Precocious cell differentiation occurs in proliferating cells in leaf primordia in Arabidopsis angustifolia3 mutant.

During leaf development, the timing of transition from cell proliferation to expansion is an important factor in determining the final organ size. However, the regulatory system involved in this transition remains less understood. To get an insight into this system, we investigated the compensation phenomenon, in which the cell number decreases while the cell size increases in organs with determinate growth. Compensation is observed in several plant species suggesting coordination between cell proliferation and expansion. In this study, we examined an Arabidopsis mutant of ANGUSTIFOLIA 3 (AN3)/GRF-INTERACTING FACTOR 1, a positive regulator of cell proliferation, which exhibits the compensation. Though the AN3 role has been extensively investigated, the mechanism underlying excess cell expansion in the an3 mutant remains unknown. Focusing on the early stage of leaf development, we performed kinematic, cytological, biochemical, and transcriptome analyses, and found that the cell size had already increased during the proliferation phase, with active cell proliferation in the an3 mutant. Moreover, at this stage, chloroplasts, vacuoles, and xylem cells developed earlier than in the wild-type cells. Transcriptome data showed that photosynthetic activity and secondary cell wall biosynthesis were activated in an3 proliferating cells. These results indicated that precocious cell differentiation occurs in an3 cells. Therefore, we suggest a novel AN3 role in the suppression of cell expansion/differentiation during the cell proliferation phase.

Neopetromin, a Cyclic Tripeptide with a C-N Cross-Link, from the Marine Sponge Neopetrosia sp., That Causes Vacuole Fragmentation in Tobacco BY-2 Cells.

HPLC-MS analysis revealed the presence of an unreported peptide in the extract of the marine sponge Neopetrosia sp. Its structure was determined as a tripeptide, named neopetromin (1), composed of two tyrosine and one tryptophan residues with a heteroaromatic C-N cross-link between side chains. The absolute configuration of amino acids was determined using Marfey's method after ozonolysis and hydrolysis of 1. Compound 1 promoted vacuole fragmentation in an actin-independent manner in tobacco BY-2 cells.

Comprehensive and quantitative analysis of intracellular structure polarization at the apical-basal axis in elongating Arabidopsis zygotes.

A comprehensive and quantitative evaluation of multiple intracellular structures or proteins is a promising approach to provide a deeper understanding of and new insights into cellular polarity. In this study, we developed an image analysis pipeline to obtain intensity profiles of fluorescent probes along the apical-basal axis in elongating Arabidopsis thaliana zygotes based on two-photon live-cell imaging data. This technique showed the intracellular distribution of actin filaments, mitochondria, microtubules, and vacuolar membranes along the apical-basal axis in elongating zygotes from the onset of cell elongation to just before asymmetric cell division. Hierarchical cluster analysis of the quantitative data on intracellular distribution revealed that the zygote may be compartmentalized into two parts, with a boundary located 43.6% from the cell tip, immediately after fertilization. To explore the biological significance of this compartmentalization, we examined the positions of the asymmetric cell divisions from the dataset used in this distribution analysis. We found that the cell division plane was reproducibly inserted 20.5% from the cell tip. This position corresponded well with the midpoint of the compartmentalized apical region, suggesting a potential relationship between the zygote compartmentalization, which begins with cell elongation, and the position of the asymmetric cell division.

Smooth Elongation of Pavement Cells Induced by RIC1 Overexpression Leads to Marginal Protrusions of the Cotyledon in Arabidopsis thaliana.

The interdigitated pavement cell shape is suggested to be mechanically rational at both the cellular and tissue levels, but the biological significance of the cell shape is not fully understood. In this study, we explored the potential importance of the jigsaw puzzle-like cell shape for cotyledon morphogenesis in Arabidopsis. We used a transgenic line overexpressing a Rho-like GTPase-interacting protein, ROP-INTERACTIVE CRIB MOTIF-CONTAINING PROTEIN 1 (RIC1), which causes simple elongation of pavement cells. Computer-assisted microscopic analyses, including virtual reality observation, revealed that RIC1 overexpression resulted in abnormal cotyledon shapes with marginal protrusions, suggesting that the abnormal organ shape might be explained by changes in the pavement cell shape. Microscopic, biochemical and mechanical observations indicated that the pavement cell deformation might be due to reduction in the cell wall cellulose content with alteration of cortical microtubule organization. To examine our hypothesis that simple elongation of pavement cells leads to an abnormal shape with marginal protrusion of the cotyledon, we developed a mathematical model that examines the impact of planar cell growth geometry on the morphogenesis of the organ that is an assemblage of the cells. Computer simulations supported experimental observations that elongated pavement cells resulted in an irregular cotyledon shape, suggesting that marginal protrusions were due to local growth variation possibly caused by stochastic bias in the direction of cell elongation cannot be explained only by polarity-based cell elongation, but that an organ-level regulatory mechanism is required.

The SYP123-VAMP727 SNARE complex delivers secondary cell wall components for root hair shank hardening in Arabidopsis.

The extended tubular shape of root hairs is established by tip growth and concomitant hardening. Here, we demonstrate that a syntaxin of plants (SYP)123-vesicle-associated membrane protein (VAMP)727-dependent secretion system delivers secondary cell wall components for hardening the subapical zone and shank of Arabidopsis (Arabidopsis thaliana) root hairs. We found increased SYP123 localization at the plasma membrane (PM) of the subapical and shank zones compared with the tip region in elongating root hairs. Inhibition of phosphatidylinositol (PtdIns)(3,5)P2 production impaired SYP123 localization at the PM and SYP123-mediated root hair shank hardening. Moreover, root hair elongation in the syp123 mutant was insensitive to a PtdIns(3,5)P2 synthesis inhibitor. SYP123 interacts with both VAMP721 and VAMP727. syp123 and vamp727 mutants exhibited reduced shank cell wall stiffness due to impaired secondary cell wall component deposition. Based on these results, we conclude that SYP123 is involved in VAMP721-mediated conventional secretion for root hair elongation as well as in VAMP727-mediated secretory functions for the delivery of secondary cell wall components to maintain root hair tubular morphology.

Microtubule detyrosination by VASH1/SVBP is regulated by the conformational state of tubulin in the lattice.

Tubulin, a heterodimer of α- and ß-tubulin, is a GTPase that assembles into microtubule (MT) polymers whose dynamic properties are intimately coupled to nucleotide hydrolysis. In cells, the organization and dynamics of MTs are further tuned by post-translational modifications (PTMs), which control the ability of MT-associated proteins (MAPs) and molecular motors to engage MTs. Detyrosination is a PTM of α-tubulin, wherein its C-terminal tyrosine residue is enzymatically removed by either the vasohibin (VASH) or MT-associated tyrosine carboxypeptidase (MATCAP) peptidases. How these enzymes generate specific patterns of MT detyrosination in cells is not known. Here, we use a novel antibody-based probe to visualize the formation of detyrosinated MTs in real time and employ single-molecule imaging of VASH1 bound to its regulatory partner small-vasohibin binding protein (SVBP) to understand the process of MT detyrosination in vitro and in cells. We demonstrate that the activity, but not binding, of VASH1/SVBP is much greater on mimics of guanosine triphosphate (GTP)-MTs than on guanosine diphosphate (GDP)-MTs. Given emerging data showing that tubulin subunits in GTP-MTs are in expanded conformation relative to tubulin subunits in GDP-MTs, we reasoned that the lattice conformation of MTs is a key factor that gates the activity of VASH1/SVBP. We show that Taxol, a drug known to expand the MT lattice, promotes MT detyrosination and that CAMSAP2 and CAMSAP3 are two MAPs that spatially regulate detyrosination in cells. Collectively, our work shows that VASH1/SVBP detyrosination is regulated by the conformational state of tubulin in the MT lattice and that this is spatially determined in cells by the activity of MAPs.

Cell polarity linked to gravity sensing is generated by LZY translocation from statoliths to the plasma membrane.

Organisms have evolved under gravitational force, and many sense the direction of gravity by means of statoliths in specialized cells. In flowering plants, starch-accumulating plastids, known as amyloplasts, act as statoliths to facilitate downstream gravitropism. The gravity-sensing mechanism has long been considered a mechanosensing process by which amyloplasts transmit forces to intracellular structures, but the molecular mechanism underlying this has not been elucidated. We show here that LAZY1-LIKE (LZY) family proteins involved in statocyte gravity signaling associate with amyloplasts and the proximal plasma membrane. This results in polar localization according to the direction of gravity. We propose a gravity-sensing mechanism by which LZY translocation to the plasma membrane signals the direction of gravity by transmitting information on the position of amyloplasts.

Arabidopsis thaliana Subclass I ACTIN DEPOLYMERIZING FACTORs Regulate Nuclear Organization and Gene Expression.

ACTIN DEPOLYMERIZING FACTOR (ADF) is a conserved protein that regulates the organization and dynamics of actin microfilaments. Eleven ADFs in the Arabidopsis thaliana genome are grouped into four subclasses, and subclass I ADFs, ADF1-4, are all expressed throughout the plant. Previously, we showed that subclass I ADFs function in the regulation of the response against powdery mildew fungus as well as in the regulation of cell size and endoreplication. Here, we report a new role of subclass I ADFs in the regulation of nuclear organization and gene expression. Through microscopic observation of epidermal cells in mature leaves, we found that the size of chromocenters in both adf4 and transgenic lines where expression of subclass I ADFs is downregulated (ADF1-4Ri) was reduced compared with that of wild-type Col-0. Arabidopsis thaliana possesses eight ACTIN (ACT) genes, among which ACT2, -7 and -8 are expressed in vegetative organs. The chromocenter size in act7, but not in the act2/8 double mutant, was enlarged compared with that in Col-0. Microarray analysis revealed that 1,818 genes were differentially expressed in adf4 and ADF1-4Ri. In particular, expression of 22 nucleotide-binding leucine-rich repeat genes, which are involved in effector-triggered plant immunity, was reduced in adf4 and ADF1-4Ri. qRT-PCR confirmed the altered expressions shown with microarray analysis. Overall, these results suggest that ADF regulates various aspects of plant physiology through its role in regulation of nuclear organization and gene expression. The mechanism how ADF and ACT regulate nuclear organization and gene expression is discussed.

Pacsin 2-dependent N-cadherin internalization regulates the migration behaviour of malignant cancer cells.

Collective cell migration is the coordinated movement of multiple cells connected by cadherin-based adherens junctions and is essential for physiological and pathological processes. Cadherins undergo dynamic intracellular trafficking, and their surface level is determined by a balance between endocytosis, recycling and degradation. However, the regulatory mechanism of cadherin turnover in collective cell migration remains elusive. In this study, we show that the Bin/amphiphysin/Rvs (BAR) domain protein pacsin 2 (protein kinase C and casein kinase substrate in neurons protein 2) plays an essential role in collective cell migration by regulating N-cadherin (also known as CDH2) endocytosis in human cancer cells. Pacsin 2-depleted cells formed cell-cell contacts enriched with N-cadherin and migrated in a directed manner. Furthermore, pacsin 2-depleted cells showed attenuated internalization of N-cadherin from the cell surface. Interestingly, GST pull-down assays demonstrated that the pacsin 2 SH3 domain binds to the cytoplasmic region of N-cadherin, and expression of an N-cadherin mutant defective in binding to pacsin 2 phenocopied pacsin 2 RNAi cells both in cell contact formation and N-cadherin endocytosis. These data support new insights into a novel endocytic route of N-cadherin in collective cell migration, highlighting pacsin 2 as a possible therapeutic target for cancer metastasis.

Ab-GALFA, A bioassay for insect gall formation using the model plant Arabidopsis thaliana.

Insect galls are abnormal plant organs formed by gall-inducing insects to provide shelter and nutrients for themselves. Although insect galls are spatialized complex structures with unique shapes and functions, the molecular mechanism of the gall formation and the screening system for the gall inducing effectors remains unknown. Here, we demonstrate that an extract of a gall-inducing aphid, Schlechtendalia chinensis, induces an abnormal structure in the root-tip region of Arabidopsis seedlings. The abnormal structure is composed of stem-like cells, vascular, and protective tissues, as observed in typical insect galls. Furthermore, we confirm similarities in the gene expression profiles between the aphid-treated seedlings and the early developmental stages of Rhus javanica galls formed by S. chinensis. Based on the results, we propose a model system for analyzing the molecular mechanisms of gall formation: the Arabidopsis-based Gall-Forming Assay (Ab-GALFA). Ab-GALFA could be used not only as a model to elucidate the mechanisms underlying gall formation, but also as a bioassay system to isolate insect effector molecules of gall-induction.

At the Nexus between Cytoskeleton and Vacuole: How Plant Cytoskeletons Govern the Dynamics of Large Vacuoles.

Large vacuoles are a predominant cell organelle throughout the plant body. They maximally account for over 90% of cell volume and generate turgor pressure that acts as a driving force of cell growth, which is essential for plant development. The plant vacuole also acts as a reservoir for sequestering waste products and apoptotic enzymes, thereby enabling plants to rapidly respond to fluctuating environments. Vacuoles undergo dynamic transformation through repeated enlargement, fusion, fragmentation, invagination, and constriction, eventually resulting in the typical 3-dimensional complex structure in each cell type. Previous studies have indicated that such dynamic transformations of plant vacuoles are governed by the plant cytoskeletons, which consist of F-actin and microtubules. However, the molecular mechanism of cytoskeleton-mediated vacuolar modifications remains largely unclear. Here we first review the behavior of cytoskeletons and vacuoles during plant development and in response to environmental stresses, and then introduce candidates that potentially play pivotal roles in the vacuole-cytoskeleton nexus. Finally, we discuss factors hampering the advances in this research field and their possible solutions using the currently available cutting-edge technologies.

Machine learning and feature analysis of the cortical microtubule organization of Arabidopsis cotyledon pavement cells.

The measurement of cytoskeletal features can provide valuable insights into cell biology. In recent years, digital image analysis of cytoskeletal features has become an important research tool for quantitative evaluation of cytoskeleton organization. In this study, we examined the utility of a supervised machine learning approach with digital image analysis to distinguish different cellular organizational patterns. We focused on the jigsaw puzzle-shaped pavement cells of Arabidopsis thaliana. Measurements of three features of cortical microtubules in these cells (parallelness, density, and the coefficient of variation of the intensity distribution of fluorescently labeled cytoskeletons [as an indicator of microtubule bundling]) were obtained from microscopic images. A random forest machine learning model was then used with these images to differentiate mutant and wild type, and Taxol-treated and control cells. Using these three metrics, we were able to distinguish wild type from bpp125 triple mutant cells, with approximately 80% accuracy; classification accuracy was 88% for control and Taxol-treated cells. Different features contributed most to the classification, namely, coefficient of variation for the wild-type/mutant cells and parallelness for the Taxol-treated/control cells. The random forest method used enabled quantitative evaluation of the contribution of features to the classification, and partial dependence plots showed the relationships between metric values and classification accuracy. While further improvements to the method are needed, our small-scale analysis shows the potential for this approach in large-scale screening analyses.

Disease-associated H58Y mutation affects the nuclear dynamics of human DNA topoisomerase IIß.

DNA topoisomerase II (TOP2) is an enzyme that resolves DNA topological problems and plays critical roles in various nuclear processes. Recently, a heterozygous H58Y substitution in the ATPase domain of human TOP2B was identified from patients with autism spectrum disorder, but its biological significance remains unclear. In this study, we analyzed the nuclear dynamics of TOP2B with H58Y (TOP2B H58Y). Although wild-type TOP2B was highly mobile in the nucleus of a living cell, the nuclear mobility of TOP2B H58Y was markedly reduced, suggesting that the impact of H58Y manifests as low protein mobility. We found that TOP2B H58Y is insensitive to ICRF-187, a TOP2 inhibitor that halts TOP2 as a closed clamp on DNA. When the ATPase activity of TOP2B was compromised, the nuclear mobility of TOP2B H58Y was restored to wild-type levels, indicating the contribution of the ATPase activity to the low nuclear mobility. Analysis of genome-edited cells harboring TOP2B H58Y showed that TOP2B H58Y retains sensitivity to the TOP2 poison etoposide, implying that TOP2B H58Y can undergo at least a part of its catalytic reactions. Collectively, TOP2 H58Y represents a unique example of the relationship between a disease-associated mutation and perturbed protein dynamics.

Bone marrow imaging reveals the migration dynamics of neonatal hematopoietic stem cells.

Hematopoietic stem cells (HSCs) are produced from the blood vessel walls and circulate in the blood during the perinatal period. However, the migration dynamics of how HSCs enter the bone marrow remain elusive. To observe the dynamics of HSCs over time, the present study develops an intravital imaging method to visualize bone marrow in neonatal long bones formed by endochondral ossification which is essential for HSC niche formation. Endogenous HSCs are labeled with tdTomato under the control of an HSC marker gene Hlf, and a customized imaging system with a bone penetrating laser is developed for intravital imaging of tdTomato-labeled neonatal HSCs in undrilled tibia, which is essential to avoid bleeding from fragile neonatal tibia by bone drilling. The migration speed of neonatal HSCs is higher than that of adult HSCs. Neonatal HSCs migrate from outside to inside the tibia via the blood vessels that penetrate the bone, which is a transient structure during the neonatal period, and settle on the blood vessel wall in the bone marrow. The results obtained from direct observations in vivo reveal the motile dynamics and colonization process of neonatal HSCs during bone marrow formation.

Two-step regulation of centromere distribution by condensin II and the nuclear envelope proteins.

The arrangement of centromeres within the nucleus differs among species and cell types. However, neither the mechanisms determining centromere distribution nor its biological significance are currently well understood. In this study, we demonstrate the importance of centromere distribution for the maintenance of genome integrity through the cytogenic and molecular analysis of mutants defective in centromere distribution. We propose a two-step regulatory mechanism that shapes the non-Rabl-like centromere distribution in Arabidopsis thaliana through condensin II and the linker of the nucleoskeleton and cytoskeleton (LINC) complex. Condensin II is enriched at centromeres and, in cooperation with the LINC complex, induces the scattering of centromeres around the nuclear periphery during late anaphase/telophase. After entering interphase, the positions of the scattered centromeres are then stabilized by nuclear lamina proteins of the CROWDED NUCLEI (CRWN) family. We also found that, despite their strong impact on centromere distribution, condensin II and CRWN proteins have little effect on chromatin organization involved in the control of gene expression, indicating a robustness of chromatin organization regardless of the type of centromere distribution.

Two Kinesin-14A Motors Oligomerize to Drive Poleward Microtubule Convergence for Acentrosomal Spindle Morphogenesis in Arabidopsis thaliana.

Plant cells form acentrosomal spindles with microtubules (MTs) converged toward two structurally undefined poles by employing MT minus end-directed Kinesin-14 motors. To date, it is unclear whether the convergent bipolar MT array assumes unified poles in plant spindles, and if so, how such a goal is achieved. Among six classes of Kinesin-14 motors in Arabidopsis thaliana, the Kinesin-14A motors ATK1 (KatA) and ATK5 share the essential function in spindle morphogenesis. To understand how the two functionally redundant Kinesin-14A motors contributed to the spindle assembly, we had ATK1-GFP and ATK5-GFP fusion proteins expressed in their corresponding null mutants and found that they were functionally comparable to their native forms. Although ATK1 was a nuclear protein and ATK5 cytoplasmic prior to nuclear envelop breakdown, at later mitotic stages, the two motors shared similar localization patterns of uniform association with both spindle and phragmoplast MTs. We found that ATK1 and ATK5 were rapidly concentrated toward unified polar foci when cells were under hyperosmotic conditions. Concomitantly, spindle poles became perfectly focused as if there were centrosome-like MT-organizing centers where ATK1 and ATK5 were highly enriched and at which kinetochore fibers pointed. The separation of ATK1/ATK5-highlighted MTs from those of kinetochore fibers suggested that the motors translocated interpolar MTs. Our protein purification and live-cell imaging results showed that ATK1 and ATK5 are associated with each other in vivo. The stress-induced spindle pole convergence was also accompanied by poleward accumulation of the MT nucleator γ-tubulin. These results led to the conclusion that the two Kinesin-14A motors formed oligomeric motor complexes that drove MT translocation toward the spindle pole to establish acentrosomal spindles with convergent poles.

EML2-S constitutes a new class of proteins that recognizes and regulates the dynamics of tyrosinated microtubules.

Tubulin post-translational modifications (PTMs) alter microtubule properties by affecting the binding of microtubule-associated proteins (MAPs). Microtubule detyrosination, which occurs by proteolytic removal of the C-terminal tyrosine from ɑ-tubulin, generates the oldest known tubulin PTM, but we lack comprehensive knowledge of MAPs that are regulated by this PTM. We developed a screening pipeline to identify proteins that discriminate between Y- and ΔY-microtubules and found that echinoderm microtubule-associated protein-like 2 (EML2) preferentially interacts with Y-microtubules. This activity depends on a Y-microtubule interaction motif built from WD40 repeats. We show that EML2 tracks the tips of shortening microtubules, a behavior not previously seen among human MAPs in vivo, and influences dynamics to increase microtubule stability. Our screening pipeline is readily adapted to identify proteins that specifically recognize a wide range of microtubule PTMs.

Migration of prospindle before the first asymmetric division in germinating spore of Marchantia polymorpha.

The development of the plant body starts with spore germination in bryophytes. In many cases, the first division of the spore occurs after germination and cell elongation of the spore. In Marchantia polymorpha, asymmetric division occurs upon spore germination to generate two daughter cells: the larger one retains the ability to divide and develops into the thallus via sporeling or protonema, while the smaller one maintains tip growth and differentiates into the first rhizoid, providing a scaffold for initial development. Although spore germination of M. polymorpha was described in the 19th century, the intracellular processes of the first asymmetric division of the spore have not been well characterized. In this study, we used live-cell imaging analyses to elucidate microtubule dynamics during the first asymmetric division concomitantly with germination. In particular, we demonstrated that the preprophase band was not formed in the spore and that the bipolar prospindle, which is a microtubule structure surrounding the nucleus during prophase, migrated from the center to the periphery in the spore, suggesting that it was the earliest visible sign of cell polarity. We also showed that the occurrence of asymmetric division depended on actin filaments. Our findings regarding the first division of the spore in M. polymorpha will lead to a better model for cell-autonomous asymmetric division in plants.

Tracking metabolites at single-cell resolution reveals metabolic dynamics during plant mitosis.

Analyzing only one cell allows the changes and characteristics of intracellular metabolites during the chromosome segregation process to be precisely captured and mitotic sub-phases to be dissected at the metabolite level.