scholarly journals Vascular disease-causing mutation R258C in ACTA2 disrupts actin dynamics and interaction with myosin

2015 ◽  
Vol 112 (31) ◽  
pp. E4168-E4177 ◽  
Author(s):  
Hailong Lu ◽  
Patricia M. Fagnant ◽  
Carol S. Bookwalter ◽  
Peteranne Joel ◽  
Kathleen M. Trybus
Keyword(s):  
Smooth Muscle ◽  
Actin Filaments ◽  
Contractile Function ◽  
Smooth Muscles ◽  
Aortic Aneurysms ◽  
Actin Dynamics ◽  
In Vitro Motility Assay ◽  
Subcortical Region ◽  
Muscle Tropomyosin

Point mutations in vascular smooth muscle α-actin (SM α-actin), encoded by the gene ACTA2, are the most prevalent cause of familial thoracic aortic aneurysms and dissections (TAAD). Here, we provide the first molecular characterization, to our knowledge, of the effect of the R258C mutation in SM α-actin, expressed with the baculovirus system. Smooth muscles are unique in that force generation requires both interaction of stable actin filaments with myosin and polymerization of actin in the subcortical region. Both aspects of R258C function therefore need investigation. Total internal reflection fluorescence (TIRF) microscopy was used to quantify the growth of single actin filaments as a function of time. R258C filaments are less stable than WT and more susceptible to severing by cofilin. Smooth muscle tropomyosin offers little protection from cofilin cleavage, unlike its effect on WT actin. Unexpectedly, profilin binds tighter to the R258C monomer, which will increase the pool of globular actin (G-actin). In an in vitro motility assay, smooth muscle myosin moves R258C filaments more slowly than WT, and the slowing is exacerbated by smooth muscle tropomyosin. Under loaded conditions, small ensembles of myosin are unable to produce force on R258C actin-tropomyosin filaments, suggesting that tropomyosin occupies an inhibitory position on actin. Many of the observed defects cannot be explained by a direct interaction with the mutated residue, and thus the mutation allosterically affects multiple regions of the monomer. Our results align with the hypothesis that defective contractile function contributes to the pathogenesis of TAAD.

scholarly journals Light chain phosphorylation regulates the movement of smooth muscle myosin on actin filaments.

1985 ◽  
Vol 101 (5) ◽  
pp. 1897-1902 ◽  
Author(s):  
J R Sellers ◽  
J A Spudich ◽  
M P Sheetz
Keyword(s):  
Smooth Muscle ◽  
Actin Filaments ◽  
Smooth Muscles ◽  
Smooth Muscle Myosin ◽  
Vitro Assay ◽  
Skeletal Muscle Myosin ◽  
Myosin Filaments ◽  
Muscle Myosin ◽  
Rate Of Movement

In smooth muscles there is no organized sarcomere structure wherein the relative movement of myosin filaments and actin filaments has been documented during contraction. Using the recently developed in vitro assay for myosin-coated bead movement (Sheetz, M.P., and J.A. Spudich, 1983, Nature (Lond.)., 303:31-35), we were able to quantitate the rate of movement of both phosphorylated and unphosphorylated smooth muscle myosin on ordered actin filaments derived from the giant alga, Nitella. We found that movement of turkey gizzard smooth muscle myosin on actin filaments depended upon the phosphorylation of the 20-kD myosin light chains. About 95% of the beads coated with phosphorylated myosin moved at velocities between 0.15 and 0.4 micron/s, depending upon the preparation. With unphosphorylated myosin, only 3% of the beads moved and then at a velocity of only approximately 0.01-0.04 micron/s. The effects of phosphorylation were fully reversible after dephosphorylation with a phosphatase prepared from smooth muscle. Analysis of the velocity of movement as a function of phosphorylation level indicated that phosphorylation of both heads of a myosin molecule was required for movement and that unphosphorylated myosin appears to decrease the rate of movement of phosphorylated myosin. Mixing of phosphorylated smooth muscle myosin with skeletal muscle myosin which moves at 2 microns/s resulted in a decreased rate of bead movement, suggesting that the more slowly cycling smooth muscle myosin is primarily determining the velocity of movement in such mixtures.

scholarly journals A simple method for measuring the relative force exerted by myosin on actin filaments in the in vitro motility assay: evidence that tropomyosin and troponin increase force in single thin filaments

2000 ◽  
Vol 350 (3) ◽  
pp. 693-699 ◽  
Author(s):  
Wu BING ◽  
Adam KNOTT ◽  
Steven B. MARSTON
Keyword(s):  
Actin Filaments ◽  
Isometric Force ◽  
Critical Concentration ◽  
Isometric Tension ◽  
Motility Assay ◽  
In Vitro Motility Assay ◽  
Simple Method ◽  
In Vitro Motility ◽  
Muscle Tropomyosin

We have studied the effect of an internal load on the movement of actin filaments over a bed of heavy meromyosin (HMM) in the invitro motility assay. Immobilized α-actinin can bind to actin filaments reversibly and ultimately stop the filaments from moving. Above a critical concentration of α-actinin, thin filament velocity rapidly diminished to zero. The fraction of thin motile filaments decreased linearly to zero with increasing α-actinin concentration. The concentration of α-actinin needed to stop all filaments from moving (0.8µg/ml with actin) was very consistent both within and between experiments. In the present study we have defined the ‘index of retardation’ as the concentration of α-actinin needed to stop all filament movement, and we propose that this index is a measure of the isometric force exerted by HMM on actin filaments. When we measured the effect of immobilized α-actinin on motility in the presence of 10mM Pi we found that the index of retardation was 0.62±0.07 (n = 3) times that in the absence of Pi. This observation is in agreement with the reduction of isometric tension in chemically-skinned muscle due to Pi. In a series of comparative experiments we observed that tropomyosin and troponin increase the index of retardation and that the degree of increase depends upon the tropomyosin isoform studied. The index of retardation of actin is increased 1.8-fold by skeletal-muscle tropomyosin, and 3-fold by both cardiac-muscle and smooth-muscle tropomyosin. In the presence of troponin the index of retardation is 2.9–3.4-fold greater than that of actin with all tropomyosin isoforms.

Targeting Matrix Metalloproteinases in Atherosclerosis and Cardiovascular Dysfunction

2019 ◽  
Vol 70 (2) ◽  
pp. 718-720
Author(s):  
Lucia Corina Dima-Cozma ◽  
Sebastian Cozma ◽  
Delia Hinganu ◽  
Cristina Mihaela Ghiciuc ◽  
Florin Mitu
Keyword(s):  
Smooth Muscle ◽  
Matrix Metalloproteinases ◽  
Cholesterol Synthesis ◽  
Balloon Dilation ◽  
Aortic Aneurysms ◽  
Arterial Lesions ◽  
Smooth Muscle Cell Migration ◽  
And Migration

Matrix metalloproteinases (MMPs) are the primary mediators of extracellular remodeling and their properties are useful in diagnostic evaluation and treatment. They are zinc-dependent proteases. MMPs have been involved in the mechanisms of atherosclerosis in various arterial areas, ischemic heart disease and myocardial infarction, atrial fibrillation and aortic aneurysms. Recently, MMP9 has been implicated in dyslipidemia and cholesterol synthesis by the liver. Increased MMP expression and activity has been associated with neointimal arterial lesions and migration of smooth muscle cells after arterial balloon dilation, while MMP inhibition decreases smooth muscle cell migration in vivo and in vitro.

scholarly journals Aging-associated changes in microRNA expression profile of internal anal sphincter smooth muscle: Role of microRNA-133a

2016 ◽  
Vol 311 (5) ◽  
pp. G964-G973 ◽  
Author(s):  
Jagmohan Singh ◽  
Ettickan Boopathi ◽  
Sankar Addya ◽  
Benjamin Phillips ◽  
Isidore Rigoutsos ◽  
...  
Keyword(s):  
Smooth Muscle ◽  
Anal Sphincter ◽  
Internal Anal Sphincter ◽  
Smooth Muscles ◽  
Smooth Muscle Contractility ◽  
Network Analyses ◽  
Rhoa Signaling

A comprehensive genomic and proteomic, computational, and physiological approach was employed to examine the (previously unexplored) role of microRNAs (miRNAs) as regulators of internal anal sphincter (IAS) smooth muscle contractile phenotype and basal tone. miRNA profiling, genome-wide expression, validation, and network analyses were employed to assess changes in mRNA and miRNA expression in IAS smooth muscles from young vs. aging rats. Multiple miRNAs, including rno-miR-1, rno-miR-340-5p, rno-miR-185, rno-miR-199a-3p, rno-miR-200c, rno-miR-200b, rno-miR-31, rno-miR-133a, and rno-miR-206, were found to be upregulated in aging IAS. qPCR confirmed the upregulated expression of these miRNAs and downregulation of multiple, predicted targets ( Eln, Col3a1, Col1a1, Zeb2, Myocd, Srf, Smad1, Smad2, Rhoa/Rock2, Fn1, Tagln v2, Klf4, and Acta2) involved in regulation of smooth muscle contractility. Subsequent studies demonstrated an aging-associated increase in the expression of miR-133a, corresponding decreases in RhoA, ROCK2, MYOCD, SRF, and SM22α protein expression, RhoA-signaling, and a decrease in basal and agonist [U-46619 (thromboxane A2analog)]-induced increase in the IAS tone. Moreover, in vitro transfection of miR-133a caused a dose-dependent increase of IAS tone in strips, which was reversed by anti-miR-133a. Last, in vivo perianal injection of anti-miR-133a reversed the loss of IAS tone associated with age. This work establishes the important regulatory effect of miRNA-133a on basal and agonist-stimulated IAS tone. Moreover, reversal of age-associated loss of tone via anti-miR delivery strongly implicates miR dysregulation as a causal factor in the aging-associated decrease in IAS tone and suggests that miR-133a is a feasible therapeutic target in aging-associated rectoanal incontinence.

Ca2+-independent contraction of longitudinal ileal smooth muscle is potentiated by a zipper-interacting protein kinase pseudosubstrate peptide

2009 ◽  
Vol 297 (2) ◽  
pp. G361-G370 ◽  
Author(s):  
Eikichi Ihara ◽  
Lori Moffat ◽  
Meredith A. Borman ◽  
Jennifer E. Amon ◽  
Michael P. Walsh ◽  
...  
Keyword(s):  
Smooth Muscle ◽  
Protein Kinase ◽  
Conformational Changes ◽  
Kinase Inhibitor ◽  
Smooth Muscles ◽  
Dominant Negative ◽  
Myosin Phosphatase ◽  
Interacting Protein ◽  
Zipper Interacting Protein Kinase

As a regulator of smooth muscle contraction, zipper-interacting protein kinase (ZIPK) can directly phosphorylate the myosin regulatory light chains (LC20) and produce contractile force. Synthetic peptides (SM-1 and AV25) derived from the autoinhibitory region of smooth muscle myosin light chain kinase can inhibit ZIPK activity in vitro. Paradoxically, treatment of Triton-skinned ileal smooth muscle strips with AV25, but not SM-1, potentiated Ca2+-independent, microcystin- and ZIPK-induced contractions. The AV25-induced potentiation was limited to ileal and colonic smooth muscles and was not observed in rat caudal artery. Thus the potentiation of Ca2+-independent contractions by AV25 appeared to be mediated by a mechanism unique to intestinal smooth muscle. AV25 treatment elicited increased phosphorylation of LC20 (both Ser-19 and Thr-18) and myosin phosphatase-targeting subunit (MYPT1, inhibitory Thr-697 site), suggesting involvement of a Ca2+-independent LC20 kinase with coincident inhibition of myosin phosphatase. The phosphorylation of the inhibitor of myosin phosphatase, CPI-17, was not affected. The AV25-induced potentiation was abolished by pretreatment with staurosporine, a broad-specificity kinase inhibitor, but specific inhibitors of Rho-associated kinase, PKC, and MAPK pathways had no effect. When a dominant-negative ZIPK [kinase-dead ZIPK(1–320)-D161A] was added to skinned ileal smooth muscle, the potentiation of microcystin-induced contraction by AV25 was blocked. Furthermore, pretreatment of skinned ileal muscle with SM-1 abolished AV25-induced potentiation. We conclude, therefore, that, even though AV25 is an in vitro inhibitor of ZIPK, activation of the ZIPK pathway occurs following application of AV25 to permeabilized ileal smooth muscle. Finally, we propose a mechanism whereby conformational changes in the pseudosubstrate region of ZIPK permit augmentation of ZIPK activity toward LC20 and MYPT1 in situ. AV25 or molecules based on its structure could be used in therapeutic situations to induce contractility in diseases of the gastrointestinal tract associated with hypomotility.

scholarly journals Myosin and gelsolin cooperate in actin filament severing and actomyosin motor activity

2020 ◽  
pp. jbc.RA120.015863
Author(s):  
Venukumar Vemula ◽  
Tamás Huber ◽  
Marko Ušaj ◽  
Beáta Bugyi ◽  
Alf Mansson
Keyword(s):  
Motor Activity ◽  
Actin Filament ◽  
Single Molecule ◽  
Actin Filaments ◽  
Actin Binding ◽  
In Vitro Motility Assay ◽  
Cooperative Effects ◽  
Myosin Motor ◽  
Filament Dynamics

Actin is a major intracellular protein with key functions in cellular motility, signaling and structural rearrangements. Its dynamic behavior, such as polymerisation and depolymerisation of actin filaments in response to intra- and extracellular cues, is regulated by an abundance of actin binding proteins. Out of these, gelsolin is one of the most potent for filament severing. However, myosin motor activity also fragments actin filaments through motor induced forces, suggesting that these two proteins could cooperate to regulate filament dynamics and motility. To test this idea, we used an in vitro motility assay, where actin filaments are propelled by surface-adsorbed heavy meromyosin (HMM) motor fragments. This allows studies of both motility and filament dynamics using isolated proteins. Gelsolin, at both nanomolar and micromolar Ca2+ concentration, appreciably enhanced actin filament severing caused by HMM-induced forces at 1 mM MgATP, an effect that was increased at higher HMM motor density. This finding is consistent with cooperativity between actin filament severing by myosin-induced forces and by gelsolin. We also observed reduced sliding velocity of the HMM-propelled filaments in the presence of gelsolin, providing further support of myosin-gelsolin cooperativity. Total internal reflection fluorescence microscopy based single molecule studies corroborated that the velocity reduction was a direct effect of gelsolin-binding to the filament and revealed different filament severing pattern of stationary and HMM propelled filaments. Overall, the results corroborate cooperative effects between gelsolin-induced alterations in the actin filaments and changes due to myosin motor activity leading to enhanced F-actin severing of possible physiological relevance.

Phenotypic Modulation of Smooth Muscle Cells after Arterial Injury Is Associated with Changes in the Distribution of Laminin and Fibronectin

1997 ◽  
Vol 45 (6) ◽  
pp. 837-846 ◽  
Author(s):  
Johan Thyberg ◽  
Karin Blomgren ◽  
Joy Roy ◽  
Phan Kiet Tran ◽  
Ulf Hedin
Keyword(s):  
Smooth Muscle ◽  
Basement Membrane ◽  
Smooth Muscle Cells ◽  
Actin Filaments ◽  
Cell Structure ◽  
Muscle Cells ◽  
Arterial Injury ◽  
Internal Elastic Lamina ◽  
Contractile Phenotype

Earlier in vitro studies suggest opposing roles of laminin and fibronectin in regulation of differentiated properties of vascular smooth muscle cells. To find out if this may also be the case in vivo, we used immunoelectron microscopy to study the distribution of these proteins during formation of intimal thickening after arterial injury. In parallel, cell structure and content of smooth muscle α-actin was analyzed. The results indicate that the cells in the normal media are in a contractile phenotype with abundant α-actin filaments and an incomplete basement membrane. Within 1 week after endothelial denudation, most cells in the innermost layer of the media convert into a synthetic phenotype, as judged by loss of actin filaments, construction of a large secretory apparatus, and destruction of the basement membrane. Some of these cells migrate through fenestrae in the internal elastic lamina and invade a fibronectin-rich network deposited on its luminal surface. Within another few weeks a thick neointima forms, newly produced matrix components replace the strands of fibronectin, and a basement membrane reappears. Simultaneously, the cells resume a contractile phenotype, recognized by disappearance of secretory organelles and restoration of α-actin filaments. These findings support the notion that laminin and other basement membrane components promote the expression of a differentiated smooth muscle phenotype, whereas fibronectin stimulates the cells to adopt a proliferative and secretory phenotype.

Effects of aging on actin sliding speed on myosin from single skeletal muscle cells of mice, rats, and humans

2001 ◽  
Vol 280 (4) ◽  
pp. C782-C788 ◽  
Author(s):  
Peter Höök ◽  
Vidyasagar Sriramoju ◽  
Lars Larsson
Keyword(s):  
Posttranslational Modifications ◽  
Actin Filaments ◽  
Mammalian Species ◽  
Type I ◽  
In Vitro Motility Assay ◽  
Sliding Speed ◽  
Myosin Heavy Chain Isoform ◽  
In Vitro Motility ◽  
Effects Of Aging

The effects of aging on the mechanical properties of myosin were measured in 87 fibers from muscles of humans ( n = 40), rats ( n = 21), and mice ( n = 26) using a single fiber in vitro motility assay. Irrespective of species, an 18–25% aging-related slowing in the speed of actin filaments was observed from 62 single fibers expressing the slow (type I) β-myosin heavy chain isoform. The mechanisms underlying the aging-related slowing of motility speed remain unknown, but it is suggested that posttranslational modifications of myosin by oxidative stress, glycation, or nitration play an important role. The aging-related slowing in the speed of actin filaments propelled by the type I myosin was confirmed in three mammalian species with an ∼3,400-fold difference in body size. Motility speed from human myosin was 3-fold slower than from myosin of the ∼3,400-fold smaller mouse and approximately twofold slower when compared with the ∼130-fold smaller rat, irrespective of age. A strong correlation was observed between the log values of actin sliding speed and body mass, suggesting that the effects of scaling is, at least in part, due to altered functional properties of the motor protein itself.

Contribution of SRF, Elk-1, and myocardin to airway smooth muscle remodeling in heaves, an asthma-like disease of horses

2015 ◽  
Vol 309 (1) ◽  
pp. L37-L45 ◽  
Author(s):  
Mylène Chevigny ◽  
Karine Guérin-Montpetit ◽  
Amandine Vargas ◽  
Josiane Lefebvre-Lavoie ◽  
Jean-Pierre Lavoie
Keyword(s):  
Smooth Muscle ◽  
Airway Smooth Muscle ◽  
Serum Response Factor ◽  
Smooth Muscles ◽  
Peripheral Airways ◽  
Myocyte Differentiation ◽  
Antigenic Exposure ◽  
Serum Response ◽  
Muscle Remodeling

Myocyte hyperplasia and hypertrophy contribute to the increased mass of airway smooth muscle (ASM) in asthma. Serum-response factor (SRF) is a transcription factor that regulates myocyte differentiation in vitro in vascular and intestinal smooth muscles. When SRF is associated with phosphorylated (p)Elk-1, it promotes ASM proliferation while binding to myocardin (MYOCD) leading to the expression of contractile elements in these tissues. The objective of this study was therefore to characterize the expression of SRF, pElk-1, and MYOCD in ASM cells from central and peripheral airways in heaves, a spontaneously occurring asthma-like disease of horses, and in controls. Six horses with heaves and five aged-matched controls kept in the same environment were studied. Nuclear protein expression of SRF, pElk-1, and MYOCD was evaluated in peripheral airways and endobronchial biopsies obtained during disease remission and after 1 and 30 days of naturally occurring antigenic exposure using immunohistochemistry and immunofluorescence techniques. Nuclear expression of SRF ( P = 0.03, remission vs. 30 days) and MYOCD ( P = 0.05, controls vs. heaves at 30 days) increased in the peripheral airways of horses with heaves during disease exacerbation, while MYOCD ( P = 0.04, remission vs. 30 days) decreased in the central airways of control horses. No changes were observed in the expression of pElk-1 protein in either tissue. In conclusion, SRF and its cofactor MYOCD likely contribute to the hypertrophy of peripheral ASM observed in equine asthmatic airways, while the remodeling of the central airways is more static or involves different transcription factors.

scholarly journals Actin-depolymerizing Factor and Cofilin-1 Play Overlapping Roles in Promoting Rapid F-Actin Depolymerization in Mammalian Nonmuscle Cells

2005 ◽  
Vol 16 (2) ◽  
pp. 649-664 ◽  
Author(s):  
Pirta Hotulainen ◽  
Eija Paunola ◽  
Maria K. Vartiainen ◽  
Pekka Lappalainen
Keyword(s):  
Cell Motility ◽  
Actin Filament ◽  
Actin Filaments ◽  
Actin Dynamics ◽  
Actin Binding ◽  
Actin Depolymerizing Factor ◽  
Cytoskeletal Dynamics ◽  
Nonmuscle Cells ◽  
In Cells

Actin-depolymerizing factor (ADF)/cofilins are small actin-binding proteins found in all eukaryotes. In vitro, ADF/cofilins promote actin dynamics by depolymerizing and severing actin filaments. However, whether ADF/cofilins contribute to actin dynamics in cells by disassembling “old” actin filaments or by promoting actin filament assembly through their severing activity is a matter of controversy. Analysis of mammalian ADF/cofilins is further complicated by the presence of multiple isoforms, which may contribute to actin dynamics by different mechanisms. We show that two isoforms, ADF and cofilin-1, are expressed in mouse NIH 3T3, B16F1, and Neuro 2A cells. Depleting cofilin-1 and/or ADF by siRNA leads to an accumulation of F-actin and to an increase in cell size. Cofilin-1 and ADF seem to play overlapping roles in cells, because the knockdown phenotype of either protein could be rescued by overexpression of the other one. Cofilin-1 and ADF knockdown cells also had defects in cell motility and cytokinesis, and these defects were most pronounced when both ADF and cofilin-1 were depleted. Fluorescence recovery after photobleaching analysis and studies with an actin monomer-sequestering drug, latrunculin-A, demonstrated that these phenotypes arose from diminished actin filament depolymerization rates. These data suggest that mammalian ADF and cofilin-1 promote cytoskeletal dynamics by depolymerizing actin filaments and that this activity is critical for several processes such as cytokinesis and cell motility.

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