Pei Wang
Basic Life Research Scientist, OHNS/Research Division
All Publications
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LOXHD1 is indispensable for maintaining TMC1 auditory mechanosensitive channels at the site of force transmission.
Nature communications
2024; 15 (1): 7865
Abstract
Hair cell bundles consist of stereocilia arranged in rows of increasing heights, connected by tip links that transmit sound-induced forces to shorter stereocilia tips. Auditory mechanotransduction channel complexes, composed of proteins TMC1/2, TMIE, CIB2, and LHFPL5, are located at the tips of shorter stereocilia. While most components can interact with the tip link in vitro, their ability to maintain the channel complexes at the tip link in vivo is uncertain. Return, using mouse models, we show that an additional component, LOXHD1, is essential for keeping TMC1-pore forming subunits at the tip link but is dispensable for TMC2. Using SUB-immunogold-SEM, we showed that TMC1 localizes near the tip link but mislocalizes without LOXHD1. LOXHD1 selectively interacts with TMC1, CIB2, LHFPL5, and tip-link protein PCDH15. Our results demonstrate that TMC1-driven mature auditory channels require LOXHD1 to stay connected to the tip link and remain functional, while TMC2-driven developmental channels do not.
View details for DOI 10.1038/s41467-024-51850-4
View details for PubMedID 39256406
View details for PubMedCentralID PMC11387651
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SUB-immunogold-SEM reveals nanoscale distribution of submembranous epitopes.
Nature communications
2024; 15 (1): 7864
Abstract
Electron microscopy paired with immunogold labeling is the most precise tool for protein localization. However, these methods are either cumbersome, resulting in small sample numbers and restricted quantification, or limited to identifying protein epitopes external to the membrane. Here, we introduce SUB-immunogold-SEM, a scanning electron microscopy technique that detects intracellular protein epitopes proximal to the membrane. We identify four critical sample preparation factors contributing to the method's sensitivity. We validate its efficacy through precise localization and high-powered quantification of cytoskeletal and transmembrane protein distribution. We evaluate the capabilities of SUB-immunogold-SEM on cells with highly differentiated apical surfaces: (i) auditory hair cells, revealing the presence of nanoscale MYO15A-L rings at the tip of stereocilia; and (ii) respiratory multiciliate cells, mapping the distribution of the SARS-CoV-2 receptor ACE2 along the motile cilia. SUB-immunogold-SEM extends the application of SEM-based nanoscale protein localization to the detection of intracellular epitopes on the exposed surfaces of any cell.
View details for DOI 10.1038/s41467-024-51849-x
View details for PubMedID 39256352
View details for PubMedCentralID PMC11387508
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SUB-Immunogold-SEM reveals nanoscale distribution of submembranous epitopes.
Research square
2024
Abstract
Electron microscopy paired with immunogold labeling is the most precise tool for protein localization. However, these methods are either cumbersome, resulting in small sample numbers and restricted quantification, or limited to identifying protein epitopes external to the membrane. Here, we introduce SUB-immunogold-SEM, a scanning electron microscopy technique that detects intracellular protein epitopes proximal to the membrane. We identified four critical sample preparation factors that contribute to the method's sensitivity and validate its efficacy through precise localization and high-powered quantification of cytoskeletal and transmembrane proteins. We evaluated the capabilities of SUB-immunogold-SEM on cells with highly differentiated apical surfaces: (i) auditory hair cells, revealing the presence of nanoscale Myosin rings at the tip of stereocilia; and (ii) respiratory multiciliate cells, mapping the distribution of the SARS-CoV-2 receptor ACE2 along the motile cilia. SUB-immunogold-SEM provides a novel solution for nanoscale protein localization at the exposed surface of any cell.
View details for DOI 10.21203/rs.3.rs-3876898/v1
View details for PubMedID 38343799
View details for PubMedCentralID PMC10854333
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LOXHD1 is indispensable for coupling auditory mechanosensitive channels to the site of force transmission.
Research square
2024
Abstract
Hearing is initiated in hair cells by the mechanical activation of ion channels in the hair bundle. The hair bundle is formed by stereocilia organized into rows of increasing heights interconnected by tip links, which convey sound-induced forces to stereocilia tips. The auditory mechanosensitive channels are complexes containing at least four protein-subunits - TMC1/2, TMIE, CIB2, and LHFPL51-16 - and are located at the tips of shorter stereocilia at a yet-undetermined distance from the lower tip link insertion point17. While multiple auditory channel subunits appear to interact with the tip link, it remains unknown whether their combined interaction alone can resist the high-frequency mechanical stimulations owing to sound. Here we show that an unanticipated additional element, LOXHD1, is indispensable for maintaining the TMC1 pore-forming channel subunits coupled to the tip link. We demonstrate that LOXHD1 is a unique element of the auditory mechanotransduction complex that selectively affects the localization of TMC1, but not its close developmental paralogue TMC2. Taking advantage of our novel immunogold scanning electron microscopy method for submembranous epitopes (SUB-immunogold-SEM), we demonstrate that TMC1 normally concentrates within 100-nm of the tip link insertion point. In LOXHD1's absence, TMC1 is instead mislocalized away from this force transmission site. Supporting this finding, we found that LOXHD1 interacts selectively in vitro with TMC1 but not with TMC2 while also binding to channel subunits CIB2 and LHFPL5 and tip-link protein PCDH15. SUB-immunogold-SEM additionally demonstrates that LOXHD1 and TMC1 are physically connected to the lower tip-link complex in situ. Our results show that the TMC1-driven mature channels require LOXHD1 to stay coupled to the tip link and remain functional, but the TMC2-driven developmental channels do not. As both tip links and TMC1 remain present in hair bundles lacking LOXHD1, it opens the possibility to reconnect them and restore hearing for this form of genetic deafness.
View details for DOI 10.21203/rs.3.rs-3752492/v1
View details for PubMedID 38260480
View details for PubMedCentralID PMC10802736
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Intracellular TMEM16A is necessary for myogenesis of skeletal muscle
ISCIENCE
2022; 25 (11): 105446
Abstract
Transmembrane protein 16A (TMEM16A) localizes at plasma membrane and controls chloride influx in various type of cells. We here showed an intracellular localization pattern of TMEM16A molecules. In myoblasts, TMEM16A was primarily localized to the cytosolic compartment and partially co-localized with intracellular organelles. The global deletion of TMEM16A led to severe skeletal muscle developmental defect. In vitro observation showed that the proliferation of Tmem16a-/- myoblasts was significantly promoted along with activated ERK1/2 and Cyclin D expression; the myogenic differentiation was impaired accompanied by the enhanced caspase 12/3 activation, implying enhanced endoplasmic reticulum (ER) stress. Interestingly, the bradykinin-induced Ca2+ release from ER calcium store was significantly enhanced after TMEM16A deletion. This suggested a suppressing role of intracellular TMEM16A in ER calcium release whereby regulating the flux of chloride ion across the ER membrane. Our findings reveal a unique location pattern of TMEM16A in undifferentiated myoblasts and its role in myogenesis.
View details for DOI 10.1016/j.isci.2022.105446
View details for Web of Science ID 000892133500003
View details for PubMedID 36388955
View details for PubMedCentralID PMC9647518
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High-resolution immunofluorescence imaging of mouse cochlear hair bundles.
STAR protocols
2022; 3 (2): 101431
Abstract
High-resolution immunofluorescence imaging of cochlear hair bundles faces many challenges due to the hair bundle's small dimensions, fragile nature, and complex organization. Here, we describe an optimized protocol for hair-bundle protein immunostaining and localization. We detail the steps and solutions for extracting and fixing the mouse inner ear and for dissecting the organ of Corti. We further emphasize the optimal permeabilization, blocking, staining, and mounting conditions as well as the parameters for high-resolution microscopy imaging. For complete details on the use and execution of this protocol, please refer to Trouillet etal. (2021).
View details for DOI 10.1016/j.xpro.2022.101431
View details for PubMedID 35669049
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Oncofusion-driven de novo enhancer assembly promotes malignancy in Ewing sarcoma via aberrant expression of the stereociliary protein LOXHD1.
Cell reports
2022; 39 (11): 110971
Abstract
Ewing sarcoma (EwS) is a highly aggressive tumor of bone and soft tissues that mostly affects children and adolescents. The pathognomonic oncofusion EWSR1::FLI1 transcription factor drives EwS by orchestrating an oncogenic transcription program through de novo enhancers. By integrative analysis of thousands of transcriptomes representing pan-cancer cell lines, primary cancers, metastasis, and normal tissues, we identify a 32-gene signature (ESS32 [Ewing Sarcoma Specific 32]) that stratifies EwS from pan-cancer. Among the ESS32, LOXHD1, encoding a stereociliary protein, is the most highly expressed gene through an alternative transcription start site. Deletion or silencing of EWSR1::FLI1 bound upstream de novo enhancer results in loss of the LOXHD1 short isoform, altering EWSR1::FLI1 and HIF1α pathway genes and resulting in decreased proliferation/invasion of EwS cells. These observations implicate LOXHD1 as a biomarker and a determinant of EwS metastasis and suggest new avenues for developing LOXHD1-targeted drugs or cellular therapies for this deadly disease.
View details for DOI 10.1016/j.celrep.2022.110971
View details for PubMedID 35705030
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Oncofusion driven de novo enhancer assembly promotes malignancy in Ewing sarcomavia aberrant expression of the stereociliary protein LOXHD1.
AMER ASSOC CANCER RESEARCH. 2021
View details for Web of Science ID 000680263506136
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Loxhd1 mutations cause mechanotransduction defects in cochlear hair cells.
The Journal of neuroscience : the official journal of the Society for Neuroscience
2021
Abstract
Sound detection happens in the inner ear via the mechanical deflection of the hair bundle of cochlear hair cells. The hair bundle is an apical specialization consisting of actin-filled membrane protrusions (called stereocilia) connected by tip links (TLs) that transfer the deflection force to gate the mechanotransduction channels. Here, we identified the hearing loss-associated Loxhd1/DFNB77 gene as being required for the mechanotransduction process. LOXHD1 consists of 15 polycystin lipoxygenase alpha-toxin (PLAT) repeats, which in other proteins can bind lipids and proteins. LOXHD1 was distributed along the length of the stereocilia. Two LOXHD1 mouse models with mutations in the 10th PLAT repeat exhibited mechanotransduction defects (in both sexes). While mechanotransduction currents in mutant inner hair cells (IHCs) were similar to wild-type (WT) levels in the first postnatal week, they were severely affected by postnatal day 11. The onset of the MET phenotype was consistent with the temporal progression of postnatal LOXHD1 expression/localization in the hair bundle. The mechanotransduction defect observed in Loxhd1-mutant IHCs was not accompanied by a morphological defect of the hair bundle or a reduction in TL number. Using immunolocalization, we found that two proteins of the upper and lower TL protein complexes (Harmonin and LHFPL5) were maintained in the mutants, suggesting that the mechanotransduction machinery was present but not activatable. This work identified a novel LOXHD1-dependent step in hair bundle development that is critical for mechanotransduction in mature hair cells as well as for normal hearing function in mice and humans.SIGNIFICANCE STATEMENT:Hair cells detect sound-induced forces via the hair bundle, which consists of membrane protrusions connected by tip links. The mechanotransduction machinery forms protein complexes at the tip-link ends. The current study showed that LOXHD1, a multi-repeat protein responsible for hearing loss in humans and mice when mutated, was required for hair-cell mechanotransduction, but only after the first postnatal week. Using immunochemistry, we demonstrated that this defect was not caused by the mislocalization of the tip-link complex proteins Harmonin or LHFPL5, suggesting that the mechanotransduction protein complexes were maintained. This work identified a new step in hair bundle development, which is critical for both hair-cell mechanotransduction and hearing.
View details for DOI 10.1523/JNEUROSCI.0975-20.2021
View details for PubMedID 33707295
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Discovery and characterization of LOXHD1 as a highly specific EWS-FLI1 driven oncogene in Ewing sarcoma.
AMER ASSOC CANCER RESEARCH. 2020: 54–55
View details for Web of Science ID 000537844900081
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ZIPK mediates endothelial cell contraction through myosin light chain phosphorylation and is required for ischemic-reperfusion injury.
FASEB journal : official publication of the Federation of American Societies for Experimental Biology
2019: fj201802052RRR
Abstract
The paracellular gap formed by endothelial cell (EC) contraction is fundamental for endothelium permeability, but the mechanism underlying EC contraction has yet to be determined. Here, we identified the zipper-interacting protein kinase (ZIPK) as the kinase for EC contraction and myosin light chain (MLC) phosphorylation. Inhibition of ZIPK activity by pharmacological inhibitors and small interfering RNAs led to a significant decrease in the mono- and diphosphorylation of MLCs along with a contractile response to thrombin, suggesting an essential role of ZIPK in EC paracellular permeability. To assess the role of ZIPK in vivo, we established mouse lines with conditional deletion of Zipk gene. The endothelium-specific deletion of Zipk led to embryonic lethality, whereas the UBC-CreERT2-mediated deletion of Zipk by tamoxifen induction at adulthood caused no apparent phenotype. The induced deletion of Zipk significantly inhibited ischemia-reperfusion-induced blood-brain barrier dysfunction and neuronal injuries from middle cerebral artery occlusion and reperfusion, as evidenced by reduced infarct and edema volume, attenuated Evans blue dye leakage, and improved neuronal behavior. We thus concluded that ZIPK and its phosphorylation of MLC were required for EC contraction and ischemic neuronal injuries. ZIPK may be a prospective therapeutic target for stroke.-Zhang, Y., Zhang, C., Zhang, H., Zeng, W., Li, S., Chen, C., Song, X., Sun, J., Sun, Z., Cui, C., Cao, X., Zheng, L., Wang, P., Zhao, W., Zhang, Z., Xu, Y., Zhu, M., Chen, H. ZIPK mediates endothelial cell contraction through myosin light chain phosphorylation and is required for ischemic-reperfusion injury.
View details for DOI 10.1096/fj.201802052RRR
View details for PubMedID 31180722
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Golgi-resident TRIO regulates membrane trafficking during neurite outgrowth.
The Journal of biological chemistry
2019
Abstract
Neurite outgrowth requires coordinated cytoskeletal rearrangements in the growth cone and directional membrane delivery from the neuronal soma. As an essential Rho guanine nucleotide exchange factor (GEF), TRIO is necessary for cytoskeletal dynamics during neurite outgrowth, but its participation in the membrane delivery is unclear. Using co-localization studies, live-cell imaging, and fluorescence recovery after photobleaching (FRAP) analysis, along with neurite outgrowth assay and various biochemical approaches, we here report that in mouse cerebellar granule neurons, TRIO protein pools at the Golgi and regulates membrane trafficking by controlling the directional maintenance of both RAB8, member RAS oncogene family (RAB8)- and RAB10-positive membrane vesicles. We found that the spectrin repeats in Golgi-resident TRIO confer RAB8 and RAB10 activation by interacting with and activating the RAB GEF Rabin8. Constitutively active RAB8 or RAB10 could partially restore the neurite outgrowth of TRIO-deficient cerebellar granule neurons, suggesting that TRIO-regulated membrane trafficking has an important functional role in neurite outgrowth. Our results also suggest a cross-talk between Rho GEF and Rab GEF in controlling both cytoskeletal dynamics and membrane trafficking during neuronal development. They further highlight how protein pools localized to specific organelles regulate crucial cellular activities and functions. In conclusion, our findings indicate that TRIO regulates membrane trafficking during neurite outgrowth in coordination with its GEF-dependent function in controlling cytoskeletal dynamics via Rho GTPases.
View details for DOI 10.1074/jbc.RA118.007318
View details for PubMedID 31152060
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CPI-17-mediated contraction of vascular smooth muscle is essential for the development of hypertension in obese mice.
Journal of genetics and genomics = Yi chuan xue bao
2019; 46 (3): 109-118
Abstract
Several factors have been implicated in obesity-related hypertension, but the genesis of the hypertension is largely unknown. In this study, we found a significantly upregulated expression of CPI-17 (C-kinase-potentiated protein phosphatase 1 inhibitor of 17 kDa) and protein kinase C (PKC) isoforms in the vascular smooth muscles of high-fat diet (HFD)-fed obese mice. The obese wild-type mice showed a significant elevation of blood pressure and enhanced calcium-sensitized contraction of vascular smooth muscles. However, the obese CPI-17-deficient mice showed a normotensive blood pressure, and the calcium-sensitized contraction was consistently reduced. In addition, the mutant muscle displayed an abolished responsive force to a PKC activator and a 30%-50% reduction in both the initial peak force and sustained force in response to various G protein-coupled receptor (GPCR) agonists. Our observations showed that CPI-17-mediated calcium sensitization is mediated through a GPCR/PKC/CPI-17/MLCP/RLC signaling pathway. We therefore propose that the upregulation of CPI-17-mediated calcium-sensitized vasocontraction by obesity contributes to the development of obesity-related hypertension.
View details for DOI 10.1016/j.jgg.2019.02.005
View details for PubMedID 30948334
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Distinct functions of Trio GEF domains in axon outgrowth of cerebellar granule neurons.
Journal of genetics and genomics = Yi chuan xue bao
2019; 46 (2): 87-96
Abstract
As a critical guanine nucleotide exchange factor (GEF) regulating neurite outgrowth, Trio coordinates multiple processes of cytoskeletal dynamics through activating Rac1, Cdc42 and RhoA small GTPases by two GEF domains, but the in vivo roles of these GEF domains and corresponding downstream effectors have not been determined yet. We established multiple lines of knockout mice and assessed the respective roles of Trio GEF domains and Rac1 in axon outgrowth. Knockout of total Trio in cerebellar granule neurons (CGNs) led to an impaired F-actin rearrangement of growth cone and hence a retarded neurite outgrowth. Such a retardation was reproduced by inhibition of GEF1 domain or knockdown of Cdc42 and restored apparently by introduction of active Cdc42. As Rac1 deficiency did not affect the neurite outgrowth of CGNs, we suggested that Trio GEF1-mediated Cdc42 activation was required for neurite outgrowth. We established a GEF2-knockout line with deletion of all Trio isoforms except a cerebella-specific Trio8, a short isoform of Trio without GEF2 domain, and used this line as a GEF2-deficient animal model. The GEF2-deficient CGNs had a normal neurite outgrowth but abolished Netrin-1-promoted growth, without affecting Netrin-1 induced Rac1 activation. We thus suggested that Trio GEF1-mediated Cdc42 activation rather than Rac1 activation drives the F-actin dynamics necessary for neurite outgrowth, while GEF2 functions in Netrin-1-promoted neurite elongation. Our results delineated the distinct roles of Trio GEF domains in neurite outgrowth, which is instructive to understand the pathogenesis of clinical Trio-related neurodevelopmental disorders.
View details for DOI 10.1016/j.jgg.2019.02.003
View details for PubMedID 30850274
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Inflammatory mediators mediate airway smooth muscle contraction through a G protein-coupled receptor-transmembrane protein 16A-voltage-dependent Ca2+ channel axis and contribute to bronchial hyperresponsiveness in asthma
JOURNAL OF ALLERGY AND CLINICAL IMMUNOLOGY
2018; 141 (4): 1259-+
Abstract
Allergic inflammation has long been implicated in asthmatic hyperresponsiveness of airway smooth muscle (ASM), but its underlying mechanism remains incompletely understood. Serving as G protein-coupled receptor agonists, several inflammatory mediators can induce membrane depolarization, contract ASM, and augment cholinergic contractile response. We hypothesized that the signal cascade integrating on membrane depolarization by the mediators might involve asthmatic hyperresponsiveness.We sought to investigate the signaling transduction of inflammatory mediators in ASM contraction and assess its contribution in the genesis of hyperresponsiveness.We assessed the capacity of inflammatory mediators to induce depolarization currents by electrophysiological analysis. We analyzed the phenotypes of transmembrane protein 16A (TMEM16A) knockout mice, applied pharmacological reagents, and measured the Ca2+ signal during ASM contraction. To study the role of the depolarization signaling in asthmatic hyperresponsiveness, we measured the synergistic contraction by methacholine and inflammatory mediators both ex vivo and in an ovalbumin-induced mouse model.Inflammatory mediators, such as 5-hydroxytryptamin, histamine, U46619, and leukotriene D4, are capable of inducing Ca2+-activated Cl- currents in ASM cells, and these currents are mediated by TMEM16A. A combination of multiple analysis revealed that a G protein-coupled receptor-TMEM16A-voltage-dependent Ca2+ channel signaling axis was required for ASM contraction induced by inflammatory mediators. Block of TMEM16A activity may significantly inhibit the synergistic contraction of acetylcholine and the mediators and hence reduces hypersensitivity.A G protein-coupled receptor-TMEM16A-voltage-dependent Ca2+ channel axis contributes to inflammatory mediator-induced ASM contraction and synergistically activated TMEM16A by allergic inflammatory mediators with cholinergic stimuli.
View details for DOI 10.1016/j.jaci.2017.05.053
View details for Web of Science ID 000429197800012
View details for PubMedID 28754608
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The molecular basis of the genesis of basal tone in internal anal sphincter
NATURE COMMUNICATIONS
2016; 7: 11358
Abstract
Smooth muscle sphincters exhibit basal tone and control passage of contents through organs such as the gastrointestinal tract; loss of this tone leads to disorders such as faecal incontinence. However, the molecular mechanisms underlying this tone remain unknown. Here, we show that deletion of myosin light-chain kinases (MLCK) in the smooth muscle cells from internal anal sphincter (IAS-SMCs) abolishes basal tone, impairing defecation. Pharmacological regulation of ryanodine receptors (RyRs), L-type voltage-dependent Ca(2+) channels (VDCCs) or TMEM16A Ca(2+)-activated Cl(-) channels significantly changes global cytosolic Ca(2+) concentration ([Ca(2+)]i) and the tone. TMEM16A deletion in IAS-SMCs abolishes the effects of modulators for TMEM16A or VDCCs on a RyR-mediated rise in global [Ca(2+)]i and impairs the tone and defecation. Hence, MLCK activation in IAS-SMCs caused by a global rise in [Ca(2+)]i via a RyR-TMEM16A-VDCC signalling module sets the basal tone. Targeting this module may lead to new treatments for diseases like faecal incontinence.
View details for DOI 10.1038/ncomms11358
View details for Web of Science ID 000374582800001
View details for PubMedID 27101932
View details for PubMedCentralID PMC4844698
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Regulation of DLK1 by the maternally expressed miR-379/miR-544 cluster may underlie callipyge polar overdominance inheritance
PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA
2015; 112 (44): 13627–32
Abstract
Inheritance of the callipyge phenotype in sheep is an example of polar overdominance inheritance, an unusual mode of inheritance. To investigate the underlying molecular mechanism, we profiled the expression of the genes located in the Delta-like 1 homolog (Dlk1)-type III iodothyronine deiodinase (Dio3) imprinting region in mice. We found that the transcripts of the microRNA (miR) 379/miR-544 cluster were highly expressed in neonatal muscle and paralleled the expression of the Dlk1. We then determined the in vivo role of the miR-379/miR-544 cluster by establishing a mouse line in which the cluster was ablated. The maternal heterozygotes of young mutant mice displayed a hypertrophic tibialis anterior muscle, extensor digitorum longus muscle, gastrocnemius muscle, and gluteus maximus muscle and elevated expression of the DLK1 protein. Reduced expression of DLK1 was mediated by miR-329, a member of this cluster. Our results suggest that maternal expression of the imprinted miR-379/miR-544 cluster regulates paternal expression of the Dlk1 gene in mice. We therefore propose a miR-based molecular working model for polar overdominance inheritance.
View details for DOI 10.1073/pnas.1511448112
View details for Web of Science ID 000364164900066
View details for PubMedID 26487685
View details for PubMedCentralID PMC4640741
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In vivo roles for myosin phosphatase targeting subunit-1 phosphorylation sites T694 and T852 in bladder smooth muscle contraction
JOURNAL OF PHYSIOLOGY-LONDON
2015; 593 (3): 681–700
Abstract
Force production and maintenance in smooth muscle is largely controlled by myosin regulatory light chain (RLC) phosphorylation, which relies on a balance between Ca(2+)/calmodulin-dependent myosin light chain kinase (MLCK) and myosin light chain phosphatase (MLCP) activities. MYPT1 is the regulatory subunit of MLCP that biochemically inhibits MLCP activity via T694 or T852 phosphorylation in vitro. Here we separately investigated the contribution of these two phosphorylation sites in bladder smooth muscles by establishing two single point mutation mouse lines, T694A and T852A, and found that phosphorylation of MYPT1 T694, but not T852, mediates force maintenance via inhibition of MLCP activity and enhancement of RLC phosphorylation in vivo. Our findings reveal the role of MYPT1 T694/T852 phosphorylation in vivo in regulation of smooth muscle contraction.Force production and maintenance in smooth muscle is largely controlled by different signalling modules that fine tune myosin regulatory light chain (RLC) phosphorylation, which relies on a balance between Ca(2+)/calmodulin-dependent myosin light chain kinase (MLCK) and myosin light chain phosphatase (MLCP) activities. To investigate the regulation of MLCP activity in vivo, we analysed the role of two phosphorylation sites on MYPT1 (regulatory subunit of MLCP) that biochemically inhibit MLCP activity in vitro. MYPT1 is constitutively phosphorylated at T694 by unidentified kinases in vivo, whereas the T852 site is phosphorylated by RhoA-associated protein kinase (ROCK). We established two mouse lines with alanine substitution of T694 or T852. Isolated bladder smooth muscle from T852A mice displayed no significant changes in RLC phosphorylation or force responses, but force was inhibited with a ROCK inhibitor. In contrast, smooth muscles containing the T694A mutation showed a significant reduction of force along with reduced RLC phosphorylation. The contractile responses of T694A mutant smooth muscle were also independent of ROCK activation. Thus, phosphorylation of MYPT1 T694, but not T852, is a primary mechanism contributing to inhibition of MLCP activity and enhancement of RLC phosphorylation in vivo. The constitutive phosphorylation of MYPT1 T694 may provide a mechanism for regulating force maintenance of smooth muscle.
View details for DOI 10.1113/jphysiol.2014.283853
View details for Web of Science ID 000349292900017
View details for PubMedID 25433069
View details for PubMedCentralID PMC4324713
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Myosin Light Chain Kinase (MLCK) Regulates Cell Migration in a Myosin Regulatory Light Chain Phosphorylation-independent Mechanism
JOURNAL OF BIOLOGICAL CHEMISTRY
2014; 289 (41): 28478–88
Abstract
Myosin light chain kinase (MLCK) has long been implicated in the myosin phosphorylation and force generation required for cell migration. Here, we surprisingly found that the deletion of MLCK resulted in fast cell migration, enhanced protrusion formation, and no alteration of myosin light chain phosphorylation. The mutant cells showed reduced membrane tether force and fewer membrane F-actin filaments. This phenotype was rescued by either kinase-dead MLCK or five-DFRXXL motif, a MLCK fragment with potent F-actin-binding activity. Pull-down and co-immunoprecipitation assays showed that the absence of MLCK led to attenuated formation of transmembrane complexes, including myosin II, integrins and fibronectin. We suggest that MLCK is not required for myosin phosphorylation in a migrating cell. A critical role of MLCK in cell migration involves regulating the cell membrane tension and protrusion necessary for migration, thereby stabilizing the membrane skeleton through F-actin-binding activity. This finding sheds light on a novel regulatory mechanism of protrusion during cell migration.
View details for DOI 10.1074/jbc.M114.567446
View details for Web of Science ID 000343765400039
View details for PubMedID 25122766
View details for PubMedCentralID PMC4192498
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Myosin Phosphatase Target Subunit 1 (MYPT1) Regulates the Contraction and Relaxation of Vascular Smooth Muscle and Maintains Blood Pressure
JOURNAL OF BIOLOGICAL CHEMISTRY
2014; 289 (32): 22512–23
Abstract
Myosin light chain phosphatase with its regulatory subunit, myosin phosphatase target subunit 1 (MYPT1) modulates Ca(2+)-dependent phosphorylation of myosin light chain by myosin light chain kinase, which is essential for smooth muscle contraction. The role of MYPT1 in vascular smooth muscle was investigated in adult MYPT1 smooth muscle specific knock-out mice. MYPT1 deletion enhanced phosphorylation of myosin regulatory light chain and contractile force in isolated mesenteric arteries treated with KCl and various vascular agonists. The contractile responses of arteries from knock-out mice to norepinephrine were inhibited by Rho-associated kinase (ROCK) and protein kinase C inhibitors and were associated with inhibition of phosphorylation of the myosin light chain phosphatase inhibitor CPI-17. Additionally, stimulation of the NO/cGMP/protein kinase G (PKG) signaling pathway still resulted in relaxation of MYPT1-deficient mesenteric arteries, indicating phosphorylation of MYPT1 by PKG is not a major contributor to the relaxation response. Thus, MYPT1 enhances myosin light chain phosphatase activity sufficient for blood pressure maintenance. Rho-associated kinase phosphorylation of CPI-17 plays a significant role in enhancing vascular contractile responses, whereas phosphorylation of MYPT1 in the NO/cGMP/PKG signaling module is not necessary for relaxation.
View details for DOI 10.1074/jbc.M113.525444
View details for Web of Science ID 000340593500060
View details for PubMedID 24951589
View details for PubMedCentralID PMC4139257
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Altered contractile phenotypes of intestinal smooth muscle in mice deficient in myosin phosphatase target subunit 1
GASTROENTEROLOGY
2013; 144 (7): 1456–U233
Abstract
The regulatory subunit of myosin light chain phosphatase, MYPT1, has been proposed to control smooth muscle contractility by regulating phosphorylation of the Ca(2+)-dependent myosin regulatory light chain. We generated mice with a smooth muscle-specific deletion of MYPT1 to investigate its physiologic role in intestinal smooth muscle contraction.We used the Cre-loxP system to establish Mypt1-floxed mice, with the promoter region and exon 1 of Mypt1 flanked by 2 loxP sites. These mice were crossed with SMA-Cre transgenic mice to generate mice with smooth muscle-specific deletion of MYPT1 (Mypt1(SMKO) mice). The phenotype was assessed by histologic, biochemical, molecular, and physiologic analyses.Young adult Mypt1(SMKO) mice had normal intestinal motility in vivo, with no histologic abnormalities. On stimulation with KCl or acetylcholine, intestinal smooth muscles isolated from Mypt1(SMKO) mice produced robust and increased sustained force due to increased phosphorylation of the myosin regulatory light chain compared with muscle from control mice. Additional analyses of contractile properties showed reduced rates of force development and relaxation, and decreased shortening velocity, compared with muscle from control mice. Permeable smooth muscle fibers from Mypt1(SMKO) mice had increased sensitivity and contraction in response to Ca(2+).MYPT1 is not essential for smooth muscle function in mice but regulates the Ca(2+) sensitivity of force development and contributes to intestinal phasic contractile phenotype. Altered contractile responses in isolated tissues could be compensated by adaptive physiologic responses in vivo, where gut motility is affected by lower intensities of smooth muscle stimulation for myosin phosphorylation and force development.
View details for DOI 10.1053/j.gastro.2013.02.045
View details for Web of Science ID 000319498500029
View details for PubMedID 23499953
View details for PubMedCentralID PMC3782749
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Role of myosin light chain kinase in regulation of basal blood pressure and maintenance of salt-induced hypertension
AMERICAN JOURNAL OF PHYSIOLOGY-HEART AND CIRCULATORY PHYSIOLOGY
2011; 301 (2): H584–H591
Abstract
Vascular tone, an important determinant of systemic vascular resistance and thus blood pressure, is affected by vascular smooth muscle (VSM) contraction. Key signaling pathways for VSM contraction converge on phosphorylation of the regulatory light chain (RLC) of smooth muscle myosin. This phosphorylation is mediated by Ca(2+)/calmodulin-dependent myosin light chain kinase (MLCK) but Ca(2+)-independent kinases may also contribute, particularly in sustained contractions. Signaling through MLCK has been indirectly implicated in maintenance of basal blood pressure, whereas signaling through RhoA has been implicated in salt-induced hypertension. In this report, we analyzed mice with smooth muscle-specific knockout of MLCK. Mesenteric artery segments isolated from smooth muscle-specific MLCK knockout mice (MLCK(SMKO)) had a significantly reduced contractile response to KCl and vasoconstrictors. The kinase knockout also markedly reduced RLC phosphorylation and developed force. We suggest that MLCK and its phosphorylation of RLC are required for tonic VSM contraction. MLCK(SMKO) mice exhibit significantly lower basal blood pressure and weaker responses to vasopressors. The elevated blood pressure in salt-induced hypertension is reduced below normotensive levels after MLCK attenuation. These results suggest that MLCK is necessary for both physiological and pathological blood pressure. MLCK(SMKO) mice may be a useful model of vascular failure and hypotension.
View details for DOI 10.1152/ajpheart.01212.2010
View details for Web of Science ID 000293443800036
View details for PubMedID 21572007
View details for PubMedCentralID PMC3154661