Incomplete-penetrant hypertrophic cardiomyopathy MYH7 G256E mutation causes hypercontractility and elevated mitochondrial respiration.

Determining the pathogenicity of hypertrophic cardiomyopathy-associated mutations in the ß-myosin heavy chain (MYH7) can be challenging due to its variable penetrance and clinical severity. This study investigates the early pathogenic effects of the incomplete-penetrant MYH7 G256E mutation on myosin function that may trigger pathogenic adaptations and hypertrophy. We hypothesized that the G256E mutation would alter myosin biomechanical function, leading to changes in cellular functions. We developed a collaborative pipeline to characterize myosin function across protein, myofibril, cell, and tissue levels to determine the multiscale effects on structure-function of the contractile apparatus and its implications for gene regulation and metabolic state. The G256E mutation disrupts the transducer region of the S1 head and reduces the fraction of myosin in the folded-back state by 33%, resulting in more myosin heads available for contraction. Myofibrils from gene-edited MYH7WT/G256E human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) exhibited greater and faster tension development. This hypercontractile phenotype persisted in single-cell hiPSC-CMs and engineered heart tissues. We demonstrated consistent hypercontractile myosin function as a primary consequence of the MYH7 G256E mutation across scales, highlighting the pathogenicity of this gene variant. Single-cell transcriptomic and metabolic profiling demonstrated upregulated mitochondrial genes and increased mitochondrial respiration, indicating early bioenergetic alterations. This work highlights the benefit of our multiscale platform to systematically evaluate the pathogenicity of gene variants at the protein and contractile organelle level and their early consequences on cellular and tissue function. We believe this platform can help elucidate the genotype-phenotype relationships underlying other genetic cardiovascular diseases.

Rab10-CAV1 mediated intraluminal vesicle transport to migrasomes.

Migrasomes, vesicular organelles generated on the retraction fibers of migrating cells, play a crucial role in migracytosis, mediating intercellular communication. The cargoes determine the functional specificity of migrasomes. Migrasomes harbor numerous intraluminal vesicles, a pivotal component of their cargoes. The mechanism underlying the transportation of these intraluminal vesicles to the migrasomes remains enigmatic. In this study, we identified that Rab10 and Caveolin-1 (CAV1) mark the intraluminal vesicles in migrasomes. Transport of Rab10-CAV1 vesicles to migrasomes required the motor protein Myosin Va and adaptor proteins RILPL2. Notably, the phosphorylation of Rab10 by the kinase LRRK2 regulated this process. Moreover, CSF-1 can be transported to migrasomes through this mechanism, subsequently fostering monocyte-macrophage differentiation in skin wound healing, which served as a proof of the physiological importance of this transporting mechanism.

Cingulin-nonmuscle myosin interaction plays a role in epithelial morphogenesis and cingulin nanoscale organization.

Cingulin (CGN) tethers nonmuscle myosin 2B (NM2B; heavy chain encoded by MYH10) to tight junctions (TJs) to modulate junctional and apical cortex mechanics. Here, we studied the role of the CGN-nonmuscle myosin 2 (NM2) interaction in epithelial morphogenesis and nanoscale organization of CGN by expressing wild-type and mutant CGN constructs in CGN-knockout Madin-Darby canine kidney (MDCK) epithelial cells. We show that the NM2-binding region of CGN is required to promote normal cyst morphogenesis of MDCK cells grown in three dimensions and to maintain the C-terminus of CGN in a distal position with respect to the ZO-2 (or TJP2)-containing TJ submembrane region, whereas the N-terminus of CGN is localized more proximal to the TJ membrane. We also show that the CGN mutant protein that causes deafness in human and mouse models is localized at TJs but does not bind to NM2B, resulting in decreased TJ membrane tortuosity. These results indicate that the interaction between CGN and NM2B regulates epithelial tissue morphogenesis and nanoscale organization of CGN and suggest that CGN regulates the auditory function of hair cells by organizing the actomyosin cytoskeleton to modulate the mechanics of the apical and junctional cortex.

Rab27a GTPase and its effector Myosin Va are host factors required for efficient Oropouche virus cell egress.

Oropouche fever, a debilitating illness common in South America, is caused by Oropouche virus (OROV), an arbovirus. OROV belongs to the Peribunyaviridae family, a large group of RNA viruses. Little is known about the biology of Peribunyaviridae in host cells, especially assembly and egress processes. Our research reveals that the small GTPase Rab27a mediates intracellular transport of OROV induced compartments and viral release from infected cells. We show that Rab27a interacts with OROV glycoproteins and colocalizes with OROV during late phases of the infection cycle. Moreover, Rab27a activity is required for OROV trafficking to the cell periphery and efficient release of infectious particles. Consistently, depleting Rab27a's downstream effector, Myosin Va, or inhibiting actin polymerization also hinders OROV compartments targeting to the cell periphery and infectious viral particle egress. These data indicate that OROV hijacks Rab27a activity for intracellular transport and cell externalization. Understanding these crucial mechanisms of OROV's replication cycle may offer potential targets for therapeutic interventions and aid in controlling the spread of Oropouche fever.

Effects of specific disease mutations in non-muscle myosin 2A on its structure and function.

Non-muscle myosin 2A (NM2A), a widely expressed class 2 myosin, is important for organizing actin filaments in cells. It cycles between a compact inactive 10S state in which its regulatory light chain (RLC) is dephosphorylated and a filamentous state in which the myosin heads interact with actin, and the RLC is phosphorylated. Over 170 missense mutations in MYH9, the gene that encodes the NM2A heavy chain, have been described. These cause MYH9 disease, an autosomal-dominant disorder that leads to bleeding disorders, kidney disease, cataracts, and deafness. Approximately two-thirds of these mutations occur in the coiled-coil tail. These mutations could destabilize the 10S state and/or disrupt filament formation or both. To test this, we determined the effects of six specific mutations using multiple approaches, including circular dichroism to detect changes in secondary structure, negative stain electron microscopy to analyze 10S and filament formation in vitro, and imaging of GFP-NM2A in fixed and live cells to determine filament assembly and dynamics. Two mutations in D1424 (D1424G and D1424N) and V1516M strongly decrease 10S stability and have limited effects on filament formation in vitro. In contrast, mutations in D1447 and E1841K, decrease 10S stability less strongly but increase filament lengths in vitro. The dynamic behavior of all mutants was altered in cells. Thus, the positions of mutated residues and their roles in filament formation and 10S stabilization are key to understanding their contributions to NM2A in disease.

Allele-Specific Suppression of Variant MHC With High-Precision RNA Nuclease CRISPR-Cas13d Prevents Hypertrophic Cardiomyopathy.

BACKGROUND: Familial hypertrophic cardiomyopathy has severe clinical complications of heart failure, arrhythmia, and sudden cardiac death. Heterozygous single nucleotide variants (SNVs) of sarcomere genes such as MYH7 are the leading cause of this type of disease. CRISPR-Cas13 (clustered regularly interspaced short palindromic repeats and their associated protein 13) is an emerging gene therapy approach for treating genetic disorders, but its therapeutic potential in genetic cardiomyopathy remains unexplored. METHODS: We developed a sensitive allelic point mutation reporter system to screen the mutagenic variants of Cas13d. On the basis of Cas13d homology structure, we rationally designed a series of Cas13d variants and obtained a high-precision Cas13d variant (hpCas13d) that specifically cleaves the MYH7 variant RNAs containing 1 allelic SNV. We validated the high precision and low collateral cleavage activity of hpCas13d through various in vitro assays. We generated 2 HCM mouse models bearing distinct MYH7 SNVs and used adenovirus-associated virus serotype 9 to deliver hpCas13d specifically to the cardiomyocytes. We performed a large-scale library screening to assess the potency of hpCas13d in resolving 45 human MYH7 allelic pathogenic SNVs. RESULTS: Wild-type Cas13d cannot distinguish and specifically cleave the heterozygous MYH7 allele with SNV. hpCas13d, with 3 amino acid substitutions, had minimized collateral RNase activity and was able to resolve various human MYH7 pathological sequence variations that cause hypertrophic cardiomyopathy. In vivo application of hpCas13d to 2 hypertrophic cardiomyopathy models caused by distinct human MYH7 analogous sequence variations specifically suppressed the altered allele and prevented cardiac hypertrophy. CONCLUSIONS: Our study unveils the great potential of CRISPR-Cas nucleases with high precision in treating inheritable cardiomyopathy and opens a new avenue for therapeutic management of inherited cardiac diseases.

Actin turnover protects the cytokinetic contractile ring from structural instability.

In common with other actomyosin contractile cellular machineries, actin turnover is required for normal function of the cytokinetic contractile ring. Cofilin is an actin-binding protein contributing to turnover by severing actin filaments, required for cytokinesis by many organisms. In fission yeast cofilin mutants, contractile rings suffer bridging instabilities in which segments of the ring peel away from the plasma membrane, forming straight bridges whose ends remain attached to the membrane. The origin of bridging instability is unclear. Here, we used molecularly explicit simulations of contractile rings to examine the role of cofilin. Simulations reproduced the experimentally observed cycles of bridging and reassembly during constriction, and the occurrence of bridging in ring segments with low density of the myosin II protein Myo2. The lack of cofilin severing produced ∼2-fold longer filaments and, consequently, ∼2-fold higher ring tensions. Simulations identified bridging as originating in the boosted ring tension, which increased centripetal forces that detached actin from Myo2, which was anchoring actin to the membrane. Thus, cofilin serves a critical role in cytokinesis by providing protection from bridging, the principal structural threat to contractile rings.

Pseudorabies virus usurps non-muscle myosin heavy chain IIA to dampen viral DNA recognition by cGAS for antagonism of host antiviral innate immunity.

Alphaherpesvirus pseudorabies virus (PRV) causes severe economic losses to the global pig industry and has garnered increasing attention due to its broad host range including humans. PRV has developed a variety of strategies to antagonize host antiviral innate immunity. However, the underlying mechanisms have not been fully elucidated. In our previous work, we demonstrated that non-muscle myosin heavy chain IIA (NMHC-IIA), a multifunctional cytoskeleton protein, attenuates innate immune responses triggered by RNA viruses. In the current study, we reported a previously unrecognized role of NMHC-IIA in counteracting PRV-induced cyclic GMP-AMP synthase (cGAS)-dependent type I interferon (IFN-I) production. Mechanistically, PRV infection led to an elevation of NMHC-IIA, strengthening the interaction between poly (ADP-ribose) polymerase 1 (PARP1) and cGAS. This interaction impeded cGAS recognition of PRV DNA and hindered downstream signaling activation. Conversely, inhibition of NMHC-IIA by Blebbistatin triggered innate immune responses and enhanced resistance to PRV proliferation both in vitro and in vivo. Taken together, our findings unveil that PRV utilizes NMHC-IIA to antagonize host antiviral immune responses via impairing DNA sensing by cGAS. This in-depth understanding of PRV immunosuppression not only provides insights for potential PRV treatment strategies but also highlights NMHC-IIA as a versatile immunosuppressive regulator usurped by both DNA and RNA viruses. Consequently, NMHC-IIA holds promise as a target for the development of broad-spectrum antiviral drugs.IMPORTANCECyclic GMP-AMP synthase (cGAS)-stimulator of interferon genes (STING) axis plays a vital role in counteracting alphaherpesvirus infections. Alphaherpesviruses exploit various strategies for antagonizing cGAS-STING-mediated antiviral immune responses. However, limited examples of pseudorabies virus (PRV)-caused immunosuppression have been documented. Our findings reveal a novel role of non-muscle myosin heavy chain IIA (NMHC-IIA) in suppressing PRV-triggered innate immune responses to facilitate viral propagation both in vitro and in vivo. In detail, NMHC-IIA recruits poly (ADP-ribose) polymerase 1 (PARP1) to augment its interaction with cGAS, which impairs cGAS recognition of PRV DNA. Building on our previous demonstration of NMHC-IIA's immunosuppressive role during RNA virus infections, these findings indicate that NMHC-IIA acts as a broad-spectrum suppressor of host antiviral innate immunity in response to both DNA and RNA viruses. Therefore, NMHC-IIA will be a promising target for the development of comprehensive antiviral strategies.

Quercetin induces a slow myofiber phenotype in engineered human skeletal muscle tissues.

Skeletal muscle comprises slow and fast myofibers, with slow myofibers excelling in aerobic metabolism and endurance. Quercetin, a polyphenol, is reported to induce slow myofibers in rodent skeletal muscle both in vitro and in vivo. However, its effect on human myofiber types remains unexplored. In this study, we evaluated quercetin's impact on slow myofiber induction using human skeletal muscle satellite cells. In a two-dimensional culture, quercetin enhanced gene expression, contributing to muscle differentiation, and significantly expanded the area of slow-type myosin heavy chain positive cells. It also elevated the gene expression of Pgc1α, an inducer of slow myofibers. Conversely, quercetin did not affect mitochondrial abundance, fission, or fusion, but it did increase the gene expression of Cox7A2L, which aids in promoting mitochondrial supercomplexity and endurance, and Mb, which contributes to oxidative phosphorylation. In a three-dimensional culture, quercetin significantly extended the time to peak tension and half relaxation time of the engineered human skeletal muscle tissues constructed on microdevices. Moreover, quercetin enhanced the muscle endurance of the tissues and curbed the rise in lactate secretion from the exercised tissues. These findings suggest that quercetin may induce slow myofibers in human skeletal muscle.

Nascent RHOH acts as a molecular brake on actomyosin-mediated effector functions of inflammatory neutrophils.

In contrast to molecular changes associated with increased inflammatory responses, little is known about intracellular counter-regulatory mechanisms that control signaling cascades associated with functional responses of neutrophils. Active RHO GTPases are typically considered as effector proteins that elicit cellular responses. Strikingly, we show here that RHOH, although being constitutively GTP-bound, limits neutrophil degranulation and the formation of neutrophil extracellular traps (NETs). Mechanistically, RHOH is induced under inflammatory conditions and binds to non-muscle myosin heavy chain IIA (NMHC IIA) in activated neutrophils in order to inhibit the transport of mitochondria and granules along actin filaments, which is partially reverted upon disruption of the interaction with NMHC IIA by introducing a mutation in RhoH at lysine 34 (RhoHK34A). In parallel, RHOH inhibits actin polymerization presumably by modulating RAC1 activity. In vivo studies using Rhoh-/- mice, demonstrate an increased antibacterial defense capability against Escherichia coli (E. coli). Collectively, our data reveal a previously undefined role of RHOH as a molecular brake for actomyosin-mediated neutrophil effector functions, which represents an intracellular regulatory axis involved in controlling the strength of an antibacterial inflammatory response.

A weak coupling mechanism for the early steps of the recovery stroke of myosin VI: A free energy simulation and string method analysis.

Myosin motors use the energy of ATP to produce force and directed movement on actin by a swing of the lever-arm. ATP is hydrolysed during the off-actin re-priming transition termed recovery stroke. To provide an understanding of chemo-mechanical transduction by myosin, it is critical to determine how the reverse swing of the lever-arm and ATP hydrolysis are coupled. Previous studies concluded that the recovery stroke of myosin II is initiated by closure of the Switch II loop in the nucleotide-binding site. Recently, we proposed that the recovery stroke of myosin VI starts with the spontaneous re-priming of the converter domain to a putative pre-transition state (PTS) intermediate that precedes Switch II closing and ATPase activation. Here, we investigate the transition from the pre-recovery, post-rigor (PR) state to PTS in myosin VI using geometric free energy simulations and the string method. First, our calculations rediscover the PTS state agnostically and show that it is accessible from PR via a low free energy transition path. Second, separate path calculations using the string method illuminate the mechanism of the PR to PTS transition with atomic resolution. In this mechanism, the initiating event is a large movement of the converter/lever-arm region that triggers rearrangements in the Relay-SH1 region and the formation of the kink in the Relay helix with no coupling to the active site. Analysis of the free-energy barriers along the path suggests that the converter-initiated mechanism is much faster than the one initiated by Switch II closure, which supports the biological relevance of PTS as a major on-pathway intermediate of the recovery stroke in myosin VI. Our analysis suggests that lever-arm re-priming and ATP hydrolysis are only weakly coupled, so that the myosin recovery stroke is initiated by thermal fluctuations and stabilised by nucleotide consumption via a ratchet-like mechanism.

Myosin VI rewrites the rules for myosin motors.

Myosin VI is the only type of myosin motor known to move toward the minus ends of actin filaments. This reversal in the direction of its movement is in part a consequence of the repositioning of its lever arm. In addition, myosin VI has a number of other specialized structural and functional adaptations that optimize performance of its unique cellular roles. Given that other classes of myosins may share some of these features, understanding the design principles of myosin VI will help guide the study of the functions of myosins that adopt similar strategies.

Switch between large hand-over-hand and small inchworm-like steps in myosin VI.

Many biological motor molecules move within cells using stepsizes predictable from their structures. Myosin VI, however, has much larger and more broadly distributed stepsizes than those predicted from its short lever arms. We explain the discrepancy by monitoring Qdots and gold nanoparticles attached to the myosin-VI motor domains using high-sensitivity nanoimaging. The large stepsizes were attributed to an extended and relatively rigid lever arm; their variability to two stepsizes, one large (72 nm) and one small (44 nm). These results suggest that there exist two tilt angles during myosin-VI stepping, which correspond to the pre- and postpowerstroke states and regulate the leading head. The large steps are consistent with the previously reported hand-over-hand mechanism, while the small steps follow an inchworm-like mechanism and increase in frequency with ADP. Switching between these two mechanisms in a strain-sensitive, ADP-dependent manner allows myosin VI to fulfill its multiple cellular tasks including vesicle transport and membrane anchoring.

Myosin-5a facilitates stress granule formation by interacting with G3BP1.

Stress granules (SGs) are non-membranous organelles composed of mRNA and proteins that assemble in the cytosol when the cell is under stress. Although the composition of mammalian SGs is both cell-type and stress-dependent, they consistently contain core components, such as Ras GTPase activating protein SH3 domain binding protein 1 (G3BP1). Upon stress, living cells rapidly assemble micrometric SGs, sometimes within a few minutes, suggesting that SG components may be actively transported by the microtubule and/or actin cytoskeleton. Indeed, SG assembly has been shown to depend on the microtubule cytoskeleton and the associated motor proteins. However, the role of the actin cytoskeleton and associated myosin motor proteins remains controversial. Here, we identified G3BP1 as a novel binding protein of unconventional myosin-5a (Myo5a). G3BP1 uses its C-terminal RNA-binding domain to interact with the middle portion of Myo5a tail domain (Myo5a-MTD). Suppressing Myo5a function in mammalian cells, either by overexpressing Myo5a-MTD, eliminating Myo5a gene expression, or treatment with myosin-5 inhibitor, inhibits the arsenite-induced formation of both small and large SGs. This is different from the effect of microtubule disruption, which abolishes the formation of large SGs but enhances the formation of small SGs under stress conditions. We therefore propose that, under stress conditions, Myo5a facilitates the formation of SGs at an earlier stage than the microtubule-dependent process.

UBR2 targets myosin heavy chain IIb and IIx for degradation: Molecular mechanism essential for cancer-induced muscle wasting.

Cancer cachexia is a lethal metabolic syndrome featuring muscle wasting with preferential loss of fast-twitching muscle mass through an undefined mechanism. Here, we show that cancer induces muscle wasting by selectively degrading myosin heavy chain (MHC) subtypes IIb and IIx through E3 ligase UBR2-mediated ubiquitylation. Induction of MHC loss and atrophy in C2C12 myotubes and mouse tibialis anterior (TA) by murine cancer cells required UBR2 up-regulation by cancer. Genetic gain or loss of UBR2 function inversely altered MHC level and muscle mass in TA of tumor-free mice. UBR2 selectively interacted with and ubiquitylated MHC-IIb and MHC-IIx through its substrate recognition and catalytic domain, respectively, in C2C12 myotubes. Elevation of UBR2 in muscle of tumor-bearing or free mice caused loss of MHC-IIb and MHC-IIx but not MHC-I and MHC-IIa or other myofibrillar proteins, including α-actin, troponin, tropomyosin, and tropomodulin. Muscle-specific knockout of UBR2 spared KPC tumor-bearing mice from losing MHC-IIb and MHC-IIx, fast-twitching muscle mass, cross-sectional area, and contractile force. The rectus abdominis (RA) muscle of patients with cachexia-prone cancers displayed a selective reduction of MHC-IIx in correlation with higher UBR2 levels. These data suggest that UBR2 is a regulator of MHC-IIb/IIx essential for cancer-induced muscle wasting, and that therapeutic interventions can be designed by blocking UBR2 up-regulation by cancer.

Predicting myosin heavy chain isoform from postdissection fiber length in human skeletal muscle fibers.

Experimental techniques in single human skeletal muscle cells require manual dissection. Unlike other mammalian species, human skeletal muscle is characterized by a heterogeneous mixture of myosin heavy chain (MHC) isoforms, typically used to define "fiber type," which profoundly influences cellular function. Therefore, it is beneficial to predict MHC isoform at the time of dissection, facilitating a more balanced fiber-type distribution from a potentially imbalanced sample. Although researchers performing single fiber dissection report predicting fiber-type based on mechanical properties of fibers upon dissection, a rigorous examination of this approach has not been performed. Therefore, we measured normalized fiber length (expressed as a % of the length of the bundle from which the fiber was dissected) in single fibers immediately following dissection. Six hundred sixty-eight individual fibers were dissected from muscle tissue samples from healthy, young adults to assess whether this characteristic could differentiate fibers containing MHC I ("slow" fiber type) or not ("fast" fiber type). Using receiver operator characteristic (ROC) curves, we found that differences in normalized fiber length (114 ± 13%, MHC I; 124 ± 17%, MHC IIA, P < 0.01) could be used to predict fiber type with excellent reliability (area under the curve = 0.72). We extended these analyses to include older adults (2 females, 1 male) to demonstrate the durability of this approach in fibers with likely different morphology and mechanical characteristics. We report that MHC isoform expression in human skeletal muscle fibers can be predicted at the time of dissection, regardless of origin.NEW & NOTEWORTHY A priori estimation of myosin heavy chain (MHC) isoform in individual muscle fibers may bias the relative abundance of fiber types in subsequent assessment. Until now, no standardized assessment approach has been proposed to characterize fibers at the time of dissection. We demonstrate an approach based on normalized fiber length that may dramatically bias a sample toward slow twitch (MHC I) or fast twitch (not MHC I) fiber populations.

IGF1 promotes human myotube differentiation toward a mature metabolic and contractile phenotype.

Skeletal muscle mediates the beneficial effects of exercise, thereby improving insulin sensitivity and reducing the risk for type 2 diabetes. Current human skeletal muscle models in vitro are incapable of fully recapitulating its physiological functions especially muscle contractility. By supplementation of insulin-like growth factor 1 (IGF1), a growth factor secreted by myofibers in vivo, we aimed to overcome these limitations. We monitored the differentiation process starting from primary human CD56-positive myoblasts in the presence/absence of IGF1 in serum-free medium in daily collected samples for 10 days. IGF1-supported differentiation formed thicker multinucleated myotubes showing physiological contraction upon electrical pulse stimulation (EPS) following day 6. Myotubes without IGF1 were almost incapable of contraction. IGF1 treatment shifted the proteome toward skeletal muscle-specific proteins that contribute to myofibril and sarcomere assembly, striated muscle contraction, and ATP production. Elevated PPARGC1A, MYH7, and reduced MYH1/2 suggest a more oxidative phenotype further demonstrated by higher abundance of proteins of the respiratory chain and elevated mitochondrial respiration. IGF1-treatment also upregulated glucose transporter (GLUT)4 and increased insulin-dependent glucose uptake compared with myotubes differentiated without IGF1. To conclude, addition of IGF1 to serum-free medium significantly improves the differentiation of human myotubes that showed enhanced myofibril formation, response to electrical pulse stimulation, oxidative respiratory capacity, and glucose metabolism overcoming limitations of previous standards. This novel protocol enables investigation of muscular exercise on a molecular level.NEW & NOTEWORTHY Human skeletal muscle models are highly valuable to study how exercise prevents type 2 diabetes without invasive biopsies. Current models did not fully recapitulate the function of skeletal muscle especially during exercise. By supplementing insulin-like growth factor 1 (IGF1), the authors developed a functional human skeletal muscle model characterized by inducible contractility and increased oxidative and insulin-sensitive metabolism. The novel protocol overcomes the limitations of previous standards and enables investigation of exercise on a molecular level.

Single-cell RNA sequencing reveals the heterogeneity of MYH11+ tumour-associated fibroblasts between left-sided and right-sided colorectal cancer.

Colorectal cancer (CRC) exhibits considerable heterogeneity on tumour location. However, there is still a lack of comprehensive annotation regarding the characteristics and differences between the left-sided (L-CRC) and right-sided (R-CRC) CRC. Here, we performed single-cell RNA sequencing (scRNA-seq) on immune and stromal cells from 12 L-CRC and 10 R-CRC patients. We found that L-CRC exhibited stronger tumour invasion and poor prognosis compared with R-CRC. In addition, functional enrichment analysis of a normal cohort showed that fibroblasts of left colon are associated with tumour-related pathways. This suggested that the heterogeneity observed in both L-CRC and R-CRC may be influenced by the specific location within the colon itself. Further, we identified a potentially novel MYH11+ cancer-associated fibroblast (CAF) subset predominantly enriched in L-CRC. Moreover, we found that MYH11+ CAFs may promote tumour migration via interacting with macrophages, and was associated with poor prognosis in CRC. In summary, our study revealed the crucial role of MYH11+ CAFs in predicting a poor prognosis, thereby contributing valuable insights to the exploration of heterogeneity in L-CRC and R-CRC.

MYH13, a superfast myosin expressed in extraocular, laryngeal and syringeal muscles.

MYH13 is a unique type of sarcomeric myosin heavy chain (MYH) first detected in mammalian extraocular (EO) muscles and later also in vocal muscles, including laryngeal muscles of some mammals and syringeal muscles of songbirds. All these muscles are specialized in generating very fast contractions while producing relatively low force, a design appropriate for muscles acting against a much lower load than most skeletal muscles inserting into the skeleton. The definition of the physiological properties of muscle fibres containing MYH13 has been complicated by the mixed fibre type composition of EO muscles and the coexistence of different MYH types within the same fibre. A major advance in this area came from studies on isolated recombinant myosin motors and the demonstration that the affinity of actin-bound human MYH13 for ADP is much weaker than those of fast-type MYH1 (type 2X) and MYH2 (type 2A). This property is consistent with a very fast detachment of myosin from actin, a major determinant of shortening velocity. The MYH13 gene arose early during vertebrate evolution but was characterized only in mammals and birds and appears to have been lost in some teleost fish. The MYH13 gene is located at the 3' end of the mammalian fast/developmental gene cluster and in a similar position to the orthologous cluster in syntenic regions of the songbird genome. MYH13 gene regulation is controlled by a super-enhancer in the mammalian locus and deletion of the neighbouring fast MYH1 and MYH4 genes leads to abnormal MYH13 expression in mouse leg muscles.

Distinct effects of two hearing loss-associated mutations in the sarcomeric myosin MYH7b.

For decades, sarcomeric myosin heavy chain proteins were assumed to be restricted to striated muscle where they function as molecular motors that contract muscle. However, MYH7b, an evolutionarily ancient member of this myosin family, has been detected in mammalian nonmuscle tissues, and mutations in MYH7b are linked to hereditary hearing loss in compound heterozygous patients. These mutations are the first associated with hearing loss rather than a muscle pathology, and because there are no homologous mutations in other myosin isoforms, their functional effects were unknown. We generated recombinant human MYH7b harboring the D515N or R1651Q hearing loss-associated mutation and studied their effects on motor activity and structural and assembly properties, respectively. The D515N mutation had no effect on steady-state actin-activated ATPase rate or load-dependent detachment kinetics but increased actin sliding velocity because of an increased displacement during the myosin working stroke. Furthermore, we found that the D515N mutation caused an increase in the proportion of myosin heads that occupy the disordered-relaxed state, meaning more myosin heads are available to interact with actin. Although we found no impact of the R1651Q mutation on myosin rod secondary structure or solubility, we observed a striking aggregation phenotype when this mutation was introduced into nonmuscle cells. Our results suggest that each mutation independently affects MYH7b function and structure. Together, these results provide the foundation for further study of a role for MYH7b outside the sarcomere.