Biology subjects

Suzuki, K. G. N.

Publications and source records attributed to Suzuki, K. G. N..

8 recordsLinked to original sources

bioRxiv2025

RNA Dynamics Regulate Transcriptional Condensate Vivacity to Drive Gene Coordination

Transcriptional condensates (TCs), enriched with Mediator, orchestrate super-enhancer (SE)-driven gene expression critical for cell identity. However, how their dynamic physical properties shape transcriptional activities remains unclear. Here, we reveal a previously unknown regulatory axis wherein dynamic features of enhancer RNA (eRNA), transcribed but rapidly degraded by the RNA exosome, maintain optimal TC fluidity and stability. Depletion of RNA exosome disrupts TC integrity, altering Mediator and Pol II colocalization, in embryonic stem cells. This TC perturbation attenuates proper transcriptional regulator loading and pause-release regulation, enhances transcriptional noise, and diminishes coordinated eRNA-mRNA expression for SE-associated genes. Multi-omics analyses, live-cell imaging, and computational modeling collectively demonstrate that RNA turnover modulates TC vivacity, facilitating widespread chromatin contacts between SE-containing active A compartments and coordinated transcriptional bursts across SE-associated genes. Our findings establish RNA-dependent condensate dynamics as an essential quality-control mechanism that fine-tunes global transcriptional coordination, maintaining cellular homeostasis and cell identity. HighlightsO_LIRNA exosome is required for the homeostasis of transcriptional condensates (TCs) C_LIO_LIRNA synthesis and decay optimize the fluidity and dynamic properties of TCs C_LIO_LITCs maintain the transcriptional consistency of super-enhancer (SE) genes C_LIO_LITCs support contacts of SE-containing A compartments for coordinated transcription C_LI

molecular biology↗

bioRxiv2024

Extracellular vesicles adhere to cells predominantly through the interaction of CD151-associated integrin heterodimers and GM1 with laminin

Tumor-derived extracellular vesicles (EVs) have attracted significant attention, yet the molecular mechanisms that govern their specific binding to recipient cells remain elusive. Our in vitro study utilizing single-particle tracking demonstrated that integrin heterodimers comprising 6{beta}4 and 6{beta}1 and ganglioside, GM1 are responsible for the binding of small-EV (sEV) subtypes to laminin. EVs derived from four distinct tumor cell lines, regardless of size, exhibited high binding affinities for laminin but not for fibronectin, although fibronectin receptors are abundant in EVs and have functional roles in EV-secreting cells. Our findings revealed that integrins in EVs bind to laminin via the conventional molecular interface, facilitated by CD151 rather than by inside-out signaling of talin-1 and kindlin-2. Super-resolution movie observation revealed that sEV integrins bind only to laminin on living recipient cells. Furthermore, sEVs bound to HUVEC and induced cell branching morphogenesis in a laminin-dependent manner. Thus, we demonstrated that EVs predominantly bind to laminin on recipient cells, which is indispensable for cell responses. SummaryQuantitative assessments using single-molecule imaging and super-resolution microscopy revealed that all extracellular vesicle subtypes derived from four distinct tumor cell lines, regardless of size, bind to laminin predominantly via CD151-facilitated integrin heterodimers and GM1, leading to response of recipient cells.

cell 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↗

bioRxiv2023

A non-toxic equinatoxin-II reveals the dynamics of sphingomyelin in the cytosolic leaflet of the plasma membrane

Super-resolution microscopic observation of a novel non-toxic sphingomyelin probe revealed the formation of dynamic small domains including sphingomyelin and cholesterol in the cytosolic leaflet of living cell plasma membranes. AbstractSphingomyelin (SM) is a major sphingolipid in mammalian cells. SM is enriched in the extracellular leaflet of the plasma membrane (PM). Besides this localization, recent electron microscopic and biochemical studies suggest the presence of SM in the cytosolic leaflet of the PM. In the present study, we generated a non-toxic SM-binding variant (NT-EqtII) based on equinatoxin-II (EqtII) from the sea anemone Actinia equina, and examined the dynamics of SM in the cytosolic leaflet of living cell PMs. NT-EqtII with two point mutations (Leu26Ala and Pro81Ala) had essentially the same specificity and affinity to SM as wild-type EqtII. NT-EqtII expressed in the cytosol was recruited to the PM in various cell lines. Super-resolution microscopic observation revealed that NT-EqtII formed tiny domains that were significantly colocalized with cholesterol and N-terminal Lyn. Meanwhile, all the examined lipid probes including NT-EqtII underwent apparent fast simple Brownian diffusion, exhibiting that SM and other lipids in the cytosolic leaflet rapidly moved in and out of domains. Thus, the novel SM-binding probe demonstrated the presence of the raft-like domain in the cytosolic leaflet of living cell PMs.

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↗

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↗