value. In this study, we present a near complete telomere-to-telomere genome assembly for C. speciosa, derived from the old growth "Sakurakkabu" tree on Izu Oshima Island. Using Illumina short-read, PacBio long-read, and Hi-C sequencing, we constructed a 269.3 Mbp genome assembly with a contig N50 of 32.0 Mbp. We examined the distribution of repetitive sequences in the assembled genome and identified regions that appeared to be centromeric. Detailed structural analysis of these putative centromeric regions revealed that the centromeric regions of C. speciosa comprised repetitive sequences with monomer lengths of 166 or 167 bp. Comparative genomic analysis with Prunus sensu lato genome revealed structural variations and conserved syntenic regions. This high-quality reference genome provides a crucial tool for studying the genetic diversity and evolutionary history of Cerasus species, facilitating advancements in horticultural research and the preservation of this iconic species.

genomics↗

bioRxiv2024

Defective interferon signaling in the circulating monocytes of type 2 diabetic mice

Type 2 diabetes mellitus (T2DM) is associated with poor outcome after stroke. Peripheral monocytes play a critical role in the secondary injury and recovery of damaged brain tissue after stroke, but the underlying mechanisms are largely unclear. To investigate transcriptome changes and molecular networks across monocyte subsets in response to T2DM and stroke, we performed single-cell RNA-sequencing (scRNAseq) from peripheral blood mononuclear cells and bulk RNA-sequencing from blood monocytes from four groups of adult mice, consisting of T2DM model db/db and normoglycemic control db/+ mice with or without ischemic stroke. Via scRNAseq we found that T2DM expands the monocyte population at the expense of lymphocytes, which was validated by flow cytometry. Among the monocytes, T2DM also disproportionally increased the inflammatory subsets with Ly6C+ and negative MHC class II expression (MO.6C+II-). Conversely, monocytes from control mice without stroke are enriched with steady-state classical monocyte subset of MO.6C+II+ but with the least percentage of MO.6C+II- subtype. Apart from enhancing inflammation and coagulation, enrichment analysis from both scRNAseq and bulk RNAseq revealed that T2DM specifically suppressed type-1 and type-2 interferon signaling pathways crucial for antigen presentation and the induction of ischemia tolerance. Preconditioning by lipopolysaccharide conferred neuroprotection against ischemic brain injury in db/+ but not in db/db mice and coincided with a lesser induction of brain Interferon-regulatory-factor-3 in the brains of the latter mice. Our results suggest that the increased diversity and altered transcriptome in the monocytes of T2DM mice underlie the worse stroke outcome by exacerbating secondary injury and potentiating stroke-induced immunosuppression. Significance StatementThe mechanisms involved in the detrimental diabetic effect on stroke are largely unclear. We show here, for the first time, that peripheral monocytes have disproportionally altered the subsets and changed transcriptome under diabetes and/or stroke conditions. Moreover, genes in the IFN-related signaling pathways are suppressed in the diabetic monocytes, which underscores the immunosuppression and impaired ischemic tolerance under the T2DM condition. Our data raise a possibility that malfunctioned monocytes may systemically and focally affect the host, leading to the poor outcome of diabetes in the setting of stroke. The results yield important clues to molecular mechanisms involved in the detrimental diabetic effect on stroke outcome.

neuroscience↗

bioRxiv2024

Evolutionary conserved cis-trans regulation machinery for diterpenoid phytoalexin production in Poaceae

O_LIMomilactones and phytocassanes are diterpenoid phytoalexins involved in plant chemical defense. These metabolites, along with biosynthetic gene clusters (BGCs), are conserved in wild rice. However, the mechanisms by which phytoalexins are regulated in wild rice are unclear. Thus, we aimed to investigate the regulatory mechanisms for biosynthetic genes within the BGCs of diterpenoid phytoalexins. C_LIO_LIWe conducted a transcriptome analysis of five wild rice species, Oryza rufipogon, Oryza punctata, Oryza officinalis, Oryza brachyantha, and Leersia perrieri, after CuCl2 treatment. C_LIO_LIAmong the CuCl2-responsive transcription factors, diterpenoid phytoalexin factor (DPF), which regulates phytoalexin production in cultivated rice (Oryza sativa), was broadly conserved in wild rice and showed phytoalexin-inducing activity when introduced into cultivated rice. Highly conserved genomic regions containing N-boxes (5'-CACGAG-3'), the potential binding motif of DPF, were found. CRISPR/Cas9 genome editing to remove these regions showed that biosynthetic gene expression and phytoalexin production were significantly attenuated after CuCl2 treatment in the leaves of the edited plants. Thus, the cis-trans factor combination of DPF and N-boxes is a key determinant of regulation. C_LIO_LIDPF has evolved as a strong cis-trans regulatory system for diterpenoid phytoalexin production, with N-boxes generated within the cluster region during the evolution from wild rice to cultivated rice. C_LI

plant biology↗

bioRxiv2024

Florigen-producing cells express FPF1-LIKE PROTEIN 1 that accelerates flowering and stem growth in long days with sunlight red/far-red ratio in Arabidopsis

Seasonal changes in spring induce flowering by expressing the florigen, FLOWERING LOCUS T (FT), in Arabidopsis. FT is expressed in unique phloem companion cells with unknown characteristics. The question of which genes are co-expressed with FT and whether they have roles in flowering remains elusive. Through tissue-specific translatome analysis, we discovered that under long-day conditions with the natural sunlight red/far-red ratio, the FT-producing cells express a gene encoding FPF1-LIKE PROTEIN 1 (FLP1). The master FT regulator, CONSTANS (CO), controls FLP1 expression, suggesting FLP1s involvement in the photoperiod pathway. FLP1 promotes early flowering independently of FT, is active in the shoot apical meristem, and induces the expression of SEPALLATA 3 (SEP3), a key E-class homeotic gene. Unlike FT, FLP1 facilitates inflorescence stem elongation. Our cumulative evidence indicates that FLP1 may act as a mobile signal. Thus, FLP1 orchestrates floral initiation together with FT and promotes inflorescence stem elongation during reproductive transitions.

plant biology↗

bioRxiv2024

The uptake of small extracellular vesicles by recipient cells is facilitated by paracrine adhesion signaling

Small extracellular vesicles (sEVs) play crucial roles in intercellular communication. However, the internalization of individual sEVs by recipient cells has not yet been directly observed. Here, we examined these mechanisms using state-of-the-art imaging techniques. Single-molecule imaging showed that tumor-derived sEVs can be classified into several subtypes. Simultaneous single-sEV particle tracking and observation of super-resolution movies of membrane invaginations in living cells revealed that all sEV subtypes were internalized via clathrin-independent endocytosis mediated by galectin-3 and LAMP-2C, while some subtypes that recruited raft markers were internalized through caveolae. Integrin {beta}1 and talin-1 accumulated in recipient cell plasma membranes beneath all sEV subtypes. Paracrine, but not autocrine, sEV binding triggered Ca2+ mobilization induced by the activation of Src family kinases and PLC{gamma}. Subsequent Ca2+-induced activation of calcineurin-dynamin promoted sEV internalization, leading to the recycling pathway. Thus, we have clarified the detailed mechanisms of sEV internalization driven by paracrine adhesion signaling.

cell biology↗

bioRxiv2024

Visualizing histone H4K20me1 in knock-in mice expressing the mCherry-tagged modification-specific intracellular antibody

During development and differentiation, histone modifications dynamically change locally and globally, associated with transcriptional regulation, DNA replication and repair, and chromosome condensation. The level of histone H4 Lys20 monomethylation (H4K20me1) increases during the G2 to M phases of the cell cycle and is enriched in facultative heterochromatin, such as inactive X chromosomes in cycling cells. To track the dynamic changes of H4K20me1 in living cells, we have developed a genetically encoded modification-specific intracellular antibody (mintbody) probe that specifically binds to the modification. Here, we report the generation of knock-in mice in which the coding sequence of the mCherry-tagged version of the H4K20me1-mintbody is inserted into the Rosa26 locus. The knock-in mice, which ubiquitously expressed the H4K20me1-mintbody, developed normally and were fertile, indicating that the expression of the probe does not disturb the cell growth, development, or differentiation. Various tissues isolated from the knock-in mice exhibited nuclear fluorescence without the need for fixation. The H4K20me1-mintbody was enriched in inactive X-chromosomes in developing embryos and in XY bodies during spermatogenesis. The knock-in mice will be useful for the histochemical analysis of H4K20me1 in any cell types.

cell biology↗

bioRxiv2023

Multiple HP1 binding motifs in SUV420H2 heterochromatic targeting module

Constitutive heterochromatin is transcriptionally repressed and densely packed chromatin, typically harboring histone H3 Lys9 trimethylation (H3K9me3) and heterochromatin protein 1 (HP1). SUV420H2, a histone H4 Lys20 methyltransferase, is recruited to heterochromatin by binding to HP1 through its Heterochromatic Targeting Module (HTM). Here, we have identified three HP1 binding motifs within the HTM. Both the full-length HTM and its N-terminal region (HTM-N), which contains the first and second motifs, stabilized HP1 on heterochromatin. The intervening region between the first and second HP1 binding motifs in HTM-N was also crucial for HP1 binding. In contrast, the C-terminal region of HTM (HTM-C), containing the third motif, destabilized HP1 on chromatin. An HTM V374D mutant, featuring a Val374 to Asp substitution in the second HP1 binding motif, localizes to heterochromatin without affecting HP1 stability. These data suggest that the second HP1 binding motif in the SUV420H2 HTM is critical for locking HP1 on H3K9me3-enriched heterochromatin. HTM V374D, tagged with a fluorescent protein, can serve as a live-cell probe to visualize HP1-bound heterochromatin.

cell biology↗

bioRxiv2023

The role of desmoplakin for neuronal function in the dentate gyrus and anxiety-related behavior

Desmoplakin (Dsp) is a component of desmosomal cell-cell junctions that interacts with the cadherin complex and cytoskeletal intermediate filaments. In addition to its function as an adhesion component, Dsp is involved in various biological processes, such as gene expression, differentiation, and migration. Dsp is specifically expressed in the hippocampal dentate gyrus (DG) in the central nervous system. However, it is unclear how Dsp impacts hippocampal function and its related behaviors. Using an adeno-associated virus knockdown system in mice, we provide evidence that Dsp in the DG maintains hippocampal functions, including neuronal activity and adult neurogenesis, and contributes to anxiolytic-like effects. Dsp protein is mostly localized in mature granule cells in the adult DG. Dsp knockdown in the DG resulted in a lowered expression of an activity-dependent transcription factor FosB, and an increased expression of mature neuronal markers, such as calbindin. In addition, the suppression of Dsp decreases serotonin responsiveness at the DG output mossy fiber synapses and alters adult neurogenic processes in the subgranular zone of the DG. Moreover, DG- specific Dsp knockdown mice showed an increase in anxiety-like behaviors. Taken together, this research uncovers an unexplored function for Dsp in the central nervous system and suggests that Dsp in the DG may function as a regulator to maintain proper neuronal activation and adult neurogenesis, and contribute to the adaptation of emotion-related behavior.

neuroscience↗

bioRxiv2023

Activity-regulated micro-exon splicing programs underlie late-onset plasticity at the axon initial segment

The axon initial segment (AIS) is a specialized neuronal compartment located at the proximal end of axons and initiates action potentials. AIS undergoes plastic changes with aging, disease, and activity levels; however, the molecular mechanisms underlying their plasticity remain unclear. We discovered that depolarization induces diffuse elongation of the AIS in cerebellar granule cells over the span of days via the Ca2+-dependent ERK/MAP kinase pathway. These structural changes were accompanied by a decrease in voltage-gated Na+ channel density, resulting in a homeostatic attenuation in neuronal excitability. Notably, we found that the late-onset AIS plasticity is associated with depolarization-induced alternative splicing of smaller exons (<100 nt) of transcripts encoding AIS-enriched proteins. In addition, depolarization-induced the skipping of the 53-nt exon19 from the transcript of the splicing protein Rbfox1. CRISPR-mediated removal of exon 19 from Rbfox1 promoted its nuclear localization and sequentially induced a series of downstream micro-exon splicing changes in several AIS proteins, recapitulating cerebellar AIS plasticity. In a Rbfox1-independent mechanism, depolarization-induced insertion of the developmentally regulated micro-exon 34 into the key AIS scaffolding protein Ankyrin G (AnkG). The constitutive insertion of exon 34 into AnkG disrupted its interaction with the AIS cytoskeletal protein {beta}IV spectrin and induced plastic changes in the AIS. Our findings provide fundamental mechanistic insights into the activity-mediated late-onset plasticity of AIS, highlighting the power of micro-scale splicing events in the homeostatic regulation of axonal remodeling.

neuroscience↗

bioRxiv2023

ISWI chromatin remodeling complexes recruit NSD2 and H3K36me2 in pericentromeric heterochromatin

Histone H3 lysine36 dimethylation (H3K36me2) is generally distributed in the gene body and euchromatic intergenic regions. However, we found that H3K36me2 is enriched in pericentromeric heterochromatin in some mouse cell lines. We here revealed the mechanism of heterochromatin targeting of H3K36me2. Among several H3K36 methyltransferases, NSD2 was responsible for inducing heterochromatic H3K36me2. Depletion and overexpression analyses of NSD2-associating proteins revealed that NSD2 recruitment to heterochromatin was mediated through the imitation switch (ISWI) chromatin remodeling complexes, such as BAZ1B-SMARCA5 (WICH), which directly binds to AT-rich DNA via a BAZ1B domain containing AT-hook-like motifs. The abundance and stoichiometry of NSD2, SMARCA5, and BAZ1B or BAZ2A could determine the localization of H3K36me2 in different cell types. To explore the physiological role of heterochromatic H3K36me2, we analyzed mouse tissues and embryos. As a result, H3K36me2 was found in heterochromatin at the 2- to 4-cell stages of mouse preimplantation embryos, suggesting its involvement in developmental regulation. SummaryThe authors discovered histone H3K36me2, which is believed to be enriched in potentially active genomic regions, is also located in transcriptionally inactive regions called heterochromatin in some cell types. The detailed molecular mechanism of its heterochromatin targeting is now revealed.

cell biology↗

bioRxiv2023

High genetic diversification in a symbiotic marine annelid is driven by microgeography and glaciation

Marine invertebrates with limited dispersal abilities exhibit high levels of genetic divergence among populations. However, the spatial extent of genetic differentiation in these species remains poorly understood because identifying natural barriers to gene flow can be challenging in the marine environment. In this study, we investigated the population genetic structure of the interstitial annelid Olavius algarvensis, a species that lays eggs in its immediate surroundings and does not have an active dispersal phase. We analyzed the mitochondrial and nuclear genome sequences of hundreds to thousands of individuals from eleven sites in the Mediterranean, spanning microgeographic scales of < 5 km to macrogeographic scales of 800 km. Comparisons of single nucleotide polymorphisms (SNPs) in mitochondrial genomes revealed a complex history of introgression events, with as many as six mitochondrial lineages co-occurring in individuals from the same site. In contrast, SNP analyses of nuclear genomes revealed clear genetic differentiation at micro- and macrographic scales, characterised by a significant isolation by distance pattern (IBD). IBD patterns further indicated the presence of a historical physical barrier to gene flow on the east coast of the island of Elba corresponding to the historical shoreline around Elba during the Last Glacial Maximum in the Late Pleistocene, and highlighting the influence of geological forces in shaping population genetic structuring in the species today. Overall, our results provide strong empirical evidence for the high genomic diversification across spatial scales in marine interstitial fauna.

evolutionary biology↗

bioRxiv2023

Micronucleus Is Not a Potent Inducer of cGAS-STING Pathway

Micronucleus (MN) has been associated with the innate immune response. The abrupt rupture of MN membranes results in the accumulation of cGAS, potentially activating STING and downstream interferon-responsive genes. However, direct evidence connecting MN and cGAS activation has been lacking. We have developed the FuVis2 reporter system, which enables the visualization of cell nucleus carrying a single sister chromatid fusion and, consequently, MN. Using this FuVis2 reporter equipped with cGAS and STING reporters, we rigorously assessed the potency of cGAS activation by MN in individual living cells. Our findings reveal that cGAS localization to membrane-ruptured MN during interphase is infrequent, with cGAS primarily capturing MN during mitosis and remaining bound to cytosolic chromatin. We found that cGAS accumulation during mitosis neither activates STING in the subsequent interphase nor triggers the interferon response. Gamma-ray irradiation activates STING independently of MN formation and cGAS localization to MN. These results suggest that cGAS accumulation in the cytosol is not a robust indicator of its activation and that MN is not the primary trigger of the cGAS/STING pathway.

cell biology↗

bioRxiv2023

Near-infrared imaging of phytochrome-derived autofluorescence in plant nuclei

Capturing images of the nuclear dynamics within live cells is an essential technique for comprehending the intricate biological processes inherent to plant cell nuclei. While various methods exist for imaging nuclei, including combining fluorescent proteins and dyes with microscopy, there is a dearth of commercially available dyes for live-cell imaging. In Arabidopsis thaliana, we discovered that nuclei emit autofluorescence in the near-infrared (NIR) range of the spectrum and devised a non-invasive technique for the visualization of live cell nuclei using this inherent NIR autofluorescence. Our studies demonstrated the capability of the NIR imaging technique to visualize the dynamic behavior of nuclei within primary roots, root hairs, and pollen tubes, which are tissues that harbor a limited number of other organelles displaying autofluorescence. We further demonstrated the applicability of NIR autofluorescence imaging in various other tissues by incorporating fluorescence lifetime imaging techniques. Nuclear autofluorescence was also detected across a wide range of plant species, enabling analyses without the need for transformation. The nuclear autofluorescence in the NIR wavelength range was not observed in animal or yeast cells. Genetic analysis revealed that this autofluorescence was caused by the phytochrome protein. Our studies demonstrated that nuclear autofluorescence imaging can be effectively employed not only in model plants but also for studying nuclei in non-model plant species.

plant biology↗

bioRxiv2023

Application of fluorescence lifetimes to multi-imaging analysis in plant cells

Multi-imaging analysis has become an indispensable technique to visualize multiple target proteins and intracellular components simultaneously. While current multi-imaging analysis relies on the differences in emission spectra of fluorescent molecules, the use of fluorescence lifetime imaging microscopy (FLIM), which exploits the differences in fluorescence lifetimes of fluorescent proteins, in multi-imaging analysis is quite limited. In this study, we successfully discriminated fluorescent proteins with similar colors but different fluorescence lifetimes in vitro and in planta. We found that four fluorescent proteins with similar emission spectra could be distinguished by FLIM. In addition, we found that FLIM could clearly separate fluorescent proteins if they differ by at least 0.2 ns. In a proof-of-concept experiments for plant live imaging, we transiently expressed fluorescent proteins with different subcellular localization tags in Physcomitrium patens by particle bombardment. Each fluorescent protein exhibited its fluorescence lifetime at the subcellular localization corresponding to the localization tag in P. patens with little or no effect of chlorophyll autofluorescence. Our results demonstrate the effectiveness of FLIM in revealing the spatiotemporal dynamics of a large number of fluorescent proteins in living plant cells.

cell biology↗