Biology subjects

Matsumoto, C.

Publications and source records attributed to Matsumoto, C..

7 recordsLinked to original sources

bioRxiv2026

Intracellular Mechanosensation in Intestinal Smooth Muscle: Piezo1 Complexes Amplify Signaling Beyond the Surface

Mechanosensation is fundamentally viewed as a plasma membrane phenomenon. We challenge this paradigm by introducing intracellular mechanosensation in intestinal smooth muscle. We hypothesized that a distinct, organelle-based signaling axis exists to amplify mechanotransduction from the inside out. To test this, we investigated whether Piezo1, a canonical plasma membrane mechanosensor, also operates within the cell. Using tissue-level wire myography, high-resolution confocal microscopy, proximity ligation assays, and patch-clamp electrophysiology on freshly dissociated cells, we identified a functional intracellular signaling hub that starts at the sarcoplasmic reticulum (SR). Unlike surface transduction, this intracellular mechanism relies on a nanoscale multiprotein complex (<40 nm) comprising an SR sensor (intra-Piezo1) and an amplifier (Ryanodine Receptor, RyR), coupled with a PM effector (large-conductance, Ca2+-activated K+ channels, i.e., BKCa channels). Activating this intracellular complex generated massive BK-mediated outward currents independent of extracellular Ca{superscript 2} but strictly dependent on internal SR Ca{superscript 2} stores, confirming intrinsic organellar mechanotransduction. Within this complex, intra-Piezo1 and RyR are positioned to operate as a coupled SR Ca{superscript 2} release unit that activates BK channels at SR-PM junctions, driving potent membrane hyperpolarization that reduces smooth muscle contractility, revealing the intra-Piezo1 complex as a molecular brake on excitation. These findings demonstrate that mechanotransduction is not confined to the cell surface. Instead, a specialized Sensor-Amplifier-Effector complex originating at intracellular organelles amplifies cellular sensitivity to physical force, providing a critical gain-control system that restrains smooth muscle excitability and regulates GI motility. Key PointsO_LIIntracellular organelles contribute to mechanosensory signaling in GI smooth muscle cells, complementing plasma membrane mechanisms. C_LIO_LIIntra-Piezo1 form a nanoscale signaling complex (<40nm) on the sarcoplasmic reticulum (SR), linking the mechanosensor Piezo1 with RyR and large conductance, Ca2+-activated K+ channels. C_LIO_LIUnlike surface sensors, this intracellular complex functions via a "Sensor-Amplifier-Effector" mechanism in which intra-Piezo1 detects mechanical stress and triggers SR Ca2+ release, thereby activating a nearby RyR and large-conductance, Ca2+-activated K+ channel. C_LIO_LIEngaging this intracellular Piezo1-mediated axis significantly dampens smooth muscle contractility, identifying a critical gain-control system essential for regulating GI motility. C_LI

physiology↗

bioRxiv2025

Beat-locked ATP microdomains in the sinoatrial node map a calcium-timed energetic hierarchy and regional pacemaker roles

Pacemaker myocytes of the sinoatrial (SA) node initiate each heartbeat through coupled voltage and Ca2+ oscillators, but whether ATP supply is regulated beat-by-beat in these cells remains unclear. Using genetically encoded sensors targeted to the cytosol and mitochondria, we tracked beat-resolved ATP dynamics in intact mouse SA node and isolated myocytes. Cytosolic ATP rose transiently with each Ca2+ transient and segregated into high- and low-gain phenotypes defined by the Ca2+-ATP coupling slope. Mitochondrial ATP flux adopted two stereotyped waveforms--Mode 1 "gains" and Mode 2 "dips." Within Mode 1 cells, ATP gains mirrored the cytosolic high/low-gain dichotomy; Mode 2 dips scaled linearly with Ca2+ load and predominated in slower-firing cells. High-gain/Mode 1 phenotypes localized to superior regions and low-gain/Mode 2 to inferior regions, paralleling gradients in rate, mitochondrial volume, and capillary density. Mechanistic dissection placed sarcoplasmic reticulum (SR) Ca2+ release upstream of ATP production, showing that Ca2+ triggers metabolic transients while membrane voltage primarily modulates their frequency. Inhibiting mitochondrial Ca2+ uptake and adenine nucleotide exchange eliminated beat-locked mito- and cyto-ATP signals, indicating that the mitochondrial Ca2+ uniporter (MCU)-adenine nucleotide translocase (ANT) machinery couples Ca2+ release to ATP fluctuations. Mode 2 recovery kinetics indicate slower ATP replenishment, which would favor low-frequency, fluctuation-rich firing in a subset of cells. Together, these findings reveal beat-locked metabolic microdomains in which Ca2+ transients time oxidative phosphorylation under a local O2 ceiling, unifying vascular architecture, mitochondrial organization, and Ca2+ signaling to match energy supply to excitability. This energetic hierarchy helps explain why some pacemaking myocytes are more likely to set the rate, whereas others may widen the bandwidth. SummaryBeat-locked cytosolic and mitochondrial ATP transients in SA-node myocytes sort into high-gain, low-gain, or consumption-dominant modes aligned with superior-inferior vascular-mitochondrial gradients. This energetic hierarchy lets high-gain cells set fast rates while low-gain/dip cells stabilize slow rhythms, broadening operating range but capping maximal bandwidth.

physiology↗

bioRxiv2025

Demonstration of Beat-to-Beat, On-Demand ATP Synthesis in Ventricular Myocytes Reveals Sex-Specific Mitochondrial and Cytosolic Dynamics.

The energetic demands on ventricular myocytes imposed by the transport of ions and cross-bridge cycling are well known, yet the spatiotemporal dynamics of ATP supply and demand remain poorly understood. Here, using confocal microscopy and genetically encoded fluorescent sensors targeted to mitochondria and cytosol, we visualized beat-to-beat ATP dynamics in ventricular myocytes from male and female mice. These probes showed fluctuations in mitochondrial ATP levels with each contraction, revealing two distinct, spatially localized waveforms--ATP "gain" and ATP "dip"--representing transient increases or decreases in matrix ATP levels, respectively. These waveforms were tightly phase-locked to intracellular Ca2+ transients and organized into energetic microdomains. Inhibition of the mitochondrial Ca2+ uniporter or the adenine nucleotide translocase attenuated these ATP transients. Although female myocytes exhibited larger mitochondrial ATP transients than their male counterparts, their mitochondrial volume was lower. Female myocytes also exhibited tighter coupling between the sarcoplasmic reticulum and mitochondria and showed a higher density of mitofusin 2 and ATP synthase catalytic -subunit per unit volume, suggesting more efficient ATP production. Cytosolic ATP transients mirrored mitochondrial waveforms and domain structure in both male and female myocytes. During faster pacing, diastolic cytosolic ATP rose more rapidly in female myocytes, whereas beat-locked ATP transients increased in both sexes but proportionally more in males than in females. These findings demonstrate that ATP is synthesized on a beat-to-beat basis in a modular, microdomain-specific manner. We propose that male myocytes rely on greater mitochondrial mass for energetic scaling, whereas female cells employ architectural precision to optimize ATP delivery. Key points summaryO_LIIt is known that each heartbeat requires precise ATP delivery to fuel ion transport and cross-bridge cycling, but the timing and spatial organization of ATP production in heart cells has been unclear. C_LIO_LIUsing advanced imaging and genetically encoded sensors, we visualized beat-to-beat ATP fluctuations in the mitochondria and cytosol of individual male and female mouse ventricular myocytes. C_LIO_LIMitochondrial ATP levels rose or fell with each beat in spatially confined regions, forming ATP "gain" or "dip" microdomains that were synchronized with Ca2+ transients. C_LIO_LIAt higher firing rates, beat-locked, diastolic ATP transients rose more quickly in female myocytes, but were larger in male myocytes, highlighting distinct sex-specific strategies for matching energy supply to contractile demand. C_LIO_LIVentricular myocytes "live paycheck-to-paycheck", producing just enough ATP on demand to fuel each beat. Male and female myocytes adopt distinct strategies to meet this demand: male myocytes scale output through greater mitochondrial mass, while female myocytes achieve energetic precision via enhanced sarcoplasmic reticulum-mitochondrial coupling. C_LI O_FIG O_LINKSMALLFIG WIDTH=192 HEIGHT=200 SRC="FIGDIR/small/663572v2_ufig1.gif" ALT="Figure 1"> View larger version (108K): org.highwire.dtl.DTLVardef@12722org.highwire.dtl.DTLVardef@1b041cdorg.highwire.dtl.DTLVardef@b007eeorg.highwire.dtl.DTLVardef@d8c3a4_HPS_FORMAT_FIGEXP M_FIG O_FLOATNOAbstract figureC_FLOATNO Beat-locked mitochondrial ATP transients reveals modular, sex-specific bioenergetic control during excitation-contraction coupling. (A) Each action potential activates L-type CaV1.2 channels, producing a Ca2+ influx that triggers Ryanodine receptors (RyR2) and elicits SR Ca2+ release. (B) The cytosolic Ca2+ signal is decoded by mitochondria into spatially distinct "high-gain" and "low-gain" regions, shaped by the extent of SR-mitochondrial tethering via mitofusin 2 (Mfn2), yielding heterogeneous mitochondrial activation rather than a uniform, cell-wide metabolic response. (C) Mitochondria generate rhythmic, phase-locked ATP transients within discrete microdomains, such that ATP increases and ATP dips can coexist within the same cell. Ca2+ entry through the outer membrane (via VDAC) and into the matrix (via MCU) stimulates oxidative phosphorylation, increasing ATP production; ATP is exported by ANT to create local cytosolic "supply bursts" aligned with beat-to-beat demand ("paycheck-to-paycheck" energetics). Female myocytes show a higher prevalence of tightly coupled, high-gain ATP-producing microdomains, whereas male myocytes display a shifted balance toward lower-gain regions, consistent with sex-dependent SR-mitochondrial coupling and ATP microdomain patterning. C_FIG

physiology↗

bioRxiv2023

Astrocyte-specific inhibition of primary cilium functions improves cognitive impairment during neuroinflammation by suppressing A1 astrocyte differentiation.

A1 astrocytes play a neurotoxic role in various neurodegenerative diseases. While inhibiting the differentiation of A1 astrocytes can slow disease progression, the mechanisms controlling A1 astrocyte differentiation are largely unknown. The primary cilium is a cellular organelle that receives extracellular signals and regulates cell proliferation, differentiation, and maturation. To elucidate the physiological function of the primary cilium in A1 astrocytes, we utilized primary astrocytes and an inflammation mouse model. We found that the length of the primary cilium was increased in astrocytes, and the inhibition of primary cilium formation inhibited their differentiation into A1 astrocytes. Since mice with systemic ciliogenesis defects exhibit embryonic lethality, the function of the primary cilium in adults has remained largely unclear. Therefore, we established conditional knockout (cKO) mice that specifically inhibit primary cilium function in astrocytes upon drug stimulation. In a neuroinflammation mouse model in which lipopolysaccharide (LPS) was intraperitoneally injected into wild-type mice, increases in A1 astrocyte number and primary cilium length were observed in the brain. In contrast, cKO mice exhibited a reduction in the proportions of A1 astrocytes and apoptotic cells in the brain. Additionally, the novel object recognition (NOR) score observed in the cKO mice was higher than that observed in the neuroinflammation model mice. These results suggest that the primary cilium in astrocytes is essential for A1 astrocyte differentiation, which leads to a decline brain function. We propose that regulating astrocyte-specific primary cilium signalling may be a novel strategy for the suppression of neuroinflammation.

neuroscience↗

bioRxiv2023

The formation of KV2.1 macro-clusters is required for sex-specific differences in L-type CaV1.2 clustering and function in arterial myocytes

In arterial myocytes, the canonical function of voltage-gated CaV1.2 and KV2.1 channels is to induce myocyte contraction and relaxation through their responses to membrane depolarization, respectively. Paradoxically, KV2.1 also plays a sex-specific role by promoting the clustering and activity of CaV1.2 channels. However, the impact of KV2.1 protein organization on CaV1.2 function remains poorly understood. We discovered that KV2.1 forms micro-clusters, which can transform into large macro-clusters when a critical clustering site (S590) in the channel is phosphorylated in arterial myocytes. Notably, female myocytes exhibit greater phosphorylation of S590, and macro-cluster formation compared to males. Contrary to current models, the activity of KV2.1 channels seems unrelated to density or macro-clustering in arterial myocytes. Disrupting the KV2.1 clustering site (KV2.1S590A) eliminated KV2.1 macro-clustering and sex-specific differences in CaV1.2 cluster size and activity. We propose that the degree of KV2.1 clustering tunes CaV1.2 channel function in a sex-specific manner in arterial myocytes.

physiology↗

bioRxiv2023

A Computational Model Predicts Sex-specific Responses to Calcium Channel Blockers in Mesenteric Vascular Smooth Muscle

The function of the smooth muscle cells lining the walls of mammalian systemic arteries and arterioles is to regulate the diameter of the vessels to control blood flow and blood pressure. Here, we describe an in-silico model, which we call the "Hernandez-Hernandez model", of electrical and Ca2+ signaling in arterial myocytes based on new experimental data indicating sex-specific differences in male and female arterial myocytes from murine resistance arteries. The model suggests the fundamental ionic mechanisms underlying membrane potential and intracellular Ca2+ signaling during the development of myogenic tone in arterial blood vessels. Although experimental data suggest that KV1.5 channel currents have similar amplitudes, kinetics, and voltage dependencies in male and female myocytes, simulations suggest that the KV1.5 current is the dominant current regulating membrane potential in male myocytes. In female cells, which have larger KV2.1 channel expression and longer time constants for activation than male myocytes, predictions from simulated female myocytes suggest that KV2.1 plays a primary role in the control of membrane potential. Over the physiological range of membrane potentials, the gating of a small number of voltage-gated K+ channels and L-type Ca2+ channels are predicted to drive sex-specific differences in intracellular Ca2+ and excitability. We also show that in an idealized computational model of a vessel, female arterial smooth muscle exhibits heightened sensitivity to commonly used Ca2+ channel blockers compared to male. In summary, we present a new model framework to investigate the potential sex-specific impact of anti-hypertensive drugs.

physiology↗

bioRxiv2022

Automated container-less cell processing method for single-cell proteomics

Single-cell genomics and transcriptomics studies enabled us to characterize cell heterogeneity in various tissues, which helped us to better understand the biological system and disease progression. Single-cell proteomics, which directly measures the protein expression level, has the potential to further enhance our knowledge by providing not only a more direct measurement but also crucial information cannot be captured by genomics or transcriptomics study, such as protein activation states and post-translation modification events. One of the main challenges of single-cell proteomics is the large sample loss during sample preparation, which is largely unavoidable using standard proteomics protocols. Protein and peptide loss to the container surface is a well-known phenomenon but often overlooked in larger-scale (>1 {micro}g) proteomics experiments. When it comes to single-cell proteomics with only picograms of protein samples, this loss becomes non-negligible and often dictates the outcomes of the experiment. More importantly, sample processing through multiple pipette tips and containers often introduces random errors, which undermine the ability to detect true heterogenous cellular events. To solve these problems and further improve the throughput and reproducibility of the single-cell proteomics experiments, we developed an automated container-less cell processing platform, utilizing acoustic levitation to process cell samples in the air. Our platform automatically performs cell lysis, protein reduction, alkylation, digestion, and peptide labeling in the air, without any sample transfer step or container. The digested and labeled peptides are then directly injected into the capillary LC-MS/MS system for analysis, eliminating manual steps and conserving most of the sample materials for proteomics analysis. Our initial test shows at least 30% improvement in peptide signals over conventional methods. This process can be performed in parallel to further improve sample processing throughput.

biochemistry↗