Biology subjects

Kusumi, A.

Publications and source records attributed to Kusumi, A..

15 recordsLinked to original sources

bioRxiv2026

A visualization framework for cell division activity and orientation in pre-anthesis ovaries of Prunus species

Fruit size and shape, which influence horticultural quality, are determined by the number and the size of the cells in the local region. In fruit trees, however, the difficulty of applying molecular genetic approaches has hindered a detailed understanding of the localization and orientation of cell division in developing fruit tissues. In this study, we established a novel framework to visualize cell division in pre-anthesis ovaries of three drupe crops, peach (Prunus persica), Japanese apricot (P. mume) and the interspecific hybrid Japanese apricot (P. salicina x P. mume), providing clear insight into the spatial distribution and orientation of dividing cells. We systematically optimized a 5-ethynyl-2'-deoxyuridine (EdU) labeling protocol for thick ovary tissues by adjusting infiltration conditions and fixation methods. In addition, electron microscopy combined with wide-view tiling visualization was applied to directly identify dividing cells, including those undergoing chromosome segregation and cell plate formation. By combining with machine learning-based detection, we efficiently and objectively identified dividing cells. Using these complementary approaches, we found that cell division activity was broadly distributed throughout pre-anthesis ovaries in all three crops, without pronounced spatial restriction. In contrast, analysis of division orientation revealed region-specific patterns: cells in the outermost exocarp divided predominantly anticlinally, whereas cells in the mesocarp divided largely periclinally, consistent with subsequent ovary (fruit) enlargement. The integrated framework presented here provides a foundation for understanding the spatial and three-dimensional regulation of fruit development and for future studies in fruit morphogenesis and horticulture.

plant biology↗

bioRxiv2025

Spatially resolved analysis of growth dynamics in pome and drupe fruits of Rosaceae using 3D Gaussian Splatting

Fruit growth has long been described using single- or double-sigmoid curves; however, these temporal models cannot fully capture the spatial heterogeneity that ultimately shapes a fruit. Here, we present a three-dimensional analysis pipeline that non-destructively tracks spatial fruit growth dynamics from field-collected imaginary. Surface landmarks were drawn, and video recordings were taken throughout development for three pome fruits, apple (Malus x domestica), Japanese pear (Pyrus pyrifolia) and European pear (Pyrus communis), and two drupe fruits, peach (Prunus persica) and Japanese apricot (Prunus mume), to track their motion. Using 3D Gaussian Splatting, we successfully reconstructed 3D models of the fruits, and the landmark displacement could be measured with high accuracy, with R2 [≥] 0.98 when compared to manual recordings. We found a common spatial growth gradient in the longitudinal growth shared in the pomes and drupes of the Rosaceae; proximal (stem-end) regions exhibited more pronounced growth than the distal (stylar) end. An exception was found in European pear Bartlett, which showed relatively vigorous growth in the distal region, explaining its distinct shape with expanded distal end. Transverse expansion varied far less than longitudinal expansion, with a possible association with initial fruit morphology. Inter-fruit growth variability peaked in the fastest-growing regions, particularly in the distal area of the European pear, highlighting the link between growth vigor and phenotypic variance. These results provide foundational insights into the developmental dynamics of both pome and drupe fruits of the Rosaceae family, contributing to the optimization of fruit size, shape, and uniformity.

plant biology↗

bioRxiv2025

Spatially resolved analysis of longitudinal fruit growth in persimmon (Diospyros kaki) via three-dimensional phenotyping

Understanding fruit development patterns is essential, as they are directly linked to fruit size and shape, which ultimately determine final yield and quality. While conventional models describe fruit development based on growth parameters for the whole fruit, it remains unclear how tissue growth at different fruit portions is coordinated to promote overall fruit development. Therefore, in this study, we investigated the relationship between differences in developmental rates among fruit portions and fruit morphology in persimmon (Diospyros kaki), a species known for its high morphological diversity. Starting two weeks after blooming (WAB), surface landmarks were routinely drawn along the calyx-apex line on fruits of four persimmon cultivars with diverse shapes, and the landmark displacement was quantified every two weeks using 3D phenotyping. Consistent with previous studies, all cultivars exhibited active development in the regions near the calyx, with the greatest difference in growth rates between the calyx and apex occurring at 6-8 WAB. However, the extent of these spatial differences varied among cultivars. In elongated fruits, a gradual growth gradient was observed from the calyx toward the apex, with relatively high growth rates even near the apex. In contrast, in flattened fruits, growth was highly concentrated near the calyx, while development in other regions remained minimal. The findings of this study provide a foundation for future research on fruit shape regulation and the elucidation of physiological disorders in persimmon. HighlightsO_LISpatially resolved 3D phenotyping quantified growth dynamics in persimmon fruit C_LIO_LIGrowth rates varied along the fruit axis, with active growth near the calyx in all tested cultivars C_LIO_LIGrowth gradients were linked to final fruit shape C_LI

plant biology↗

bioRxiv2025

SynGAP forms biocondensates at sub-micromolar concentrations and recruits PSD95 and receptor oligomers, functioning as a key initiator of PSD formation

A key issue in neuronal circuit regulation is how synapse formation is initiated. Synapse formation could start when one or more synaptic scaffold proteins that can initiate synapse formation reach certain threshold concentrations in the dendritic shaft, which might lead to their oligomerization or even liquid-liquid phase separation (LLPS). By combining in vitro reconstitution of purified proteins with live-cell single-molecule and confocal imaging, we demonstrated that SynGAP alone forms assemblies of nanoscale clusters containing several to several tens of molecules at 10-nM order concentrations and micron-scale LLPS hydrogel-like condensates at submicromolar concentrations. The trimers of SynGAP's intrinsically disordered region (IDR) induced by its coiled-coil domain are responsible for SynGAP condensation. CaMKII-mediated phosphorylation moderately suppresses SynGAP condensation, and also increases condensate liquidity. While PSD95 fails to form assemblies under these conditions, it is recruited to SynGAP condensates by specifically binding to the PDZ-binding motif of SynGAP. SynGAP[PSD95] condensates selectively immobilize postsynaptic transmembrane proteins, Neuroligin1 and AMPAR-TARP2 complexes, in a manner dependent on their oligomerization state, indicating cooperative recruitment dynamics among SynGAP, PSD95, and transmembrane components, which might mimic initial PSD assembly. These findings suggest that SynGAP may act as a primary nucleator of postsynaptic density assembly, challenging the PSD95-centered models.

neuroscience↗

bioRxiv2024

Single-molecule detection of transient dimerization of opioid receptors 1: Homodimers' effect on signaling and internalization

Opioid receptors (ORs) are critical for endogenous and synthetic analgesics. Their homodimerization is considered important for their pharmacological diversities, but whether they form homodimers remains controversial. Here, we established that the three classical ORs, -, {kappa}-, and {delta}-ORs (MOR, KOR, and DOR, respectively) undergo repeated transient (120-180 ms) homodimerizations every few seconds. This was done by using single-molecule imaging and developing theories for analyzing single-molecule colocalization data, which provide the key parameters, homodimer-monomer dissociation equilibrium constants and rate constants. Their 9-26 amino-acid C-terminal cytoplasmic domains, without sequence similarities, are involved in specific homodimerization, whereas the transmembrane domains provide less specific affinities. Using the membrane-permeable peptides mimicking the C-terminal homodimerization sequences which block homodimerizations, functions of monomers and homodimers were dissected. KOR and DOR homodimers, but not MOR homodimers, activate downstream G-proteins differently from monomers upon agonist addition, without influencing OR internalization. These findings could guide strategies to enhance OR-based analgesia.

biophysics↗

bioRxiv2024

Single-molecule detection of transient dimerization of opioid receptors 2: Heterodimer blockage reduces morphine tolerance

Heterodimerization of opioid receptors (ORs), MOR, KOR, and DOR, is implied in their functional regulation and diversification, and thus its understanding is crucial for developing better analgesic treatments. However, our knowledge on OR heterodimerization/heterodimers remains limited. Here, using single-molecule imaging and functional analysis, we found that MOR, the main morphine receptor, repeatedly forms transient ({approx}250 ms) heterodimers with DOR every 1-10 seconds, but not with KOR, whereas DOR and KOR also form transient heterodimers. We obtained all the heterodimer-monomer equilibrium constants and rate constants with/without agonists. We identified the critical heterodimer binding sites in the extracellular domains, in addition to the less-specific transmembrane domains, and developed soluble peptide blockers for MOR-DOR and DOR-KOR heterodimerization, using amino-acid sequences mimicking the extracellular binding sites. With these peptide blockers, we dissected the monomer/dimer roles in OR internalization and signaling. The soluble MOR-DOR heterodimer blocker reduced the development of long-term morphine tolerance in mice.

biophysics↗

bioRxiv2023

Three-dimensional fruit growth analysis clarifies developmental mechanisms underlying complex shape diversity in persimmon fruit

How fruit size and shape are determined is of research interest in agriculture and developmental biology. Fruit typically exhibits three-dimensional structures with genotype-dependent geometric features. Although minor developmental variations have been recognized, little research has fully visualized and measured these variations throughout fruit growth. In this study, a high-resolution 3D scanner was used to investigate the fruit development of 51 persimmon (Diospyros kaki) cultivars with various complex shapes. We obtained 2,380 3D fruit models that fully represented fruit appearance, and enabled precise and automated measurements of unique geometric features throughout fruit development. The 3D fruit model analysis identified key stages that determined the shape attributes at maturity. Typically, genetic diversity in vertical groove development was found, and such grooves can be filled by tissue expansion in the carpal fusion zone during fruit development. Furthermore, transcriptome analysis of fruit tissues from groove/non-groove tissues revealed gene co-expression networks that were highly associated with groove depth variation. The presence of YABBY homologs was most closely associated with groove depth and indicated the possibility that this pathway is a key molecular contributor to vertical groove depth variation. These results demonstrate the validity of fruit 3D growth analysis, which is a powerful tool for identifying the developmental mechanisms of fruit shape variation and the molecular basis of this diversity.

plant biology↗

bioRxiv2021

iTRVZ: liquid nano-platform for signal integration on the plasma membrane

Cell survival in immune-competent organisms relies on both growth and immune evasion, yet these processes have been studied as independent cellular programs. Here, using super-resolution single-molecule imaging, we identify GEM (Growth and Evasion Metastable hub), a {approx}34-nm, metastable, nano-liquid, plasma membrane hub that physically integrates receptor-type tyrosine kinase (RTK) inputs with CD59-mediated immune-evasion signaling within the same assembly. GEM is formed by LLPS-like protein clustering on PI(4,5)P2-containing raft-nanodomains. Upon concurrent RTK and CD59 stimulations, GEM recruits both receptors and downstream kinases, enabling reciprocal activation via nano-liquid confinement that supralinearly amplifies PLC{gamma}-IP3-Ca2+ and PI3K-Akt survival signaling outputs. In mice, disrupting GEM suppresses tumor growth in vivo, establishing nano-liquid GEM as an integrator that physically couples growth and CD59-mediated immune-evasion signaling for cell survival.

cell biology↗

bioRxiv2021

Development of ultrafast camera-based imaging of single fluorescent molecules and live-cell PALM

The spatial resolution of fluorescence microscopy has recently been greatly improved. However, its temporal resolution has not been improved much, despite its importance for examining living cells. Here, by developing an ultrafast camera system, we achieved the worlds highest time resolutions for single fluorescent-molecule imaging of 33 (100) {micro}s (multiple single molecules simultaneously) with a single-molecule localization precision of 34 (20) nm for Cy3 (best dye found), and for PALM data acquisition of a view-field of 640x640 pixels at 1 kHz with a single-molecule localization precision of 29 nm for mEos3.2. Both are considered the ultimate rates with available probes. This camera system (1) successfully detected fast hop diffusion of membrane molecules in the plasma membrane, detectable previously only by using less preferable 40-nm gold probes and bright-field microscopy, and (2) enabled PALM imaging of the entire live cell, while revealing meso-scale dynamics and structures, caveolae and paxillin islands in the focal adhesion, proving its usefulness for cell biology research. SummaryAn ultrafast camera developed by Fujiwara et al. allows single fluorescent-molecule imaging every 33 s with a localization precision of 34 nm (every 100 s; 20 nm), and enables ultrafast PALM imaging of whole live cells.

cell biology↗

bioRxiv2021

Focal adhesion membrane is dotted with protein islands and partitioned for molecular hop diffusion

Using the ultrafast camera system and new theories for hop diffusion described in the companion paper, we for the first time demonstrated that membrane molecules undergo hop diffusion among the compartments in the bulk basal plasma membrane (PM), with virtually the same compartment sizes (108 nm) as those in the bulk apical PM and the same dwell lifetimes within a compartment (10 and 24 ms for the phospholipid and transferrin receptor, respectively), suggesting that the basic structures and molecular dynamics are very similar in the bulk regions of the apical and basal PMs. Ultrafast PALM and single-molecule imaging revealed that the focal adhesion (FA) is mostly a fluid membrane, partitioned into [~]74-nm compartments where transferrin receptor and {beta}3 integrin undergo hop diffusion, and that the FA membrane is sparsely dotted with 51-nm diameter paxillin islands, where many other FA proteins probably assemble (compartmentalized archipelago model). {beta}3 integrin intermittently associates with the paxillin islands, dynamically linking them to the extracellular matrix. SummaryAn ultrafast camera with single fluorescent-molecule sensitivities developed by Fujiwara et al. reveals that the focal adhesion membrane is dotted with protein islands and partitioned for molecular hop diffusion, and integrin {beta}3 molecules become temporarily immobilized at the islands.

cell biology↗

bioRxiv2020

High-speed single-molecule imaging reveals signal transduction by induced transbilayer raft phases

Using single-molecule imaging with enhanced time resolutions down to 5 ms, we found that CD59-cluster rafts and GM1-cluster rafts stably induced in the outer leaflet of the plasma membrane (PM), which triggered the activation of Lyn, H-Ras, and ERK, continually recruited Lyn and H-Ras right beneath them in the inner leaflet, with dwell lifetimes of <0.1 s. The detection was possible due to the enhanced time resolutions employed here. The recruitment depended on the PM cholesterol and saturated alkyl chains of Lyn and H-Ras, whereas it was blocked by the non-raftophilic transmembrane protein moiety and unsaturated alkyl chains linked to the inner-leaflet molecules. Since GM1-cluster rafts recruited Lyn and H-Ras as efficiently as CD59-cluster rafts, and the protein moieties of Lyn and H-Ras were not required for the recruitment, we conclude that the transbilayer raft phases induced by the outer-leaflet stabilized rafts recruit lipid-anchored signaling molecules by lateral raft-lipid interactions, and thus serve as a key signal transduction platform. SummaryHigh-speed single-molecule imaging indicated that CD59-cluster rafts and GM1-cluster rafts stably induced in the plasma membrane outer leaflet generated nano-scale transbilayer raft phases, which continually and transiently recruited Lyn and H-Ras in the inner leaflet by cooperative raft-lipid interactions.

cell biology↗

bioRxiv2020

Live cell imaging of single neurotrophin receptor molecules on human neuron in Alzheimer's disease

The changes in the receptor dynamics such as the surface movement of the receptor molecules on the plasma membrane are essential to receptor function. However, whether the receptor dynamics are affected by disease conditions is unknown. Neurotrophin receptors such as TrkA and p75NTR play a critical role in neuronal survival and their functions are highly affected in Alzheimers disease (AD). Using live-cell single-molecule imaging of neurotrophin receptors we examined the surface trafficking of TrKA and p75NTR molecules on human induced pluripotent stem cells (hiPSCs) derived live neurons from presenilin 1 (PSEN1) mutant AD patients and healthy subjects. Here we report that surface trafficking of p75NTR molecules on neurites is faster than that of TrkA molecules in healthy controls. The surface dynamics of TrkA molecules were elevated in AD patients compared to healthy individuals. In contrast, the surface movement of p75NTR was significantly smaller in AD patients compared to healthy individuals. Interestingly, amyloid beta1-42 (A{beta}1-42) administration increased the surface trafficking of both TrkA and p75NTR in healthy hiPSCs neurons. These findings provides the first evidence that the surface diffusion of TrkA and p75NTR molecules are altered in patients suffering from AD. Our data also suggest that A{beta}1-42 may responsible for the alteration of the surface movements of TrkA but not for p75NTR. One Sentence SummaryThe surface movements of neurotrophin receptors such as TrkA and p75NTR are altered in neurons derived from patients suffering from familial Alzheimers disease.

neuroscience↗

bioRxiv2020

Metastable GPCR dimers trigger the basal signal by recruiting G-proteins

G-protein-coupled receptors (GPCRs) constitute the largest family of integral membrane proteins in the human genome and are responsible for various important signaling pathways for vision, olfaction, gustation, emotion, cell migration, etc. A distinct feature of the GPCR-family proteins is that many GPCRs, including the prototypical GPCR, {beta}2-adrenergic receptor ({beta}2AR), elicit low levels of basal constitutive signals without agonist stimulation, which function in normal development and various diseases1-3. However, how the basal signals are induced is hardly known. Another general distinctive feature of GPCRs is to form metastable homo-dimers, with lifetimes on the order of 0.1 s, even in the resting state. Here, our single-molecule-based quantification4 determined the dissociation constant of {beta}2AR homo-dimers in the PM (1.6 {+/-} 0.29 copies/m2) and their lifetimes (83.2 {+/-} 6.4 ms), and furthermore found that, in the resting state, trimeric G-proteins were recruited to both {beta}2AR monomers and homo-dimers. Importantly, inverse agonists, which suppress the GPCRs basal constitutive activity, specifically blocked the G-protein recruitment to GPCR homo-dimers, without affecting that to monomers. These results indicate that the G-proteins recruited to transient GPCR homo-dimers are responsible for inducing their basic constitutive signals. These results suggest novel drug development strategies to enhance or suppress GPCR homo-dimer formation.

biochemistry↗

bioRxiv2019

Dynamic actin-mediated nano-scale clustering of CD44 regulates its meso-scale organization at the plasma membrane

Transmembrane adhesion receptors at the cell surface, such as CD44, are often equipped with modules to interact with the extracellular-matrix(ECM) and the intra-cellular cytoskeletal machinery. CD44 has been recently shown to compartmentalize the membrane into domains by acting as membrane pickets, facilitating the function of signaling receptors. While spatial organization and diffusion studies of membrane proteins are usually conducted separately, here we combine observations of organization and diffusion by using high spatio-temporal resolution imaging on living cells to reveal a hierarchical organization of CD44. CD44 is present in a meso-scale meshwork pattern where it exhibits enhanced confinement and is enriched in nano-clusters of CD44 along its boundaries. This nanoclustering is orchestrated by the underlying cortical actin dynamics. Interaction with actin is mediated by specific segments of the intracellular-domain(ICD). This influences the organization of the protein at the nano-scale, generating a selective requirement for formin over Arp2/3-based actin-nucleation machinery. The extracellular-domain(ECD) and its interaction with elements of ECM do not influence the meso-scale organization, but may serve to reposition the meshwork with respect to the ECM. Taken together, our results capture the hierarchical nature of CD44 organization at the cell surface, with active cytoskeleton-templated nano-clusters localized to a meso-scale meshwork pattern.

cell biology↗

bioRxiv2019

Single-molecule analysis reveals the rapid effect of estradiol on the surface movement of AMPAR in live neurons

The gonadal steroid 17{beta}-estradiol (E2) rapidly alters glutamatergic neurotransmission, but its direct effect on the AMPA receptor (AMPAR) remains unknown. Live-cell single-molecule imaging experiments revealed that E2 rapidly and dose-dependently alters the surface movement of AMPAR via membrane estrogen receptors with distinct effects on somas and neurites. The effect of E2 on the surface mobility of AMPAR depends on the integrity of the cortical actin network.

neuroscience↗