Biology subjects

Murayama, T.

Publications and source records attributed to Murayama, T..

18 recordsLinked to original sources

bioRxiv2026

Highly plastic macrophage niches orchestrate acquired quiescence and reactivation in breast-cancer bone metastasis

Recurrence and metastasis remain major causes of cancer mortality, sustained by therapy-resistant micrometastatic cells. Bone is a frequent site of breast-cancer relapse, yet the cues that reawaken disseminated cells remain poorly defined. We identify a previously unrecognized, highly plastic CXCL16 macrophage population that integrates tumor-associated macrophage programs found in distant metastatic sites such as lung and brain with non-tumor disease-associated traits in bone marrow. These CXCL16 macrophages establish a transient niche that restrains disseminated cancer-cell proliferation. Single-cell transcriptomics delineate functional remodeling of myeloid niches within the bone metastatic microenvironment: a CXCL16 macrophage niche that transiently constrains metastatic growth, and G-CSF macrophage and neutrophil niches that reignite tumor outgrowth. In primary tumors, cancer-associated fibroblasts (CAFs) aberrantly secrete G-CSF in response to cancer-cell signals, expanding G-CSF-receptor-positive subset of cancer cells with high metastatic potential. In advanced human bone metastases, CXCL16 macrophages localize to CAF-rich stroma but are excluded from cancer-cell clusters, indicating immune evasion. Together, these findings uncover CAF-bone-marrow cross-talk as a therapeutic target linking stromal inflammation, immune remodeling, and metastatic progression.

cancer biology↗

bioRxiv2026

Developmental emergence of spatiotemporal coordination in cerebellar Purkinje cell populations

Coordinated activity across neuronal populations is fundamental to brain function, yet how such network-level organization emerges during development remains incompletely understood. Here, we performed whole-cerebellar calcium imaging at cellular scale in zebrafish larvae to examine the developmental maturation of Purkinje cell population dynamics. Visual stimulation recruited large, spatially organized populations of Purkinje cells whose responses depended on inferior olive input and were associated with optokinetic behavior. Notably, in the absence of stimuli, Purkinje cells formed transient assemblies exhibiting distance-dependent coordination. During development, long-range coordination progressively emerged, extending initially local correlations into distributed population-wide coordination. Early enucleation, but not dark rearing, disrupted the developmental refinement of long-range coordination and induced aberrant population clustering, suggesting that early retina-dependent signals contribute to cerebellar network development. Together, these findings reveal key organizational features underlying the developmental emergence of coordinated cerebellar population dynamics and suggest that early retina-dependent signals shape population-level organization.

neuroscience↗

bioRxiv2026

Structural basis for Ca2+-dependent, RyR2-selective stabilization by Ryanozole in CPVT

The cardiac ryanodine receptor (RyR2) is a Ca{superscript 2} release channel essential for excitation-contraction coupling. RyR2 mutations cause severe arrhythmogenic disorders, including catecholaminergic polymorphic ventricular tachycardia (CPVT), through gain-of-function (GOF) effects leading to aberrant Ca{superscript 2} release. We have recently developed Ryanozole, a potential therapeutic compound for CPVT, which selectively stabilizes RyR2 in a Ca2+-dependent manner. Here, we define the mechanism of action of Ryanozole by combining high-resolution cryo-electron microscopy, targeted mutagenesis and functional assays. Ryanozole binds to the interface between the Ca{superscript 2}-binding site and the pore-forming S6 helix, interfering with conformational changes required for Ca{superscript 2}-induced channel opening at low Ca2+. We identified key residues for the binding and isoform-specific modulation of Ryanozole. Notably, Ryanozole-bound RyR2 retains its ability to open at high Ca2+ via unique conformational changes. These findings provide a structural basis for CPVT-targeted therapy and redefine the paradigm of small molecule-based regulation of large ion channels.

biochemistry↗

bioRxiv2025

Baculovirus-mediated gene transfer enables functional expression of PIEZO1 ion channel in isolated muscle satellite cells

Primary tissue stem cells are useful not only for basic cell biological research but also for therapeutic applications: however, their broader utility is often limited by technical challenges, such as a low efficiency of exogenous gene expression. PIEZO1 is a large mechanosensitive ion channel that plays an important role in muscle-resident stem cells, known as muscle satellite cells (MuSCs), during muscle regeneration. In this study, we developed a method for the ectopic expression of PIEZO1 in isolated MuSCs. Using a baculovirus vector system, we expressed PIEZO1 in myoblast C2C12 cells. Following optimization of the infection condition, we achieved robust PIEZO1 expression in isolated MuSCs during activated and differentiated states, with appropriate subcellular localization and ion channel activity. Importantly, the baculovirus-mediated PIEZO1 expression restored the reduced proliferative capacity of Piezo1-deficient MuSCs to a level comparable to wild-type cells, indicating that the exogenously expressed PIEZO1 is functionally equivalent to the endogenous protein. Overall, we established an efficient method for the transfer of the Piezo1 gene into isolated MuSCs, which should provide a versatile platform to study other large proteins in MuSCs. Summary StatementA baculovirus-based method was developed, enabling robust and functional PIEZO1 expression in isolated muscle satellite cells and providing a platform to study large proteins in isolated stem cells.

cell biology↗

bioRxiv2024

A novel RyR2-selective stabilizer prevents and ameliorates severe arrhythmias in stress-induced ventricular tachycardia

Background and PurposeAberrant activation of the type 2 ryanodine receptor (RyR2) causes lethal arrhythmias, such as catecholaminergic polymorphic ventricular tachycardia (CPVT). Developing drugs that suppress RyR2 hyperactivation may be key to novel arrhythmia treatments. This study evaluated the antiarrhythmic potential of Ryanozole, a recently developed novel RyR2 modulator with high affinity and selectivity, using CPVT mouse models harboring mutant RyR2s. Experimental approachIn vitro effects of Ryanozole were evaluated by ER Ca2+-based assay and [3H]Ryanodine binding assay using RyR2-expressing HEK293 cells. Two lines of mice with different arrhythmia severity, RyR2-R420W and -K4750Q, were employed for in vivo assessments. Intracellular Ca2+ signals were analyzed in isolated cardiomyocytes using Cal520. Antiarrhythmic effects were evaluated by ECG under catecholaminergic challenge in anesthetized mice and during spontaneous arrhythmias in conscious mice. ECG and echocardiographic parameters were evaluated before and after drug administration. Key resultsRyanozole inhibited both wild type and mutant RyR2s with similar IC50 of 15-40 nM. The inhibition was more potent at lower cytosolic Ca2+ concentrations. It suppressed Ca2+ waves and Ca2+ sparks without affecting action potential-evoked Ca2+ transients. Ryanozole effectively prevented adrenaline-induced arrhythmias and rapidly terminated spontaneous arrhythmias during daily activity. Importantly, Ryanozole did not impair cardiac conduction or contractility, unlike conventional antiarrhythmic drugs. Conclusions and implicationsRyanozole strongly suppresses RyR2 under diastolic Ca2+ conditions, thereby preventing the arrhythmogenic trigger of aberrant Ca2+ release. This mechanism likely provides potent antiarrhythmic effects while preserving cardiac function. Ryanozole is a promising therapeutic candidate treating RyR2-mediated arrhythmias, such as CPVT.

pharmacology and toxicology↗

bioRxiv2024

Dantrolene inhibition of ryanodine receptor 1 carrying the severe malignant hyperthermia mutation Y522S visualized by cryo-EM

Mutations in the skeletal isoform of the ryanodine receptor (RyR1) pose grave risks during anesthesia or treatment with succinylcholine muscle relaxants. These can trigger a potentially lethal malignant hyperthermia (MH) episode via intracellular calcium increase mainly from RyR1 channel leakage. Dantrolene is the primary antidote to prevent death. The main target of dantrolene is RyR1, however little is known about the mechanism of inhibition. Cryo-EM of dantrolene bound to the severe MH Y522S RyR1 mutant in the closed and open states at 2.5-3.3 [A] resolution revealed that the drug binds to the channels cytoplasmic assembly, far from the ion gate, interacting with residues W882, W996 and R1000 in the P1 domain. The finding was validated by Ca2+ imaging and [3H]ryanodine binding in WT and alanine mutants. Dantrolene reduced channel opening probability by restricting the central activation module, "cooling down" the primed conformation caused by the mutation. These findings shed light on dantrolenes mechanism against MH and suggest avenues for developing more effective therapeutics.

biochemistry↗

bioRxiv2024

Non-physiologic interaction of MG53 with insulin receptor and lack of evidence for MG53's role in controlling insulin-stimulated Akt phosphorylation in muscle, heart and liver tissues.

RationaleMG53s known function in facilitating tissue repair and anti-inflammation has broad applications to regenerative medicine. There is controversy regarding MG53s role in the development of type 2 diabetes mellitus (T2DM). ObjectiveThis study aims to address this controversy - whether MG53s myokine function contributes to inhibition of insulin signaling in muscle, heart, and liver tissues. Study DesignWe determined the binding affinity of the recombinant human MG53 (rhMG53) to the insulin receptor extracellular domain (IR-ECD) and found low affinity of interaction with Kd (>480 nM). Using cultured C2C12 myotubes and HepG2 cells, we found no effect of rhMG53 on insulin-stimulated Akt phosphorylation (p-Akt). We performed in vivo assay with C57BL/6J mice subjected to insulin stimulation (1 U/kg, intraperitoneal injection) and observed no effect of rhMG53 on insulin-stimulated p-Akt in muscle, heart and liver tissues. ConclusionOverall, our data suggest that rhMG53 can bind to the IR-ECD, however has a low likelihood of a physiologic role, as the Kd for binding is [~]10,000 higher than the physiologic level of MG53 present in the serum of rodents and humans ([~]10 pM). Our findings question the notion proposed by Xiao and colleagues - whether targeting circulating MG53 opens a new therapeutic avenue for T2DM and its complications.

physiology↗

bioRxiv2023

Carvedilol suppresses ryanodine receptor-dependent Ca2+ bursts in human neurons bearing PSEN1 variants found in early onset Alzheimer's disease

Seizures are increasingly being recognized as the hallmark of Alzheimers disease (AD). Neuronal hyperactivity can be a consequence of neuronal damage caused by abnormal amyloid {beta} (A{beta}) depositions. However, it can also be a cell-autonomous phenomenon causing AD by A{beta}-independent mechanisms. Indeed, various studies using animal models showed that Ca2+ releases from the endoplasmic reticulum (ER) via type 1 inositol triphosphate receptors (InsP3R1s) and ryanodine receptors (RyRs). To investigate which is the main pathophysiological mechanism in human neurons, we measured Ca2+ signaling in neural cells derived from three early-onset AD patients harboring variants of Presenilin-1 (PSEN1 p.A246E, p.L286V, and p.M146L). Of these, it has been reported that PSEN1 p.A246E and p.L286V did not produce a significant amount of abnormal A{beta}. We found that all PSEN1-mutant neurons, but not wild-type, caused abnormal Ca2+-bursts in a manner dependent on the calcium channel, Ryanodine Receptor 2 (RyR2). Indeed, carvedilol, anRyR2 inhibitor, and VK-II-86, an analog of carvedilol without the {beta}-blocking effects, sufficiently eliminated the abnormal Ca2+ bursts. In contrast, Dantrolene, a RyR1 inhibitor, and Xestospongin c, an IP3R inhibitor, did not attenuate the Ca2+-bursts. The RNA-Seq data revealed that ER-stress responsive genes were increased, and mitochondrial Ca2+-transporter genes were decreased in PSEN1A246E cells compared to the WT neurons. Thus, we propose that aberrant Ca2+ signaling is a key link between human pathogenic PSEN1 variants and cell-intrinsic hyperactivity prior to deposition of abnormal A{beta}, offering prospects for the development of targeted prevention strategies for at-risk individuals. One Sentence SummaryAberrant Ca2+-signaling causes PSEN1-related early onset Alzheimers disease.

neuroscience↗

bioRxiv2023

Screening for novel RyR2 inhibitors by ER Ca2+ monitoring

Type 2 ryanodine receptor (RyR2) is a Ca2+ release channel on the endoplasmic/sarcoplasmic reticulum (ER/SR) that plays a central role in the excitation-contraction coupling in the heart. Hyperactivity of RyR2 has been linked to ventricular arrhythmias in patients with catecholaminergic polymorphic ventricular tachycardia (CPVT) and heart failure, where spontaneous Ca2+ release via hyperactivated RyR2 depolarizes diastolic membrane potential to induce triggered activity. In such cases, drugs that suppress RyR2 activity are expected to prevent the arrhythmias, but there is no clinically available RyR2 inhibitors at present. In this study, we searched for RyR2 inhibitors from a well-characterized compound library using a recently developed ER Ca2+-based assay, where the inhibition of RyR2 activity was detected by the increase in ER Ca2+ signals from R-CEPIA1er, a genetically encoded ER Ca2+ indicator, in RyR2-expressing HEK293 cells. By screening 1535 compounds in the library, we identified three compounds (chloroxylenol, methyl orsellinate and riluzole) that greatly increased the ER Ca2+ signal. All of the three compounds suppressed spontaneous Ca2+ oscillations in RyR2-expressing HEK293 cells and correspondingly reduced the Ca2+-dependent [3H]ryanodine binding activity. In cardiomyocytes from RyR2-mutant mice, the three compounds effectively suppressed abnormal Ca2+ waves without substantial effects on the action-potential-induced Ca2+ transients. These results confirm that ER Ca2+-based screening is useful for identifying modulators of ER Ca2+ release channels and suggest that RyR2 inhibitors have potential to be developed as a new category of antiarrhythmic drugs. Significance statementWe successfully identified three compounds having RyR2 inhibitory action from a well-characterized compound library using an ER Ca2+-based assay, and demonstrated that these compounds suppressed arrhythmogenic Ca2+ wave generation without substantially affecting physiological action-potential induced Ca2+ transients in cardiomyocytes. This study will facilitate the development of RyR2 specific inhibitors as a potential new class of drugs for life-threatening arrhythmias induced by hyperactivation of RyR2.

pharmacology and toxicology↗

bioRxiv2022

High-throughput platform for depolarization-induced Ca2+ release for skeletal muscle disease mutation validation and drug discovery

In skeletal muscle excitation-contraction (E-C) coupling, depolarization of the plasma membrane triggers Ca2+ release from the sarcoplasmic reticulum (SR), referred to as depolarization-induced Ca2+ release (DICR). DICR occurs via the type 1 ryanodine receptor (RyR1), which physically interacts with the dihydropyridine receptor Cav1.1 subunit in specific machinery formed with additional essential components including {beta}1a, Stac3 adaptor protein and junctophilins. Exome sequencing has accelerated the discovery of many novel mutations in genes encoding DICR machinery in various skeletal muscle diseases. However, functional validation is time-consuming because it must be performed in a skeletal muscle environment. In this study, we established a platform of the reconstituted DICR in HEK293 cells. The essential components were effectively transduced into HEK293 cells expressing RyR1 using baculovirus vectors, and Ca2+ release was quantitatively measured with R-CEPIA1er, a fluorescent ER Ca2+ indicator, without contaminant of extracellular Ca2+ influx. In these cells, [K+]-dependent Ca2+ release was triggered by chemical depolarization with the aid of inward rectifying potassium channel, indicating a successful reconstitution of DICR. Using the platform, we evaluated several Cav1.1 mutations that are implicated in malignant hyperthermia and myopathy. We also tested several RyR1 inhibitors; whereas dantrolene and Cpd1 inhibited DICR, procaine had no effect. Furthermore, twitch potentiators such as perchlorate and thiocyanate shifted the voltage dependence of DICR to more negative potentials without affecting Ca2+-induced Ca2+ release. These results well reproduced the findings with the muscle fibers and the cultured myotubes. Since the procedure is simple and reproducible, the reconstituted DICR platform will be highly useful for validation of mutations and drug discovery for skeletal muscle diseases. SummaryMutations in essential components for depolarization-induced Ca2+ release (DICR) are implicated into various skeletal muscle diseases. Murayama et al. establish a reconstituted DICR platform in nonmuscle cells for evaluation of disease-causing mutations and drug discovery.

physiology↗

bioRxiv2021

Enhanced CICR activity reduces ER Ca2+ level in cells expressing CPVT-linked mutant RyR2

Type 2 ryanodine receptor (RyR2) is a cardiac Ca2+ release channel in the endoplasmic reticulum (ER). Mutations in RyR2 are linked to catecholaminergic polymorphic ventricular tachycardia (CPVT), which is considered to be associated with enhanced spontaneous Ca2+ release. This spontaneous Ca2+ release tends to occur when ER Ca2+ ([Ca2+]ER) reaches a certain threshold level, and CPVT mutations are reported to lower this threshold. There are two explanations for this lowered threshold: the mutations increase sensitivity to luminal Ca2+ or they enhance cytosolic [Ca2+] ([Ca2+]cyt)-induced Ca2+ release (CICR) activity. However, no quantitative analysis of this issue has been performed so far. Here, we quantitatively explored how the change in CICR activity of RyR2 affects the threshold [Ca2+]ER experimentally and by model-based simulation. Wild-type (WT) and CPVT-linked mutant RyR2s were expressed in HEK293 cells. [Ca2+]cyt and [Ca2+]ER measurements with Ca2+ indicators revealed that CPVT RyR2 cells showed higher oscillation frequency and lower threshold [Ca2+]ER in a mutation-specific manner compared with WT cells. The CICR activity of mutant RyR2s was assessed by Ca2+-dependent [3H]ryanodine binding and parameter analysis. CICR activity at resting [Ca2+]cyt, A7.0, was higher in CPVT mutants than in WT and a strong inverse correlation was found between threshold [Ca2+]ER and A7.0. Interestingly, lowering RyR2 expression increased threshold [Ca2+]ER, suggesting that the threshold [Ca2+]ER depends on net Ca2+ release rate via RyR2, a product of A7.0 for each mutant and the density of RyR2 molecules. A model-based simulation successfully reproduced the [Ca2+]cyt and [Ca2+]ER changes. Interestingly, the CICR activity associated with specific mutations correlated well with the age of onset of the disease in CPVT patients carrying the mutations. Our data suggest that the reduction in threshold [Ca2+]ER for spontaneous Ca2+ release by CPVT mutation is explained by enhanced CICR activity without considering a change in the [Ca2+]ER sensitivity of RyR2. SummaryCPVT-linked RyR2 mutations are prone to induce spontaneous Ca2+ release from ER, which is strongly associated with arrhythmias. Kurebayashi et al. quantitatively explore how the changes in CICR activity by RyR2 mutations affect spontaneous Ca2+ experimentally and by model simulation.

physiology↗

bioRxiv2020

Gating mechanism of cardiac ryanodine receptor 2 upon calcium ion binding

Cardiac ryanodine receptor (RyR2) is a large Ca2+ release channel in the sarcoplasmic reticulum and indispensable for excitation-contraction coupling in the heart. RyR2 is activated by Ca2+ and RyR2 mutations are implicated in severe arrhythmogenic diseases. Yet, the structural basis underlying channel opening and how mutations affect the channel remain unknown. Here, we addressed gating mechanism of RyR2 by combining high-resolution structures determined by cryo-electron microscopy with quantitative functional analysis of channels carrying various mutations in specific residues. We demonstrated two fundamental mechanisms for channel gating: interactions close to the channel pore stabilize the channel to prevent hyperactivity and a series of interactions in the surrounding regions is necessary for channel opening upon Ca2+ binding. Mutations at the residues involved in the former and the latter mechanisms cause gain-of-function and loss-of-function, respectively. Our results reveal gating mechanisms of the RyR2 channel and alterations by pathogenic mutations at the atomic level.

biochemistry↗

bioRxiv2020

Heat hypersensitivity of ryanodine receptor type 1 mutants implicated in malignant hyperthermia

Cellular heat-sensing is a universal strategy for avoiding thermal damage and adapting to environments by regulating thermogenic activities. If heat-sensing results in the acceleration of processes governing cellular thermogenesis, hyperthermia can occur. However, how this positive feedback loop contributes to hyperthermia development, especially the gap between heat-sensing and thermogenesis, remains largely unknown. Here, we show that an optically controlled local heat pulse induces an intracellular Ca2+ burst in cultured HEK 293 cells overexpressing ryanodine-receptor-type-1 (RyR1) mutants related to the life-threatening illness malignant hyperthermia (MH), and that the Ca2+ burst originates from heat-induced Ca2+-release (HICR) because of the mutant channels heat hypersensitivity. Furthermore, the heat hypersensitivity of the four RyR1 mutants was ranked, highlighting the complexity of MH. Our findings reveal the novel cellular heat-sensing mechanism, HICR, is essential for the functional positive feedback loop causing MH, suggesting a well-tuned HICR is fundamental for heat-mediated intracellular signaling.

biophysics↗

bioRxiv2020

Zinc-binding motif acts as an oxidative stress sensor to regulate TRPM7 channel activity

TRPM7 channel activity is negatively regulated by intracellular Mg2+. We previously reported that TRPM7 was inhibited by oxidative stress due to an enhancement of the inhibition by intracellular Mg2+. In the present study, we aimed to clarify the precise mechanism underlying the TRPM7 inhibition by oxidative stress induced by hydrogen peroxide (H2O2). Site-directed mutagenesis on full-length TRPM7 revealed that none of the cysteines other than C1809 and C1813 within the zinc-binding motif of the TRPM7 kinase domain were involved in the H2O2-induced TRPM7 inhibition. When C1809 or C1813 was mutated, full-length TRPM7 was not expressed on the plasma membrane. We, therefore, developed a novel approach in which the full-length TRPM7 is functionally reconstituted by co-expressing the TRPM7 channel domain (M7cd) and the TRPM7 kinase domain (M7kd) as separate individual proteins in HEK293 cells. When M7cd was expressed alone, the current was inhibited by intracellular Mg2+ more strongly than in full-length TRPM7. Co-expression of M7cd and M7kd attenuated the current inhibition by intracellular Mg2+, and the current was sensitive to oxidative stress, indicating successful reconstitution of a full-length TRPM7-like current. A similar current reconstitution was observed when M7cd was co-expressed with the kinase inactive mutant M7kd-K1645R. Thus, it is suggested that the kinase activity is not essential for the reconstitution. Co-expression of M7cd and M7kd carrying a mutation at C1809 or C1813 failed to restore the full-length TRPM7-like current. No reconstitution was also observed with M7kd carrying a mutation at H1750 and H1807, which are involved in the zinc-binding motif formation together with C1809 and C1813. These data suggest that the zinc-binding motif is essential for the intracellular Mg2+-dependent regulation of the TRPM7 channel activity by M7kd, and the cysteines in the zinc-binding motif might play a role in the oxidative stress response of TRPM7.

physiology↗

bioRxiv2020

MCM10 compensates for Myc-induced DNA replication stress in breast cancer stem-like cells

Cancer stem-like cells (CSCs) are responsible for the drug resistance of tumors and recurrence while they experience DNA replication stress. However, the underlying mechanisms that cause DNA replication stress in CSCs and how they compensate for this stress remain unclear. Here we provide evidence that upregulated c-Myc expression induces stronger DNA replication stress in patient-derived breast CSCs than in differentiated cancer cells. Our results suggest critical roles for mini-chromosome maintenance protein 10 (MCM10), which is a firing (activating) factor of the DNA replication origins, to compensate for the DNA replication stress. Expression levels of MCM10 are upregulated in CSCs and maintained by c-Myc. c-Myc-dependent collisions may take place between RNA transcription and DNA replication machinery in nuclei, thereby causing DNA replication stress. MCM10 may activate dormant replication origins close to the collisions to ensure replication progression. Moreover, patient-derived breast CSCs were dependent on MCM10 for their maintenance even after enrichment for CSCs that were resistant to paclitaxel, the standard chemotherapeutic agent. In addition, MCM10 depletion decreased the growth of cancer cells but not normal cells. Therefore, MCM10 is likely to robustly compensate for DNA replication stress and facilitate genome duplication in the S-phase in cancer cells, which is more pronounced in CSCs. We provide a preclinical rationale to target the c-Myc-MCM10 axis to prevent drug resistance and recurrence.

cancer biology↗

bioRxiv2020

A novel saline-soluble, rapidly-metabolized RyR1 inhibitor rescues volatile anesthesia-induced death and environmental heat stroke in a mouse model relevant to malignant hyperthermia

Mutations in the type 1 ryanodine receptor (RyR1), a Ca2+ release channel in skeletal muscle, hyperactivate the channel to cause malignant hyperthermia (MH) and are implicated in severe heat stroke. Dantrolene, the only approved drug for MH, has the disadvantages of having very poor water solubility and long plasma half-life. We show here that a novel RyR1-selective inhibitor, 6,7-(methylenedioxy)-1-octyl-4-quinolone-3-carboxylic acid (Compound 1, Cpd1), effectively prevents and treats MH and heat stroke in several mouse models relevant to MH. Cpd1 reduced resting intracellular Ca2+, inhibited halothane- and isoflurane-induced Ca2+ release, suppressed caffeine-induced contracture in skeletal muscle, reduced sarcolemmal cation influx, and prevented or reversed the fulminant MH crisis induced by isoflurane anesthesia and rescued animals from heat stroke caused by environmental heat stress. Notably, Cpd1 has great advantages of better water solubility and rapid clearance in vivo over dantrolene. Cpd1 has the potential to be a promising new candidate for effective treatment of patients carrying RyR1 mutations.

pharmacology and toxicology↗

bioRxiv2018

Domain organization and conformational change of dynactin p150

Dynactin is a principal regulator of the minus-end directed microtubule motor dynein. The sidearm of dynactin is essential for binding to microtubules and regulation of dynein activity. Although our understanding of the structure of the dynactin backbone (Arp1 rod) has greatly improved recently, structural details of the sidearm part remain elusive. Here, electron microscopy of individual molecules of the dynactin complex revealed that the sidearm was highly flexible and exhibited diverse morphologies. Utilizing mutants for nanogold labeling and deletion analysis, we determined the domain organization of the largest subunit p150 and identified a filamentous structure protruding from the head domain of the sidearm as the coiled-coil 1 (CC1), the dynein-binding domain, in p150. Furthermore, the protrusion formed by CC1 exhibited either a folded or an extended form, suggesting that CC1 works as an extending \"arm\". These findings provide clues to understand how dynactin binds to microtubules and regulates dynein.

biophysics↗

bioRxiv2017

Dynactin has two antagonistic regulatory domains and exerts opposing effects on dynein motility

Dynactin is a dynein-regulating protein that increases the processivity of dynein movement on microtubules. Recent studies have shown that a tripartite complex of dynein-dynactin-Bicaudal D2 is essential for highly processive movement. To elucidate the regulation of dynein motility by dynactin, we focused on two isoforms (A and B) of dynactin 1 (DCTN1), the largest subunit of dynactin that contains both microtubule- and dynein-binding domains. The only difference between the primary structures of the two isoforms is that DCTN1B lacks the K-rich domain, a cluster of basic residues. We measured dynein motility by single molecule observation of recombinant dynein and dynactin. Whereas the tripartite complex containing DCTN1A exhibited highly processive movement, the complex containing DCTN1B dissociated from microtubules with no apparent processive movement. This inhibitory effect of DCTN1B was caused by reductions of the microtubule-binding affinities of both dynein and dynactin, which is attributed to the coiled-coil 1 domain of DCTN1. In DCTN1A, the K-rich domain antagonized these inhibitory effects. Therefore, dynactin has two antagonistic domains and promotes or suppresses dynein motility to accomplish correct localization and functions of dynein within a cell.

biophysics↗