scholarly journals Mechanisms of thin filament assembly in embryonic chick cardiac myocytes: tropomodulin requires tropomyosin for assembly.

1995 ◽  
Vol 129 (3) ◽  
pp. 683-695 ◽  
Author(s):  
C C Gregorio ◽  
V M Fowler
Keyword(s):  
Actin Filaments ◽  
Thin Filament ◽  
Striated Muscle ◽  
Cell Model ◽  
Significant Proportion ◽  
Embryonic Chick ◽  
Permeabilized Cell ◽  
Thin Filaments ◽  
Filament Assembly ◽  
Cell Biol

Tropomodulin is a pointed end capping protein for tropomyosin-coated actin filaments that is hypothesized to play a role in regulating the precise lengths of striated muscle thin filaments (Fowler, V. M., M. A. Sussman, P. G. Miller, B. E. Flucher, and M. P. Daniels. 1993. J. Cell Biol. 120:411-420; Weber, A., C. C. Pennise, G. G. Babcock, and V. M. Fowler. 1994, J. Cell Biol. 127:1627-1635). To gain insight into the mechanisms of thin filament assembly and the role of tropomodulin therein, we have characterized the temporal appearance, biosynthesis and mechanisms of assembly of tropomodulin onto the pointed ends of thin filaments during the formation of striated myofibrils in primary embryonic chick cardiomyocyte cultures. Our results demonstrate that tropomodulin is not assembled coordinately with other thin filament proteins. Double immunofluorescence staining and ultrastructural immunolocalization demonstrate that tropomodulin is incorporated in its characteristic sarcomeric location at the pointed ends of the thin filaments after the thin filaments have become organized into periodic I bands. In fact, tropomodulin assembles later than all other well characterized myofibrillar proteins studied including: actin, tropomyosin, alpha-actinin, titin, myosin and C-protein. Nevertheless, at steady state, a significant proportion (approximately 39%) of tropomodulin is present in a soluble pool throughout myofibril assembly. Thus, the absence of tropomodulin in some striated myofibrils is not due to limiting quantities of the protein. In addition, kinetic data obtained from [35S]methionine pulse-chase experiments indicate that tropomodulin assembles more slowly into myofibrils than does tropomyosin. This observation, together with results obtained using a novel permeabilized cell model for thin filament assembly, indicate that tropomodulin assembly is dependent on the prior association of tropomyosin with actin filaments. We conclude that tropomodulin is a late marker for the assembly of striated myofibrils in cardiomyocytes; its assembly appears to be linked to their maturity. We propose that tropomodulin is involved in maintaining and stabilizing the final lengths of thin filaments after they are assembled.

scholarly journals Tropomodulin Capping of Actin Filaments in Striated Muscle Development and Physiology

2011 ◽  
Vol 2011 ◽  
pp. 1-16 ◽  
Author(s):  
David S. Gokhin ◽  
Velia M. Fowler
Keyword(s):  
Actin Filament ◽  
Actin Filaments ◽  
Thin Filament ◽  
Muscle Development ◽  
Striated Muscle ◽  
Expression Patterns ◽  
Contractile Apparatus ◽  
Thin Filaments ◽  
Filament Assembly ◽  
Abundant Evidence

Efficient striated muscle contraction requires precise assembly and regulation of diverse actin filament systems, most notably the sarcomeric thin filaments of the contractile apparatus. By capping the pointed ends of actin filaments, tropomodulins (Tmods) regulate actin filament assembly, lengths, and stability. Here, we explore the current understanding of the expression patterns, localizations, and functions of Tmods in both cardiac and skeletal muscle. We first describe the mechanisms by which Tmods regulate myofibril assembly and thin filament lengths, as well as the roles of closely related Tmod family variants, the leiomodins (Lmods), in these processes. We also discuss emerging functions for Tmods in the sarcoplasmic reticulum. This paper provides abundant evidence that Tmods are key structural regulators of striated muscle cytoarchitecture and physiology.

scholarly journals Knockout of Lmod2 results in shorter thin filaments followed by dilated cardiomyopathy and juvenile lethality

2015 ◽  
Vol 112 (44) ◽  
pp. 13573-13578 ◽  
Author(s):  
Christopher T. Pappas ◽  
Rachel M. Mayfield ◽  
Christine Henderson ◽  
Nima Jamilpour ◽  
Cathleen Cover ◽  
...  
Keyword(s):  
Dilated Cardiomyopathy ◽  
Thin Filament ◽  
Striated Muscle ◽  
Heart Function ◽  
Contractile Force ◽  
Actin Binding ◽  
Actin Assembly ◽  
Thin Filaments ◽  
Filament Assembly ◽  
Muscle Thin Filament

Leiomodin 2 (Lmod2) is an actin-binding protein that has been implicated in the regulation of striated muscle thin filament assembly; its physiological function has yet to be studied. We found that knockout of Lmod2 in mice results in abnormally short thin filaments in the heart. We also discovered that Lmod2 functions to elongate thin filaments by promoting actin assembly and dynamics at thin filament pointed ends. Lmod2-KO mice die as juveniles with hearts displaying contractile dysfunction and ventricular chamber enlargement consistent with dilated cardiomyopathy. Lmod2-null cardiomyocytes produce less contractile force than wild type when plated on micropillar arrays. Introduction of GFP-Lmod2 via adeno-associated viral transduction elongates thin filaments and rescues structural and functional defects observed in Lmod2-KO mice, extending their lifespan to adulthood. Thus, to our knowledge, Lmod2 is the first identified mammalian protein that functions to elongate actin filaments in the heart; it is essential for cardiac thin filaments to reach a mature length and is required for efficient contractile force and proper heart function during development.

Orientation of actin filaments during motion in motility assay

1995 ◽  
Vol 53 ◽  
pp. 52-53
Author(s):  
J. Borejdo ◽  
S. Burlacu
Keyword(s):  
Actin Filaments ◽  
Thin Filament ◽  
Striated Muscle ◽  
Myosin Head ◽  
Classical Method ◽  
Polarization Of Fluorescence ◽  
Single Protein ◽  
Intracellular Organelles ◽  
Change Orientation ◽  
Active Entity

Polarization of fluorescence is a classical method to assess orientation or mobility of macromolecules. It has been a common practice to measure polarization of fluorescence through a microscope to characterize orientation or mobility of intracellular organelles, for example anisotropic bands in striated muscle. Recently, we have extended this technique to characterize single protein molecules. The scientific question concerned the current problem in muscle motility: whether myosin heads or actin filaments change orientation during contraction. The classical view is that the force-generating step in muscle is caused by change in orientation of myosin head (subfragment-1 or SI) relative to the axis of thin filament. The molecular impeller which causes this change resides at the interface between actin and SI, but it is not clear whether only the myosin head or both SI and actin change orientation during contraction. Most studies assume that observed orientational change in myosin head is a reflection of the fact that myosin is an active entity and actin serves merely as a passive "rail" on which myosin moves.

scholarly journals Tropomodulin caps the pointed ends of actin filaments.

1994 ◽  
Vol 127 (6) ◽  
pp. 1627-1635 ◽  
Author(s):  
A Weber ◽  
C R Pennise ◽  
G G Babcock ◽  
V M Fowler
Keyword(s):  
Red Blood Cells ◽  
Blood Cells ◽  
Actin Filaments ◽  
Striated Muscle ◽  
Critical Concentration ◽  
Thin Filaments ◽  
Capping Protein ◽  
End Capping ◽  
Pointed End ◽  
A Site

Many proteins have been shown to cap the fast growing (barbed) ends of actin filaments, but none have been shown to block elongation and depolymerization at the slow growing (pointed) filament ends. Tropomodulin is a tropomyosin-binding protein originally isolated from red blood cells that has been localized by immunofluorescence staining to a site at or near the pointed ends of skeletal muscle thin filaments (Fowler, V. M., M. A., Sussman, P. G. Miller, B. E. Flucher, and M. P. Daniels. 1993. J. Cell Biol. 120: 411-420). Our experiments demonstrate that tropomodulin in conjunction with tropomyosin is a pointed end capping protein: it completely blocks both elongation and depolymerization at the pointed ends of tropomyosin-containing actin filaments in concentrations stoichiometric to the concentration of filament ends (Kd < or = 1 nM). In the absence of tropomyosin, tropomodulin acts as a "leaky" cap, partially inhibiting elongation and depolymerization at the pointed filament ends (Kd for inhibition of elongation = 0.1-0.4 microM). Thus, tropomodulin can bind directly to actin at the pointed filament end. Tropomodulin also doubles the critical concentration at the pointed ends of pure actin filaments without affecting either the rate of extent of polymerization at the barbed filament ends, indicating that tropomodulin does not sequester actin monomers. Our experiments provide direct biochemical evidence that tropomodulin binds to both the terminal tropomyosin and actin molecules at the pointed filament end, and is the long sought-after pointed end capping protein. We propose that tropomodulin plays a role in maintaining the narrow length distributions of the stable, tropomyosin-containing actin filaments in striated muscle and in red blood cells.

Characterization of the actin filament capping state in human erythrocyte ghost and cytoskeletal preparations

2000 ◽  
Vol 349 (1) ◽  
pp. 105-111 ◽  
Author(s):  
Philip A. KUHLMAN
Keyword(s):  
Actin Filament ◽  
Human Erythrocyte ◽  
Actin Filaments ◽  
Striated Muscle ◽  
Length Distribution ◽  
Shearing Stress ◽  
Bivalent Cation ◽  
Thin Filaments ◽  
Cation Concentration ◽  
Bivalent Cations

The narrow Gaussian-length-distribution of actin filaments forming the cytoskeleton of the human erythrocyte indicates the existence of strict mechanisms for length determination and maintenance. A similar regulation is achieved in striated muscle by the capping of both the ends of the thin filaments, which consequently prevents monomer exchange. However, the ability of erythroid cytoskeletal preparations to nucleate actin polymerization has led to the proliferation of the idea that at least the barbed ends of the actin filaments are uncapped. The mechanism by which the length of the filaments is thus maintained has been left open to debate. In an effort to resolve any doubt regarding length-maintenance in human erythrocytes we have characterized the capping state of the actin filaments in a number of different ghost and cytoskeletal preparations. Under conditions of sufficiently high bivalent-cation concentration the actin filaments retain functional caps at both the barbed and pointed ends. Hence filament capping at both ends prevents redistribution of the actin monomer in a similar manner to that proposed for the thin filaments of striated muscle. Actin filament uncapping is apparently caused by the centrifugal shearing stress imposed during ghost preparation. The uncapping is more pronounced when the bivalent-cation concentration is reduced or when the membrane is removed by detergents. The effects of bivalent cations seem to be mediated through the erythroid protein spectrin, consistent with the hypothesis of Wallis et al. [Wallis, Babitch and Wenegieme (1993) Biochemistry 32, 5045-5050] that the ability of spectrin to resist shearing stress is dependent on the degree of bound bivalent cations.

scholarly journals Nebulin Interacts with CapZ and Regulates Thin Filament Architecture within the Z-Disc

2008 ◽  
Vol 19 (5) ◽  
pp. 1837-1847 ◽  
Author(s):  
Christopher T. Pappas ◽  
Nandini Bhattacharya ◽  
John A. Cooper ◽  
Carol C. Gregorio
Keyword(s):  
Actin Filaments ◽  
Thin Filament ◽  
Actin Polymerization ◽  
Striated Muscle ◽  
Solid Phase ◽  
Molecular Model ◽  
Direct Interaction ◽  
Tryptophan Fluorescence ◽  
Actin Binding

The barbed ends of actin filaments in striated muscle are anchored within the Z-disc and capped by CapZ; this protein blocks actin polymerization and depolymerization in vitro. The mature lengths of the thin filaments are likely specified by the giant “molecular ruler” nebulin, which spans the length of the thin filament. Here, we report that CapZ specifically interacts with the C terminus of nebulin (modules 160–164) in blot overlay, solid-phase binding, tryptophan fluorescence, and SPOTs membrane assays. Binding of nebulin modules 160–164 to CapZ does not affect the ability of CapZ to cap actin filaments in vitro, consistent with our observation that neither of the two C-terminal actin binding regions of CapZ is necessary for its interaction with nebulin. Knockdown of nebulin in chick skeletal myotubes using small interfering RNA results in a reduction of assembled CapZ, and, strikingly, a loss of the uniform alignment of the barbed ends of the actin filaments. These data suggest that nebulin restricts the position of thin filament barbed ends to the Z-disc via a direct interaction with CapZ. We propose a novel molecular model of Z-disc architecture in which nebulin interacts with CapZ from a thin filament of an adjacent sarcomere, thus providing a structural link between sarcomeres.

scholarly journals Analysis of myofibrillar structure and assembly using fluorescently labeled contractile proteins.

1984 ◽  
Vol 98 (3) ◽  
pp. 825-833 ◽  
Author(s):  
J W Sanger ◽  
B Mittal ◽  
J M Sanger
Keyword(s):  
Actin Filaments ◽  
Striated Muscle ◽  
Contractile Proteins ◽  
Actin Binding ◽  
Thin Filaments ◽  
In Vitro System ◽  
Self Association ◽  
Cross Bridges

To study how contractile proteins become organized into sarcomeric units in striated muscle, we have exposed glycerinated myofibrils to fluorescently labeled actin, alpha-actinin, and tropomyosin. In this in vitro system, alpha-actinin bound to the Z-bands and the binding could not be saturated by prior addition of excess unlabeled alpha-actinin. Conditions known to prevent self-association of alpha-actinin, however, blocked the binding of fluorescently labeled alpha-actinin to Z-bands. When tropomyosin was removed from the myofibrils, alpha-actinin then added to the thin filaments as well as the Z-bands. Actin bound in a doublet pattern to the regions of the myosin filaments where there were free cross-bridges i.e., in that part of the A-band free of interdigitating native thin filaments but not in the center of the A-band which lacks cross-bridges. In the presence of 0.1-0.2 mM ATP, no actin binding occurred. When unlabeled alpha-actinin was added first to myofibrils and then labeled actin was added fluorescence occurred not in a doublet pattern but along the entire length of the myofibril. Tropomyosin did not bind to myofibrils unless the existing tropomyosin was first removed, in which case it added to the thin filaments in the l-band. Tropomyosin did bind, however, to the exogenously added tropomyosin-free actin that localizes as a doublet in the A-band. These results indicate that the alpha-actinin present in Z-bands of myofibrils is fully complexed with actin, but can bind exogenous alpha-actinin and, if actin is added subsequently, the exogenous alpha-actinin in the Z-band will bind the newly formed fluorescent actin filaments. Myofibrillar actin filaments did not increase in length when G-actin was present under polymerizing conditions, nor did they bind any added tropomyosin. These observations are discussed in terms of the structure and in vivo assembly of myofibrils.

scholarly journals Molecular-scale visualization of sarcomere contraction within native cardiomyocytes

2020 ◽  
Author(s):  
Laura Burbaum ◽  
Jonathan Schneider ◽  
Sarah Scholze ◽  
Ralph T Böttcher ◽  
Wolfgang Baumeister ◽  
...  
Keyword(s):  
Thin Filament ◽  
Striated Muscle ◽  
Hexagonal Lattice ◽  
Muscular Contraction ◽  
Neonatal Rat ◽  
Molecular Architecture ◽  
Thin Filaments ◽  
Rat Cardiomyocytes ◽  
Thick Filaments ◽  
Activated State

Sarcomeres, the basic contractile units of striated muscle, produce the forces driving muscular contraction through cross-bridge interactions between actin-containing thin filaments and myosin II-based thick filaments. Until now, direct visualization of the molecular architecture underlying sarcomere contractility has remained elusive. Here, we use in situ cryo-electron to-mography to unveil sarcomere contraction in frozen-hydrated neonatal rat cardiomyocytes. We show that the hexagonal lattice of the thick filaments is already established at the neonatal stage, with an excess of thin filaments outside the trigonal positions. Structural assessment of actin polarity by subtomogram averaging reveals that thin filaments in the fully activated state form overlapping arrays of opposite polarity in the center of the sarcomere. Our approach provides direct evidence for thin filament sliding during muscle contraction and may serve as a basis for structural understanding of thin filament activation and actomyosin interactions inside unperturbed cellular environments.

scholarly journals Reactivities of Actin as a Contractile Protein

1967 ◽  
Vol 50 (6) ◽  
pp. 119-133 ◽  
Author(s):  
Teru Hayashi
Keyword(s):  
Complex Formation ◽  
Molecular Interaction ◽  
Actin Filaments ◽  
Molecular Basis ◽  
Striated Muscle ◽  
Thin Filaments ◽  
Contraction Process ◽  
Tentative Hypothesis ◽  
Myosin Interaction

The molecular basis for the mechanism of contraction in striated muscle, with primary emphasis on the interaction between the thick and thin filaments and the role of the thin (actin) filaments, is the theme presented. Recent information relating to actin-myosin interaction points up the fact that definitive statements cannot be made regarding the molecular interaction(s) that lead to contraction. Nevertheless, the properties of actin indicate that (a) actin in the monomeric state has properties differing markedly from actin in the polymer (filament) state; (b) these property differences may be significant in the contractile process, for they include changes in the reactivity of the bound nucleotide and actin-myosin complex formation; (c) the bound nucleotide seems to be required in the contraction process. For these, and other, reasons discussed, the tentative hypothesis is advanced that the contraction reaction involves local changes in the actin filament providing local monomer or monomer-like actin units in the reaction with myosin.

scholarly journals CAP2 is a regulator of actin pointed end dynamics and myofibrillogenesis in cardiac muscle

2021 ◽  
Vol 4 (1) ◽  
Author(s):  
Mert Colpan ◽  
Jessika Iwanski ◽  
Carol C. Gregorio
Keyword(s):  
Muscle Contraction ◽  
Cardiac Muscle ◽  
Adenylyl Cyclase ◽  
Thin Filament ◽  
Essential Role ◽  
Striated Muscle ◽  
Actin Dynamics ◽  
Actin Assembly ◽  
Thin Filaments ◽  
Pointed End

AbstractThe precise assembly of actin-based thin filaments is crucial for muscle contraction. Dysregulation of actin dynamics at thin filament pointed ends results in skeletal and cardiac myopathies. Here, we discovered adenylyl cyclase-associated protein 2 (CAP2) as a unique component of thin filament pointed ends in cardiac muscle. CAP2 has critical functions in cardiomyocytes as it depolymerizes and inhibits actin incorporation into thin filaments. Strikingly distinct from other pointed-end proteins, CAP2’s function is not enhanced but inhibited by tropomyosin and it does not directly control thin filament lengths. Furthermore, CAP2 plays an essential role in cardiomyocyte maturation by modulating pre-sarcomeric actin assembly and regulating α-actin composition in mature thin filaments. Identification of CAP2’s multifunctional roles provides missing links in our understanding of how thin filament architecture is regulated in striated muscle and it reveals there are additional factors, beyond Tmod1 and Lmod2, that modulate actin dynamics at thin filament pointed ends.

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