The coiled-coil domain of MURC/cavin-4 is involved in membrane trafficking of caveolin-3 in cardiomyocytes

2015 ◽  
Vol 309 (12) ◽  
pp. H2127-H2136 ◽  
Author(s):  
Daisuke Naito ◽  
Takehiro Ogata ◽  
Tetsuro Hamaoka ◽  
Naohiko Nakanishi ◽  
Kotaro Miyagawa ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Cardiac Function ◽  
Membrane Trafficking ◽  
Protein Level ◽  
Interstitial Fibrosis ◽  
Coiled Coil ◽  
Cardiomyocyte Hypertrophy ◽  
Coiled Coil Domain ◽  
Cytoplasmic Proteins ◽  
And Function

Muscle-restricted coiled-coil protein (MURC), also referred to as cavin-4, is a member of the cavin family that works cooperatively with caveolins in caveola formation and function. Cavins are cytoplasmic proteins with coiled-coil domains and form heteromeric complexes, which are recruited to caveolae in cells expressing caveolins. Among caveolins, caveolin-3 (Cav3) is exclusively expressed in muscle cells, similar to MURC/cavin-4. In the heart, Cav3 overexpression contributes to cardiac protection, and its deficiency leads to progressive cardiomyopathy. Mutations in the MURC/cavin-4 gene have been identified in patients with dilated cardiomyopathy. In the present study, we show the role of MURC/cavin-4 as a caveolar component in the heart. In H9c2 cells, MURC/cavin-4 was localized at the plasma membrane, whereas a MURC/cavin-4 mutant lacking the coiled-coil domain (ΔCC) was primarily localized to the cytoplasm. ΔCC bound to Cav3 and impaired membrane localization of Cav3 in cardiomyocytes. Additionally, although ΔCC did not alter Cav3 mRNA expression, ΔCC decreased the Cav3 protein level. MURC/cavin-4 and ΔCC similarly induced cardiomyocyte hypertrophy; however, ΔCC showed higher hypertrophy-related fetal gene expression than MURC/cavin-4. ΔCC induced ERK activation in cardiomyocytes. Transgenic mice expressing ΔCC in the heart (ΔCC-Tg mice) showed impaired cardiac function accompanied by cardiomyocyte hypertrophy and marked interstitial fibrosis. Hearts from ΔCC-Tg mice showed a reduction of the Cav3 protein level and activation of ERK. These results suggest that MURC/cavin-4 requires its coiled-coil domain to target the plasma membrane and to stabilize Cav3 at the plasma membrane of cardiomyocytes and that MURC/cavin-4 functions as a crucial caveolar component to regulate cardiac function.

Abstract 15441: MURC/Cavin-4 Requires the Coiled-coil Domain for Membrane Trafficking of Caveolin-3 in Cardiomyocytes

Circulation ◽  
2015 ◽  
Vol 132 (suppl_3) ◽  
Author(s):  
Masahiro Nishi ◽  
Takehiro Ogata ◽  
Daisuke Naito ◽  
Tetsuro Hamaoka ◽  
Naohiko Nakanishi ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Cardiac Function ◽  
Mrna Expression ◽  
Membrane Trafficking ◽  
Protein Level ◽  
Interstitial Fibrosis ◽  
Coiled Coil ◽  
Cardiomyocyte Hypertrophy ◽  
Lung Weight ◽  
Coiled Coil Domain

[Background] Muscle-restricted coiled-coil protein (MURC), also referred to as Cavin-4, is a member of the cavin family that is cooperated with caveolins in caveola formation and function. Cavins are cytoplasmic proteins that have coiled-coil domains and form heteromeric complexes, which are recruited to caveolae in cells expressing caveolins. Among caveolins, caveolin-3 (Cav3) is exclusively expressed in muscle cells, similar to MURC/Cavin-4. In the heart, Cav3 overexpression contributes to cardiac protection, while its deficiency leads to progressive cardiomyopathy. Mutations in the MURC/Cavin-4 gene have been identified in patients with dilated cardiomyopathy. MURC/Cavin-4 has the coiled-coil domain; however, its functional significance remains unknown. [Methods and Results] MURC/Cavin-4 was localized at the plasma membrane in H9c2 cells, while a MURC/Cavin-4 mutant lacking the coiled-coil domain (ΔCC) was primarily localized to the cytoplasm. ΔCC bound to Cav3 and impaired membrane localization of Cav3 in cardiomyocytes. In addition, although ΔCC did not influence Cav3 mRNA expression, it decreased the Cav3 protein level in cardiomyocytes. MURC/Cavin-4 and ΔCC similarly induced cardiomyocytes hypertrophy; however, ΔCC showed higher hypertrophy-related fetal gene expression, such as ANP, BNP, and skeletal α-actin (SkA) mRNA expression and the βMHC/αMHC ratio, than MURC/Cavin-4. ΔCC induced ERK activation in cardiomyocytes. Cardiac function assessed by echocardiography was significantly impaired in transgenic mice expressing ΔCC in the heart (ΔCC-Tg mice) compared with non-transgenic (NTg) mice (% FS; 24.6 ± 2.4 % in ΔCC-Tg mice vs. 48.8 ± 2.5 % in NTg mice, p<0.01), which was accompanied by cardiomyocyte hypertrophy, increased interstitial fibrosis, and the increase of lung weight-to-tibial-length ratio (LW/TL; 18.04 ± 2.41 in ΔCC-Tg mice vs. 7.51 ± 0.81 in NTg mice, p<0.01). ΔCC-Tg hearts showed reduction of the Cav3 protein level and the activation of ERK. [Conclusions] These results reveal that MURC/Cavin-4 requires its coiled-coil domain to target the plasma membrane and Cav3 trafficking to the plasma membrane of cardiomyocytes, and that MURC/Cavin-4 functions as a crucial caveolar component to regulate cardiac function.

scholarly journals Direct regulation of Arp2/3 complex activity and function by the actin binding protein coronin

2002 ◽  
Vol 159 (6) ◽  
pp. 993-1004 ◽  
Author(s):  
Christine L. Humphries ◽  
Heath I. Balcer ◽  
Jessica L. D'Agostino ◽  
Barbara Winsor ◽  
David G. Drubin ◽  
...  
Keyword(s):  
Binding Protein ◽  
Coiled Coil ◽  
Actin Binding ◽  
Complex Activity ◽  
Actin Binding Protein ◽  
Coiled Coil Domain ◽  
Allele Specific ◽  
And Function

Mechanisms for activating the actin-related protein 2/3 (Arp2/3) complex have been the focus of many recent studies. Here, we identify a novel mode of Arp2/3 complex regulation mediated by the highly conserved actin binding protein coronin. Yeast coronin (Crn1) physically associates with the Arp2/3 complex and inhibits WA- and Abp1-activated actin nucleation in vitro. The inhibition occurs specifically in the absence of preformed actin filaments, suggesting that Crn1 may restrict Arp2/3 complex activity to the sides of filaments. The inhibitory activity of Crn1 resides in its coiled coil domain. Localization of Crn1 to actin patches in vivo and association of Crn1 with the Arp2/3 complex also require its coiled coil domain. Genetic studies provide in vivo evidence for these interactions and activities. Overexpression of CRN1 causes growth arrest and redistribution of Arp2 and Crn1p into aberrant actin loops. These defects are suppressed by deletion of the Crn1 coiled coil domain and by arc35-26, an allele of the p35 subunit of the Arp2/3 complex. Further in vivo evidence that coronin regulates the Arp2/3 complex comes from the observation that crn1 and arp2 mutants display an allele-specific synthetic interaction. This work identifies a new form of regulation of the Arp2/3 complex and an important cellular function for coronin.

Multimerization and tubulin binding are required for the SPIRAL2 protein to localize to and stabilize microtubule minus ends

2021 ◽  
Author(s):  
YUANWEI FAN ◽  
Natasha Bilkey ◽  
Ram Dixit
Keyword(s):  
Coiled Coil ◽  
Spatial Arrangement ◽  
In Planta ◽  
Structural Determinants ◽  
Coiled Coil Domain ◽  
Tubulin Binding ◽  
And Function ◽  
Necessary And Sufficient ◽  
Basic Region

Accruing evidence points to the control of microtubule minus-end dynamics as being crucial for the spatial arrangement and function of the microtubule cytoskeleton. In plants, the SPIRAL2 (SPR2) protein has emerged as a microtubule minus-end regulator that is structurally distinct from the animal minus-end regulators. Previously, SPR2 was shown to autonomously localize to microtubule minus ends and decrease their depolymerization rate. Here, we used in vitro and in planta experiments to identify the structural determinants required for SPR2 to recognize and stabilize microtubule minus ends. We show that SPR2 contains a single N-terminal TOG domain that binds to soluble tubulin. The TOG domain, a basic region, and coiled-coil domain are necessary and sufficient to target and stabilize microtubule minus ends. We demonstrate that the coiled-coil domain mediates multimerization of SPR2 that provides avidity for microtubule binding and is essential for binding to soluble tubulin. While TOG domain-containing proteins are traditionally thought to function as microtubule plus-end regulators, our results reveal that nature has repurposed the TOG domain of SPR2 to regulate microtubule minus ends.

scholarly journals Optimized filopodia formation requires myosin tail domain cooperation

2019 ◽  
Vol 116 (44) ◽  
pp. 22196-22204
Author(s):  
Ashley L. Arthur ◽  
Livia D. Songster ◽  
Helena Sirkia ◽  
Akash Bhattacharya ◽  
Carlos Kikuti ◽  
...  
Keyword(s):  
Coiled Coil ◽  
Tail Domain ◽  
Tail Region ◽  
Lever Arm ◽  
Coiled Coil Domain ◽  
And Function

Filopodia are actin-filled protrusions employed by cells to interact with their environment. Filopodia formation in Amoebozoa and Metazoa requires the phylogenetically diverse MyTH4-FERM (MF) myosins DdMyo7 and Myo10, respectively. While Myo10 is known to form antiparallel dimers, DdMyo7 lacks a coiled-coil domain in its proximal tail region, raising the question of how such divergent motors perform the same function. Here, it is shown that the DdMyo7 lever arm plays a role in both autoinhibition and function while the proximal tail region can mediate weak dimerization, and is proposed to be working in cooperation with the C-terminal MF domain to promote partner-mediated dimerization. Additionally, a forced dimer of the DdMyo7 motor is found to weakly rescue filopodia formation, further highlighting the importance of the C-terminal MF domain. Thus, weak dimerization activity of the DdMyo7 proximal tail allows for sensitive regulation of myosin activity to prevent inappropriate activation of filopodia formation. The results reveal that the principles of MF myosin-based filopodia formation are conserved via divergent mechanisms for dimerization.

scholarly journals Num1 anchors mitochondria to the plasma membrane via two domains with different lipid binding specificities

2016 ◽  
Vol 213 (5) ◽  
pp. 513-524 ◽  
Author(s):  
Holly A. Ping ◽  
Lauren M. Kraft ◽  
WeiTing Chen ◽  
Amy E. Nilles ◽  
Laura L. Lackner
Keyword(s):  
Plasma Membrane ◽  
Specific Binding ◽  
Coiled Coil ◽  
Lipid Binding ◽  
General Mechanism ◽  
Cell Cortex ◽  
Pleckstrin Homology Domain ◽  
Binding Domains ◽  
Coiled Coil Domain ◽  
Mitochondrial Distribution

The mitochondria–ER cortex anchor (MECA) is required for proper mitochondrial distribution and functions by tethering mitochondria to the plasma membrane. The core component of MECA is the multidomain protein Num1, which assembles into clusters at the cell cortex. We show Num1 adopts an extended, polarized conformation. Its N-terminal coiled-coil domain (Num1CC) is proximal to mitochondria, and the C-terminal pleckstrin homology domain is associated with the plasma membrane. We find that Num1CC interacts directly with phospholipid membranes and displays a strong preference for the mitochondria-specific phospholipid cardiolipin. This direct membrane interaction is critical for MECA function. Thus, mitochondrial anchoring is mediated by a protein that interacts directly with two different membranes through lipid-specific binding domains, suggesting a general mechanism for interorganelle tethering.

scholarly journals Model for the architecture of caveolae based on a flexible, net-like assembly of Cavin1 and Caveolin discs

2016 ◽  
Vol 113 (50) ◽  
pp. E8069-E8078 ◽  
Author(s):  
Miriam Stoeber ◽  
Pascale Schellenberger ◽  
C. Alistair Siebert ◽  
Cedric Leyrat ◽  
Ari Helenius ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Complex Formation ◽  
Membrane Binding ◽  
Coiled Coil ◽  
Structural Basis ◽  
Membrane Domains ◽  
Coiled Coil Domain ◽  
Regular Dodecahedron ◽  
Plasma Membrane Domains ◽  
Electron Cryotomography

Caveolae are invaginated plasma membrane domains involved in mechanosensing, signaling, endocytosis, and membrane homeostasis. Oligomers of membrane-embedded caveolins and peripherally attached cavins form the caveolar coat whose structure has remained elusive. Here, purified Cavin1 60S complexes were analyzed structurally in solution and after liposome reconstitution by electron cryotomography. Cavin1 adopted a flexible, net-like protein mesh able to form polyhedral lattices on phosphatidylserine-containing vesicles. Mutating the two coiled-coil domains in Cavin1 revealed that they mediate distinct assembly steps during 60S complex formation. The organization of the cavin coat corresponded to a polyhedral nano-net held together by coiled-coil segments. Positive residues around the C-terminal coiled-coil domain were required for membrane binding. Purified caveolin 8S oligomers assumed disc-shaped arrangements of sizes that are consistent with the discs occupying the faces in the caveolar polyhedra. Polygonal caveolar membrane profiles were revealed in tomograms of native caveolae inside cells. We propose a model with a regular dodecahedron as structural basis for the caveolae architecture.

scholarly journals Ketogenic Diet Suppressed T-Regulatory Cells and Promoted Cardiac Fibrosis via Reducing Mitochondria-Associated Membranes and Inhibiting Mitochondrial Function

2021 ◽  
Vol 2021 ◽  
pp. 1-15
Author(s):  
Jun Tao ◽  
Hao Chen ◽  
Ya-Jing Wang ◽  
Jun-Xiong Qiu ◽  
Qing-Qi Meng ◽  
...  
Keyword(s):  
Fatty Acid ◽  
Cardiac Function ◽  
Ketogenic Diet ◽  
Mitochondrial Respiration ◽  
Cardiac Fibrosis ◽  
Interstitial Fibrosis ◽  
Diabetic Patients ◽  
T Regulatory Cell ◽  
Metabolic Indices ◽  
And Function

Ketogenic diet (KD) is popular in diabetic patients but its cardiac safety and efficiency on the heart are unknown. The aim of the present study is to determine the effects and the underlined mechanisms of KD on cardiac function in diabetic cardiomyopathy (DCM). We used db/db mice to model DCM, and different diets (regular or KD) were used. Cardiac function and interstitial fibrosis were determined. T-regulatory cell (Treg) number and functions were evaluated. The effects of ketone body (KB) on fatty acid (FA) and glucose metabolism, mitochondria-associated endoplasmic reticulum membranes (MAMs), and mitochondrial respiration were assessed. The mechanisms via which KB regulated MAMs and Tregs were addressed. KD improved metabolic indices in db/db mice. However, KD impaired cardiac diastolic function and exacerbated ventricular fibrosis. Proportions of circulatory CD4+CD25+Foxp3+ cells in whole blood cells and serum levels of IL-4 and IL-10 were reduced in mice fed with KD. KB suppressed the differentiation to Tregs from naive CD4+ T cells. Cultured medium from KB-treated Tregs synergically activated cardiac fibroblasts. Meanwhile, KB inhibited Treg proliferation and productions of IL-4 and IL-10. Treg MAMs, mitochondrial respiration and respiratory complexes, and FA synthesis and oxidation were all suppressed by KB while glycolytic levels were increased. L-carnitine reversed Treg proliferation and function inhibited by KB. Proportions of ST2L+ cells in Tregs were reduced by KB, as well as the production of ST2L ligand, IL-33. Reinforcement expressions of ST2L in Tregs counteracted the reductions in MAMs, mitochondrial respiration, and Treg proliferations and productions of Treg cytokines IL-4 and IL-10. Therefore, despite the improvement of metabolic indices, KD impaired Treg expansion and function and promoted cardiac fibroblast activation and interstitial fibrosis. This could be mainly mediated by the suppression of MAMs and fatty acid metabolism inhibition via blunting IL-33/ST2L signaling.

scholarly journals Stac adaptor proteins regulate trafficking and function of muscle and neuronal L-type Ca2+ channels

2014 ◽  
Vol 112 (2) ◽  
pp. 602-606 ◽  
Author(s):  
Alexander Polster ◽  
Stefano Perni ◽  
Hicham Bichraoui ◽  
Kurt G. Beam
Keyword(s):  
Skeletal Muscle ◽  
Plasma Membrane ◽  
Membrane Trafficking ◽  
Mammalian Cells ◽  
Ryanodine Receptors ◽  
Adaptor Proteins ◽  
Surface Expression ◽  
Ec Coupling ◽  
And Function

Excitation–contraction (EC) coupling in skeletal muscle depends upon trafficking of CaV1.1, the principal subunit of the dihydropyridine receptor (DHPR) (L-type Ca2+ channel), to plasma membrane regions at which the DHPRs interact with type 1 ryanodine receptors (RyR1) in the sarcoplasmic reticulum. A distinctive feature of this trafficking is that CaV1.1 expresses poorly or not at all in mammalian cells that are not of muscle origin (e.g., tsA201 cells), in which all of the other nine CaV isoforms have been successfully expressed. Here, we tested whether plasma membrane trafficking of CaV1.1 in tsA201 cells is promoted by the adapter protein Stac3, because recent work has shown that genetic deletion of Stac3 in skeletal muscle causes the loss of EC coupling. Using fluorescently tagged constructs, we found that Stac3 and CaV1.1 traffic together to the tsA201 plasma membrane, whereas CaV1.1 is retained intracellularly when Stac3 is absent. Moreover, L-type Ca2+ channel function in tsA201 cells coexpressing Stac3 and CaV1.1 is quantitatively similar to that in myotubes, despite the absence of RyR1. Although Stac3 is not required for surface expression of CaV1.2, the principle subunit of the cardiac/brain L-type Ca2+ channel, Stac3 does bind to CaV1.2 and, as a result, greatly slows the rate of current inactivation, with Stac2 acting similarly. Overall, these results indicate that Stac3 is an essential chaperone of CaV1.1 in skeletal muscle and that in the brain, Stac2 and Stac3 may significantly modulate CaV1.2 function.

scholarly journals Lrrcc1 and Ccdc61 are conserved effectors of multiciliated cell function

2021 ◽  
Author(s):  
Aude Nommick ◽  
Camille Boutin ◽  
Olivier Rosnet ◽  
Elsa Bazellières ◽  
Virginie Thomé ◽  
...  
Keyword(s):  
Pathogenic Bacteria ◽  
Cell Function ◽  
Coiled Coil ◽  
Fluid Flows ◽  
Basal Bodies ◽  
Animal Evolution ◽  
Ciliary Beating ◽  
Coiled Coil Domain ◽  
Multiciliated Cells ◽  
And Function

AbstractCiliated epithelia perform a variety of essential functions across animal evolution, ranging from locomotion of marine organisms to mucociliary clearance of airways in mammals. These epithelia are composed of multiciliated cells (MCCs) harbouring myriads of motile cilia, which rest on modified centrioles called basal bodies (BBs), and beat coordinately to generate directed fluid flows. Thus, BB biogenesis and organization is central to MCC function. In basal eukaryotes, the coiled-coil domain proteins Lrrcc1 and Ccdc61 were shown to be required for proper BB construction and function. Here, we used the Xenopus embryonic ciliated epidermis to characterize Lrrcc1 and Ccdc61 in vertebrate MCCs. We found that they both encode BB components, with a prominent association to striated rootlets. Knocking down either gene caused defects in BB docking, spacing, and polarization. Moreover, their depletion impaired the apical cytoskeleton, and altered ciliary beating. Consequently, cilia-powered fluid flow was greatly reduced in morphant tadpoles, which displayed enhanced mortality when exposed to pathogenic bacteria. This work illustrates how integration across organizational scales make elementary BB components essential for the emergence of the physiological function of ciliated epithelia.

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