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Science Advances Oct 2023Pancreatic ductal adenocarcinoma (PDAC) has a very poor prognosis because of its high propensity to metastasize and its immunosuppressive microenvironment. Using a panel...
Pancreatic ductal adenocarcinoma (PDAC) has a very poor prognosis because of its high propensity to metastasize and its immunosuppressive microenvironment. Using a panel of pancreatic cancer cell lines, three-dimensional (3D) invasion systems, microarray gene signatures, microfluidic devices, mouse models, and intravital imaging, we demonstrate that ROCK-Myosin II activity in PDAC cells supports a transcriptional program conferring amoeboid invasive and immunosuppressive traits and in vivo metastatic abilities. Moreover, we find that immune checkpoint CD73 is highly expressed in amoeboid PDAC cells and drives their invasive, metastatic, and immunomodulatory traits. Mechanistically, CD73 activates RhoA-ROCK-Myosin II downstream of PI3K. Tissue microarrays of human PDAC biopsies combined with bioinformatic analysis reveal that rounded-amoeboid invasive cells with high CD73-ROCK-Myosin II activity and their immunosuppressive microenvironment confer poor prognosis to patients. We propose targeting amoeboid PDAC cells as a therapeutic strategy.
Topics: Animals; Humans; Mice; Adenocarcinoma; Amoeba; Carcinoma, Pancreatic Ductal; Cell Line, Tumor; Cell Movement; Cytoskeletal Proteins; Immunosuppression Therapy; Myosin Type II; Pancreatic Neoplasms; Tumor Microenvironment
PubMed: 37851808
DOI: 10.1126/sciadv.adi0244 -
Circulation. Genomic and Precision... Aug 2023The 2 sarcomere genes most commonly associated with hypertrophic cardiomyopathy (HCM), MYBPC3 (myosin-binding protein C3) and MYH7 (β-myosin heavy chain), are... (Review)
Review
BACKGROUND
The 2 sarcomere genes most commonly associated with hypertrophic cardiomyopathy (HCM), MYBPC3 (myosin-binding protein C3) and MYH7 (β-myosin heavy chain), are indistinguishable at presentation, and genotype-phenotype correlations have been elusive. Based on molecular and pathophysiological differences, however, it is plausible to hypothesize a different behavior in myocardial performance, impacting lifetime changes in left ventricular (LV) function.
METHODS
We reviewed the initial and final echocardiograms of 402 consecutive HCM patients with pathogenic or likely pathogenic MYBPC3 (n=251) or MYH7 (n=151) mutations, followed over 9±8 years.
RESULTS
At presentation, MYBPC3 patients were less frequently obstructive (15% versus 26%; =0.005) and had lower LV ejection fraction compared with MYH7 (66±8% versus 68±8%, respectively; =0.03). Both HCM patients harboring MYBPC3 and MYH7 mutations exhibited a small but significant decline in LV systolic function during follow-up; however, new onset of severe LV systolic dysfunction (LV ejection fraction, <50%) was greater among MYBPC3 patients (15% versus 5% among MYH7; =0.013). Prevalence of grade II/III diastolic dysfunction at final evaluation was comparable between MYBPC3 and MYH7 patients (=0.509). In a Cox multivariable analysis, MYBPC3-positive status (hazard ratio, 2.53 [95% CI, 1.09-5.82]; =0.029), age (hazard ratio, 1.03 [95% CI, 1.00-1.06]; =0.027), and atrial fibrillation (hazard ratio, 2.39 [95% CI, 1.14-5.05]; =0.020) were independent predictors of severe systolic dysfunction. No statistically significant differences occurred with regard to incidence of atrial fibrillation, heart failure, appropriate implanted cardioverter defibrillator shock, or cardiovascular death.
CONCLUSIONS
MYBPC3-related HCM showed increased long-term prevalence of systolic dysfunction compared with MYH7, in spite of similar outcome. Such observations suggest different pathophysiology of clinical progression in the 2 subsets and may prove relevant for understanding of genotype-phenotype correlations in HCM.
Topics: Humans; Atrial Fibrillation; Prevalence; Phenotype; Cardiomyopathy, Hypertrophic; Mutation; Cytoskeletal Proteins; Myosin Heavy Chains; Cardiac Myosins
PubMed: 37409452
DOI: 10.1161/CIRCGEN.122.003832 -
Current Biology : CB Jul 2023Collective cell movements contribute to tissue development and repair and spread metastatic disease. In epithelia, cohesive cell movements require reorganization of...
Collective cell movements contribute to tissue development and repair and spread metastatic disease. In epithelia, cohesive cell movements require reorganization of adherens junctions and the actomyosin cytoskeleton. However, the mechanisms that coordinate cell-cell adhesion and cytoskeletal remodeling during collective cell migration in vivo are unclear. We investigated the mechanisms of collective cell migration during epidermal wound healing in Drosophila embryos. Upon wounding, the cells adjacent to the wound internalize cell-cell adhesion molecules and polarize actin and the motor protein non-muscle myosin II to form a supracellular cable around the wound that coordinates cell movements. The cable anchors at former tricellular junctions (TCJs) along the wound edge, and TCJs are reinforced during wound closure. We found that the small GTPase Rap1 was necessary and sufficient for rapid wound repair. Rap1 promoted myosin polarization to the wound edge and E-cadherin accumulation at TCJs. Using embryos expressing a mutant form of the Rap1 effector Canoe/Afadin that cannot bind Rap1, we found that Rap1 signals through Canoe for adherens junction remodeling, but not for actomyosin cable assembly. Instead, Rap1 was necessary and sufficient for RhoA/Rho1 activation at the wound edge. The RhoGEF Ephexin localized to the wound edge in a Rap1-dependent manner, and Ephexin was necessary for myosin polarization and rapid wound repair, but not for E-cadherin redistribution. Together, our data show that Rap1 coordinates the molecular rearrangements that drive embryonic wound healing, promoting actomyosin cable assembly through Ephexin-Rho1, and E-cadherin redistribution through Canoe, thus enabling rapid collective cell migration in vivo.
Topics: Animals; Actomyosin; Cell Adhesion; Drosophila melanogaster; Drosophila Proteins; Cell Movement; Myosins; Adherens Junctions; Cadherins
PubMed: 37244252
DOI: 10.1016/j.cub.2023.05.009 -
Nature Nov 2023Pumping of the heart is powered by filaments of the motor protein myosin that pull on actin filaments to generate cardiac contraction. In addition to myosin, the...
Pumping of the heart is powered by filaments of the motor protein myosin that pull on actin filaments to generate cardiac contraction. In addition to myosin, the filaments contain cardiac myosin-binding protein C (cMyBP-C), which modulates contractility in response to physiological stimuli, and titin, which functions as a scaffold for filament assembly. Myosin, cMyBP-C and titin are all subject to mutation, which can lead to heart failure. Despite the central importance of cardiac myosin filaments to life, their molecular structure has remained a mystery for 60 years. Here we solve the structure of the main (cMyBP-C-containing) region of the human cardiac filament using cryo-electron microscopy. The reconstruction reveals the architecture of titin and cMyBP-C and shows how myosin's motor domains (heads) form three different types of motif (providing functional flexibility), which interact with each other and with titin and cMyBP-C to dictate filament architecture and function. The packing of myosin tails in the filament backbone is also resolved. The structure suggests how cMyBP-C helps to generate the cardiac super-relaxed state; how titin and cMyBP-C may contribute to length-dependent activation; and how mutations in myosin and cMyBP-C might disturb interactions, causing disease. The reconstruction resolves past uncertainties and integrates previous data on cardiac muscle structure and function. It provides a new paradigm for interpreting structural, physiological and clinical observations, and for the design of potential therapeutic drugs.
Topics: Humans; Cardiac Myosins; Carrier Proteins; Connectin; Cryoelectron Microscopy; Myocardium
PubMed: 37914935
DOI: 10.1038/s41586-023-06691-4 -
Nature Communications Oct 2023Myosin VI (Myo6) is the only minus-end directed nanomotor on actin, allowing it to uniquely contribute to numerous cellular functions. As for other nanomotors, the...
Myosin VI (Myo6) is the only minus-end directed nanomotor on actin, allowing it to uniquely contribute to numerous cellular functions. As for other nanomotors, the proper functioning of Myo6 relies on precise spatiotemporal control of motor activity via a poorly defined off-state and interactions with partners. Our structural, functional, and cellular studies reveal key features of myosin regulation and indicate that not all partners can activate Myo6. TOM1 and Dab2 cannot bind the off-state, while GIPC1 binds Myo6, releases its auto-inhibition and triggers proximal dimerization. Myo6 partners thus differentially recruit Myo6. We solved a crystal structure of the proximal dimerization domain, and show that its disruption compromises endocytosis in HeLa cells, emphasizing the importance of Myo6 dimerization. Finally, we show that the L926Q deafness mutation disrupts Myo6 auto-inhibition and indirectly impairs proximal dimerization. Our study thus demonstrates the importance of partners in the control of Myo6 auto-inhibition, localization, and activation.
Topics: Humans; HeLa Cells; Dimerization; Actins; Myosin Heavy Chains
PubMed: 37872146
DOI: 10.1038/s41467-023-42376-2 -
JCI Insight Nov 2023Myosin heavy chains encoded by MYH7 and MYH2 are abundant in human skeletal muscle and important for muscle contraction. However, it is unclear how mutations in these...
Myosin heavy chains encoded by MYH7 and MYH2 are abundant in human skeletal muscle and important for muscle contraction. However, it is unclear how mutations in these genes disrupt myosin structure and function leading to skeletal muscle myopathies termed myosinopathies. Here, we used multiple approaches to analyze the effects of common MYH7 and MYH2 mutations in the light meromyosin (LMM) region of myosin. Analyses of expressed and purified MYH7 and MYH2 LMM mutant proteins combined with in silico modeling showed that myosin coiled coil structure and packing of filaments in vitro are commonly disrupted. Using muscle biopsies from patients and fluorescent ATP analog chase protocols to estimate the proportion of myosin heads that were super-relaxed, together with x-ray diffraction measurements to estimate myosin head order, we found that basal myosin ATP consumption was increased and the myosin super-relaxed state was decreased in vivo. In addition, myofiber mechanics experiments to investigate contractile function showed that myofiber contractility was not affected. These findings indicate that the structural remodeling associated with LMM mutations induces a pathogenic state in which formation of shutdown heads is impaired, thus increasing myosin head ATP demand in the filaments, rather than affecting contractility. These key findings will help design future therapies for myosinopathies.
Topics: Humans; Muscular Diseases; Myosins; Muscle, Skeletal; Mutation; Adenosine Triphosphate
PubMed: 37788100
DOI: 10.1172/jci.insight.172322 -
Cell Death & Disease Oct 2023Emerging evidence indicates that DNA methylation plays an important role in the initiation and progression of nasopharyngeal carcinoma (NPC). DNAJA4 is hypermethylated...
Emerging evidence indicates that DNA methylation plays an important role in the initiation and progression of nasopharyngeal carcinoma (NPC). DNAJA4 is hypermethylated in NPC, while its role in regulating NPC progression remains unclear. Here, we revealed that the promoter of DNAJA4 was hypermethylated and its expression was downregulated in NPC tissues and cells. Overexpression of DNAJA4 significantly suppressed NPC cell migration, invasion, and EMT in vitro, and markedly inhibited the inguinal lymph node metastasis and lung metastatic colonization in vivo, while it did not affect NPC cell viability and proliferation capability. Mechanistically, DNAJA4 facilitated MYH9 protein degradation via the ubiquitin-proteasome pathway by recruiting PSMD2. Furthermore, the suppressive effects of DNAJA4 on NPC cell migration, invasion, and EMT were reversed by overexpression of MYH9 in NPC cells. Clinically, a low level of DNAJA4 indicated poor prognosis and an increased probability of distant metastasis in NPC patients. Collectively, DNAJA4 serves as a crucial driver for NPC invasion and metastasis, and the DNAJA4-PSMD2-MYH9 axis might contain potential targets for NPC treatments.
Topics: Humans; Nasopharyngeal Carcinoma; Epithelial-Mesenchymal Transition; Signal Transduction; Cell Movement; Nasopharyngeal Neoplasms; Cell Line, Tumor; Cell Proliferation; Gene Expression Regulation, Neoplastic; Neoplasm Invasiveness; TNF Receptor-Associated Factor 2; Myosin Heavy Chains; HSP40 Heat-Shock Proteins
PubMed: 37875476
DOI: 10.1038/s41419-023-06225-w -
Nature Communications Nov 2023Direct modulation of cardiac myosin function has emerged as a therapeutic target for both heart disease and heart failure. However, the development of myosin-based...
Direct modulation of cardiac myosin function has emerged as a therapeutic target for both heart disease and heart failure. However, the development of myosin-based therapeutics has been hampered by the lack of targeted in vitro screening assays. In this study we use Artificial Intelligence-based virtual high throughput screening (vHTS) to identify novel small molecule effectors of human β-cardiac myosin. We test the top scoring compounds from vHTS in biochemical counter-screens and identify a novel chemical scaffold called 'F10' as a cardiac-specific low-micromolar myosin inhibitor. Biochemical and biophysical characterization in both isolated proteins and muscle fibers show that F10 stabilizes both the biochemical (i.e. super-relaxed state) and structural (i.e. interacting heads motif) OFF state of cardiac myosin, and reduces force and left ventricular pressure development in isolated myofilaments and Langendorff-perfused hearts, respectively. F10 is a tunable scaffold for the further development of a novel class of myosin modulators.
Topics: Humans; Cardiac Myosins; Artificial Intelligence; Myosins; Muscle Fibers, Skeletal; Heart Failure
PubMed: 38001148
DOI: 10.1038/s41467-023-43538-y -
The Journal of Cell Biology Nov 2023Asymmetric meiotic divisions in oocytes rely on spindle positioning in close vicinity to the cortex. In metaphase II mouse oocytes, eccentric spindle positioning...
Asymmetric meiotic divisions in oocytes rely on spindle positioning in close vicinity to the cortex. In metaphase II mouse oocytes, eccentric spindle positioning triggers cortical polarization, including the build-up of an actin cap surrounded by a ring of activated myosin II. While the role of the actin cap in promoting polar body formation is established, ring myosin II activation mechanisms and functions have remained elusive. Here, we show that ring myosin II activation requires myotonic dystrophy kinase-related Cdc42-binding kinase (MRCK), downstream of polarized Cdc42. MRCK inhibition resulted in spindle rotation defects during anaphase II, precluding polar body extrusion. Remarkably, disengagement of segregated chromatids from the anaphase spindle could rescue rotation. We further show that the MRCK/myosin II pathway is activated in the fertilization cone and is required for male pronucleus migration toward the center of the zygote. These findings provide novel insights into the mechanism of myosin II activation in oocytes and its role in orchestrating asymmetric division and pronucleus centration.
Topics: Animals; Male; Mice; Actin Cytoskeleton; Actins; Cytoskeletal Proteins; Myosin Type II; Oocytes; Rotation; Female; Protein Serine-Threonine Kinases; Spindle Poles; Anaphase
PubMed: 37651121
DOI: 10.1083/jcb.202211029 -
Circulation Research Aug 2023Modulating myosin function is a novel therapeutic approach in patients with cardiomyopathy. Danicamtiv is a novel myosin activator with promising preclinical data that...
BACKGROUND
Modulating myosin function is a novel therapeutic approach in patients with cardiomyopathy. Danicamtiv is a novel myosin activator with promising preclinical data that is currently in clinical trials. While it is known that danicamtiv increases force and cardiomyocyte contractility without affecting calcium levels, detailed mechanistic studies regarding its mode of action are lacking.
METHODS
Permeabilized porcine cardiac tissue and myofibrils were used for X-ray diffraction and mechanical measurements. A mouse model of genetic dilated cardiomyopathy was used to evaluate the ability of danicamtiv to correct the contractile deficit.
RESULTS
Danicamtiv increased force and calcium sensitivity via increasing the number of myosins in the ON state and slowing cross-bridge turnover. Our detailed analysis showed that inhibition of ADP release results in decreased cross-bridge turnover with cross bridges staying attached longer and prolonging myofibril relaxation. Danicamtiv corrected decreased calcium sensitivity in demembranated tissue, abnormal twitch magnitude and kinetics in intact cardiac tissue, and reduced ejection fraction in the whole organ.
CONCLUSIONS
As demonstrated by the detailed studies of Danicamtiv, increasing myosin recruitment and altering cross-bridge cycling are 2 mechanisms to increase force and calcium sensitivity in cardiac muscle. Myosin activators such as Danicamtiv can treat the causative hypocontractile phenotype in genetic dilated cardiomyopathy.
Topics: Mice; Animals; Swine; Cardiomyopathy, Dilated; Calcium; Myocardium; Myosins; Myocytes, Cardiac; Cardiotonic Agents
PubMed: 37470183
DOI: 10.1161/CIRCRESAHA.123.322629