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.

CLE3 and its homologs share overlapping functions in the modulation of lateral root formation through CLV1 and BAM1 in Arabidopsis thaliana.

Lateral roots are important for a wide range of processes, including uptake of water and nutrients. The CLAVATA3 (CLV3)/EMBRYO SURROUNDING REGION-RELATED (CLE) 1 ~ 7 peptide family and their cognate receptor CLV1 have been shown to negatively regulate lateral root formation under low-nitrate conditions. However, little is known about how CLE signaling regulates lateral root formation. A persistent obstacle in CLE peptide research is their functional redundancies, which makes functional analyses difficult. To address this problem, we generate the cle1 ~ 7 septuple mutant (cle1 ~ 7-cr1, cr stands for mutant allele generated with CRISPR/Cas9). cle1 ~ 7-cr1 exhibits longer lateral roots under normal conditions. Specifically, in cle1 ~ 7-cr1, the lateral root density is increased, and lateral root primordia initiation is found to be accelerated. Further analysis shows that cle3 single mutant exhibits slightly longer lateral roots. On the other hand, plants that overexpress CLE2 and CLE3 exhibit decreased lateral root lengths. To explore cognate receptor(s) of CLE2 and CLE3, we analyze lateral root lengths in clv1 barely any meristem 1(bam1) double mutant. Mutating both the CLV1 and BAM1 causes longer lateral roots, but not in each single mutant. In addition, genetic analysis reveals that CLV1 and BAM1 are epistatic to CLE2 and CLE3. Furthermore, gene expression analysis shows that the LATERAL ORGAN BOUNDARIES DOMAIN/ASYMMETRIC LEAVES2-LIKE (LBD/ASL) genes, which promote lateral root formation, are upregulated in cle1 ~ 7-cr1 and clv1 bam1. We therefore propose that CLE2 and CLE3 peptides are perceived by CLV1 and BAM1 to mediate lateral root formation through LBDs regulation.

An ABCB transporter regulates anisotropic cell expansion via cuticle deposition in the moss Physcomitrium patens.

Anisotropic cell expansion is crucial for the morphogenesis of land plants, as cell migration is restricted by the rigid cell wall. The anisotropy of cell expansion is regulated by mechanisms acting on the deposition or modification of cell wall polysaccharides. Besides the polysaccharide components in the cell wall, a layer of hydrophobic cuticle covers the outer cell wall and is subjected to tensile stress that mechanically restricts cell expansion. However, the molecular machinery that deposits cuticle materials in the appropriate spatiotemporal manner to accommodate cell and tissue expansion remains elusive. Here, we report that PpABCB14, an ATP-binding cassette transporter in the moss Physcomitrium patens, regulates the anisotropy of cell expansion. PpABCB14 localized to expanding regions of leaf cells. Deletion of PpABCB14 resulted in impaired anisotropic cell expansion. Unexpectedly, the cuticle proper was reduced in the mutants, and the cuticular lipid components decreased. Moreover, induced PpABCB14 expression resulted in deformed leaf cells with increased cuticle lipid accumulation on the cell surface. Taken together, PpABCB14 regulates the anisotropy of cell expansion via cuticle deposition, revealing a regulatory mechanism for cell expansion in addition to the mechanisms acting on cell wall polysaccharides.

Two atypical ANGUSTIFOLIA without a plant-specific C-terminus regulate gametophore and sporophyte shapes in the moss Physcomitrium (Physcomitrella) patens.

ANGUSTIFOLIA (AN) is a plant-specific subfamily of the CtBP/BARS/AN family, characterized by a plant-specific C-terminal domain of approximately 200 amino acids. Previously, we revealed that double knockout (DKO) lines of Physcomitrium (Physcomitrella) patens ANGUSTIFOLIA genes (PpAN1-1 and PpAN1-2) show defects in gametophore height and the lengths of the seta and foot region of sporophytes, by reduced cell elongation. In addition to two canonical ANs, the genome of P. patens has two atypical ANs without a coding region for a plant-specific C-terminus (PpAN2-1 and PpAN2-2); these were investigated in this study. Similar to PpAN1s, both promoters of the PpAN2 genes were highly active in the stems of haploid gametophores and in the middle-to-basal region of young diploid sporophytes that develop into the seta and foot. Analyses of PpAN2-1/2-2 DKO and PpAN quadruple knockout (QKO) lines implied that these four AN genes have partially redundant functions to regulate cell elongation in their expression regions. Transgenic strains harboring P. patens α-tubulin fused to green fluorescent protein, which were generated from a QKO line, showed that the orientation of the microtubules in the gametophore tips in the PpAN QKO lines was unchanged from the wild-type and PpAN1-1/1-2 DKO plants. In addition to both PpAN2-1 and PpAN2-2, short Arabidopsis AN without the C-terminus of 200 amino acids could rescue the Arabidopsis thaliana an-1 phenotypes, implying AN activity is dependent on the N-terminal regions.

A photostable fluorescent marker for the superresolution live imaging of the dynamic structure of the mitochondrial cristae.

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.

Two ANGUSTIFOLIA genes regulate gametophore and sporophyte development in Physcomitrella patens.

In Arabidopsis thaliana the ANGUSTIFOLIA (AN) gene regulates the width of leaves by controlling the diffuse growth of leaf cells in the medio-lateral direction. In the genome of the moss Physcomitrella patens, we found two normal ANs (PpAN1-1 and 1-2). Both PpAN1 genes complemented the A. thaliana an-1 mutant phenotypes. An analysis of spatiotemporal promoter activity of each PpAN1 gene, using transgenic lines that contained each PpAN1-promoter- uidA (GUS) gene, showed that both promoters are mainly active in the stems of haploid gametophores and in the middle to basal region of the young sporophyte that develops into the seta and foot. Analyses of the knockout lines for PpAN1-1 and PpAN1-2 genes suggested that these genes have partially redundant functions and regulate gametophore height by controlling diffuse cell growth in gametophore stems. In addition, the seta and foot were shorter and thicker in diploid sporophytes, suggesting that cell elongation was reduced in the longitudinal direction, whereas no defects were detected in tip-growing protonemata. These results indicate that both PpAN1 genes in P. patens function in diffuse growth of the haploid and diploid generations but not in tip growth. To visualize microtubule distribution in gametophore cells of P. patens, transformed lines expressing P. patens α-tubulin fused to sGFP were generated. Contrary to expectations, the orientation of microtubules in the tips of gametophores in the PpAN1-1/1-2 double-knockout lines was unchanged. The relationships among diffuse cell growth, cortical microtubules and AN proteins are discussed.

Role of Reelin in cell positioning in the cerebellum and the cerebellum-like structure in zebrafish.

The cerebellum and the cerebellum-like structure in the mesencephalic tectum in zebrafish contain multiple cell types, including principal cells (i.e., Purkinje cells and type I neurons) and granule cells, that form neural circuits in which the principal cells receive and integrate inputs from granule cells and other neurons. It is largely unknown how these cells are positioned and how neural circuits form. While Reelin signaling is known to play an important role in cell positioning in the mammalian brain, its role in the formation of other vertebrate brains remains elusive. Here we found that zebrafish with mutations in Reelin or in the Reelin-signaling molecules Vldlr or Dab1a exhibited ectopic Purkinje cells, eurydendroid cells (projection neurons), and Bergmann glial cells in the cerebellum, and ectopic type I neurons in the tectum. The ectopic Purkinje cells and type I neurons received aberrant afferent fibers in these mutants. In wild-type zebrafish, reelin transcripts were detected in the internal granule cell layer, while Reelin protein was localized to the superficial layer of the cerebellum and the tectum. Laser ablation of the granule cell axons perturbed the localization of Reelin, and the mutation of both kif5aa and kif5ba, which encode major kinesin I components in the granule cells, disrupted the elongation of granule cell axons and the Reelin distribution. Our findings suggest that in zebrafish, (1) Reelin is transported from the granule cell soma to the superficial layer by axonal transport; (2) Reelin controls the migration of neurons and glial cells from the ventricular zone; and (3) Purkinje cells and type I neurons attract afferent axons during the formation of the cerebellum and the cerebellum-like structure.

The Effect of Branching on the One- and Two-Photon Absorption, Cell Viability, and Localization of Cationic Triarylborane Chromophores with Dipolar versus Octupolar Charge Distributions for Cellular Imaging.

Two different chromophores, namely a dipolar and an octupolar system, were prepared and their linear and nonlinear optical properties as well as their bioimaging capabilities were compared. Both contain triphenylamine as the donor and a triarylborane as the acceptor, the latter modified with cationic trimethylammonio groups to provide solubility in aqueous media. The octupolar system exhibits a much higher two-photon brightness, and also better cell viability and enhanced selectivity for lysosomes compared with the dipolar chromophore. Furthermore, both dyes were applied in two-photon excited fluorescence (TPEF) live-cell imaging.

Cytoskeleton dynamics control the first asymmetric cell division in Arabidopsis zygote.

The asymmetric cell division of the zygote is the initial and crucial developmental step in most multicellular organisms. In flowering plants, whether zygote polarity is inherited from the preexisting organization in the egg cell or reestablished after fertilization has remained elusive. How dynamically the intracellular organization is generated during zygote polarization is also unknown. Here, we used a live-cell imaging system with Arabidopsis zygotes to visualize the dynamics of the major elements of the cytoskeleton, microtubules (MTs), and actin filaments (F-actins), during the entire process of zygote polarization. By combining image analysis and pharmacological experiments using specific inhibitors of the cytoskeleton, we found features related to zygote polarization. The preexisting alignment of MTs and F-actin in the egg cell is lost on fertilization. Then, MTs organize into a transverse ring defining the zygote subapical region and driving cell outgrowth in the apical direction. F-actin forms an apical cap and longitudinal arrays and is required to position the nucleus to the apical region of the zygote, setting the plane of the first asymmetrical division. Our findings show that, in flowering plants, the preexisting cytoskeletal patterns in the egg cell are lost on fertilization and that the zygote reorients the cytoskeletons to perform directional cell elongation and polar nuclear migration.

ClearSee: a rapid optical clearing reagent for whole-plant fluorescence imaging.

Imaging techniques for visualizing and analyzing precise morphology and gene expression patterns are essential for understanding biological processes during development in all organisms. With the aid of chemical screening, we developed a clearing method using chemical solutions, termed ClearSee, for deep imaging of morphology and gene expression in plant tissues. ClearSee rapidly diminishes chlorophyll autofluorescence while maintaining fluorescent protein stability. By adjusting the refractive index mismatch, whole-organ and whole-plant imaging can be performed by both confocal and two-photon excitation microscopy in ClearSee-treated samples. Moreover, ClearSee is applicable to multicolor imaging of fluorescent proteins to allow structural analysis of multiple gene expression. Given that ClearSee is compatible with staining by chemical dyes, the technique is useful for deep imaging in conjunction with genetic markers and for plant species not amenable to transgenic approaches. This method is useful for whole imaging for intact morphology and will help to accelerate the discovery of new phenomena in plant biological research.

Type IV Collagen Controls the Axogenesis of Cerebellar Granule Cells by Regulating Basement Membrane Integrity in Zebrafish.

Granule cells (GCs) are the major glutamatergic neurons in the cerebellum, and GC axon formation is an initial step in establishing functional cerebellar circuits. In the zebrafish cerebellum, GCs can be classified into rostromedial and caudolateral groups, according to the locations of their somata in the corresponding cerebellar lobes. The axons of the GCs in the caudolateral lobes terminate on crest cells in the dorsal hindbrain, as well as forming en passant synapses with Purkinje cells in the cerebellum. In the zebrafish mutant shiomaneki, the caudolateral GCs extend aberrant axons. Positional cloning revealed that the shiomaneki (sio) gene locus encodes Col4a6, a subunit of type IV collagen, which, in a complex with Col4a5, is a basement membrane (BM) component. Both col4a5 and col4a6 mutants displayed similar abnormalities in the axogenesis of GCs and retinal ganglion cells (RGCs). Although type IV collagen is reported to control axon targeting by regulating the concentration gradient of an axonal guidance molecule Slit, Slit overexpression did not affect the GC axons. The structure of the BM surrounding the tectum and dorsal hindbrain was disorganized in the col4a5 and col4a6 mutants. Moreover, the abnormal axogenesis of the caudolateral GCs and the RGCs was coupled with aberrant BM structures in the type IV collagen mutants. The regrowth of GC axons after experimental ablation revealed that the original and newly formed axons displayed similar branching and extension abnormalities in the col4a6 mutants. These results collectively suggest that type IV collagen controls GC axon formation by regulating the integrity of the BM, which provides axons with the correct path to their targets.

A Highly Photostable Near-Infrared Labeling Agent Based on a Phospha-rhodamine for Long-Term and Deep Imaging.

Various fluorescence microscopy techniques require bright NIR-emitting fluorophores with high chemical and photostability. Now, the significant performance improvement of phosphorus-substituted rhodamine dyes (PORs) upon substitution at the 9-position with a 2,6-dimethoxyphenyl group is reported. The thus obtained dye PREX 710 was used to stain mitochondria in living cells, which allowed long-term and three-color imaging in the vis-NIR range. Moreover, the high fluorescence longevity of PREX 710 allows tracking a dye-labeled biomolecule by single-molecule microscopy under physiological conditions. Deep imaging of blood vessels in mice brain has also been achieved using the bright NIR-emitting PREX 710-dextran conjugate.

A Water-Soluble Warped Nanographene: Synthesis and Applications for Photoinduced Cell Death.

Nanographene, a small piece of graphene, has attracted unprecedented interest across diverse scientific disciplines particularly in organic electronics. The biological applications of nanographenes, such as bioimaging, cancer therapies and drug delivery, provide significant opportunities for breakthroughs in the field. However, the intrinsic aggregation behavior and low solubility of nanographenes, which stem from their flat structures, hamper their development for bioapplications. Herein, we report a water-soluble warped nanographene (WNG) that can be easily synthesized by sequential regioselective C-H borylation and cross-coupling reactions of the saddle-shaped WNG core structure. The saddle-shaped structure and hydrophilic tetraethylene glycol chains impart high water solubility to the WNG. The water-soluble WNG possesses a range of promising properties including good photostability and low cytotoxicity. Moreover, the water-soluble WNG was successfully internalized into HeLa cells and promoted photoinduced cell death.

Super-Photostable Phosphole-Based Dye for Multiple-Acquisition Stimulated Emission Depletion Imaging.

As stimulated emission depletion (STED) microscopy can provide structural details of cells with an optical resolution beyond the diffraction limit, it has become an indispensable tool in cell biology. However, the intense STED laser beam usually causes rapid photobleaching of the employed fluorescent dyes, which significantly limits the utility of STED microscopy from a practical perspective. Herein we report a new design of super-photostable dye, PhoxBright 430 (PB430), comprising a fully ring-fused π-conjugated skeleton with an electron-accepting phosphole P-oxide unit. We previously developed a super-photostable dye C-Naphox by combining the phosphole unit with an electron-donating triphenylamine moiety. In PB430, removal of the amino group alters the transition type from intramolecular charge transfer character to π-π* transition character, which gives rise to intense fluorescence insensitive to molecular environment in terms of fluorescence colors and intensity, and bright fluorescence even in aqueous media. PB430 also furnishes high solubility in water, and is capable of labeling proteins with maintaining high fluorescence quantum yields. This dye exhibits outstanding resistance to photoirradiation even under the STED conditions and allows continuous acquisition of STED images. Indeed, using a PB430-conjugated antibody, we succeed in attaining a 3-D reconstruction of super-resolution STED images as well as photostability-based multicolor STED imaging of fluorescently labeled cytoskeletal structures.

WOX13-like genes are required for reprogramming of leaf and protoplast cells into stem cells in the moss Physcomitrella patens.

Many differentiated plant cells can dedifferentiate into stem cells, reflecting the remarkable developmental plasticity of plants. In the moss Physcomitrella patens, cells at the wound margin of detached leaves become reprogrammed into stem cells. Here, we report that two paralogous P. patens WUSCHEL-related homeobox 13-like (PpWOX13L) genes, homologs of stem cell regulators in flowering plants, are transiently upregulated and required for the initiation of cell growth during stem cell formation. Concordantly, Δppwox13l deletion mutants fail to upregulate genes encoding homologs of cell wall loosening factors during this process. During the moss life cycle, most of the Δppwox13l mutant zygotes fail to expand and initiate an apical stem cell to form the embryo. Our data show that PpWOX13L genes are required for the initiation of cell growth specifically during stem cell formation, in analogy to WOX stem cell functions in seed plants, but using a different cellular mechanism.

Kinesins have a dual function in organizing microtubules during both tip growth and cytokinesis in Physcomitrella patens.

Microtubules (MTs) play a crucial role in the anisotropic deposition of cell wall material, thereby affecting the direction of growth. A wide range of tip-growing cells display highly polarized cell growth, and MTs have been implicated in regulating directionality and expansion. However, the molecular machinery underlying MT dynamics in tip-growing plant cells remains unclear. Here, we show that highly dynamic MT bundles form cyclically in the polarized expansion zone of the moss Physcomitrella patens caulonemal cells through the coalescence of growing MT plus ends. Furthermore, the plant-specific kinesins (KINID1) that are is essential for the proper MT organization at cytokinesis also regulate the turnover of the tip MT bundles as well as the directionality and rate of cell growth. The plus ends of MTs grow toward the expansion zone, and KINID1 is necessary for the stability of a single coherent focus of MTs in the center of the zone, whose formation coincides with the accumulation of KINID1. We propose that KINID-dependent MT bundling is essential for the correct directionality of growth as well as for promoting growth per se. Our findings indicate that two localized cell wall deposition processes, tip growth and cytokinesis, previously believed to be functionally and evolutionarily distinct, share common and plant-specific MT regulatory components.

A Macrocyclic Fluorophore Dimer with Flexible Linkers: Bright Excimer Emission with a Long Fluorescence Lifetime.

Bright fluorescent molecules with long fluorescence lifetimes are important for the development of lifetime-based fluorescence imaging techniques. Herein, a molecular design is described for simultaneously attaining long fluorescence lifetime (τ) and high brightness (ΦF ×ɛ) in a system that features macrocyclic dimerization of fluorescent π-conjugated skeletons with flexible linkers. An alkylene-linked macrocyclic dimer of bis(thienylethynyl)anthracene was found to show excimer emission with a long fluorescence lifetime (τ≈19 ns) in solution, while maintaining high brightness. A comparison with various relevant derivatives revealed that the macrocyclic structure and the length of the alkylene chains play crucial roles in attaining these properties. In vitro time-gated imaging experiments were conducted as a proof-of-principle for the superiority of this macrocyclic fluorophore relative to the commercial fluorescent dye Alexa Fluor 488.

A Phosphole Oxide Based Fluorescent Dye with Exceptional Resistance to Photobleaching: A Practical Tool for Continuous Imaging in STED Microscopy.

The development of stimulated emission depletion (STED) microscopy represented a major breakthrough in cellular and molecular biology. However, the intense laser beams required for both excitation and STED usually provoke rapid photobleaching of fluorescent molecular probes, which significantly limits the performance and practical utility of STED microscopy. We herein developed a photoresistant fluorescent dye C-Naphox as a practical tool for STED imaging. With excitation using either a λ=405 or 488 nm laser in protic solvents, C-Naphox exhibited an intense red/orange fluorescence (quantum yield ΦF >0.7) with a large Stokes shift (circa 5900 cm(-1) ). Even after irradiation with a Xe lamp (300 W, λex =460 nm, full width at half maximum (FWHM)=11 nm) for 12 hours, 99.5 % of C-Naphox remained intact. The high photoresistance of C-Naphox allowed repeated STED imaging of HeLa cells. Even after recording 50 STED images, 83 % of the initial fluorescence intensity persisted.

Environment-sensitive fluorescent probe: a benzophosphole oxide with an electron-donating substituent.

Electron-donating aryl groups were attached to electron-accepting benzophosphole skeletons. Among several derivatives thus prepared, one benzophosphole oxide was particularly interesting, as it retained high fluorescence quantum yields even in polar and protic solvents. This phosphole-based compound exhibited a drastic color change of its fluorescence spectrum as a function of the solvent polarity, while the absorption spectra remained virtually unchanged. Capitalizing on these features, this phosphole-based compound was used to stain adipocytes, in which the polarity of subcellular compartments could then be discriminated on the basis of the color change of the fluorescence emission.

Physcomitrella cyclin-dependent kinase A links cell cycle reactivation to other cellular changes during reprogramming of leaf cells.

During regeneration, differentiated plant cells can be reprogrammed to produce stem cells, a process that requires coordination of cell cycle reactivation with acquisition of other cellular characteristics. However, the factors that coordinate the two functions during reprogramming have not been determined. Here, we report a link between cell cycle reactivation and the acquisition of new cell-type characteristics through the activity of cyclin-dependent kinase A (CDKA) during reprogramming in the moss Physcomitrella patens. Excised gametophore leaf cells of P. patens are readily reprogrammed, initiate tip growth, and form chloronema apical cells with stem cell characteristics at their first cell division. We found that leaf cells facing the cut undergo CDK activation along with induction of a D-type cyclin, tip growth, and transcriptional activation of protonema-specific genes. A DNA synthesis inhibitor, aphidicolin, inhibited cell cycle progression but prevented neither tip growth nor protonemal gene expression, indicating that cell cycle progression is not required for acquisition of protonema cell-type characteristics. By contrast, treatment with a CDK inhibitor or induction of dominant-negative CDKA;1 protein inhibited not only cell cycle progression but also tip growth and protonemal gene expression. These findings indicate that cell cycle progression is coordinated with other cellular changes by the concomitant regulation through CDKA;1.