Thin filaments elongate from their pointed ends during myofibril assembly in Drosophila indirect flight muscle

Tropomodulin (Tmod) is an actin pointed-end capping protein that regulates actin dynamics at thin filament pointed ends in striated muscle. Although pointed-end capping by Tmod controls thin filament lengths in assembled myofibrils, its role in length specification during de novo myofibril assembly is not established. We used the Drosophila Tmod homologue, sanpodo (spdo), to investigate Tmod's function during muscle development in the indirect flight muscle. SPDO was associated with the pointed ends of elongating thin filaments throughout myofibril assembly. Transient overexpression of SPDO during myofibril assembly irreversibly arrested elongation of preexisting thin filaments. However, the lengths of thin filaments assembled after SPDO levels had declined were normal. Flies with a preponderance of abnormally short thin filaments were unable to fly. We conclude that: (a) thin filaments elongate from their pointed ends during myofibril assembly; (b) pointed ends are dynamically capped at endogenous levels of SPDO so as to allow elongation; (c) a transient increase in SPDO levels during myofibril assembly converts SPDO from a dynamic to a permanent cap; and (d) developmental regulation of pointed-end capping during myofibril assembly is crucial for specification of final thin filament lengths, myofibril structure, and muscle function.

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

Communications Biology ◽

10.1038/s42003-021-01893-w ◽

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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Tropomodulin isoforms regulate thin filament pointed-end capping and skeletal muscle physiology

The Journal of Cell Biology ◽

10.1083/jcb.201001125 ◽

2010 ◽

Vol 189 (1) ◽

pp. 95-109 ◽

Cited By ~ 53

Author(s):

David S. Gokhin ◽

Raymond A. Lewis ◽

Caroline R. McKeown ◽

Roberta B. Nowak ◽

Nancy E. Kim ◽

...

Keyword(s):

Skeletal Muscle ◽

Thin Filament ◽

Striated Muscle ◽

Fiber Type ◽

Actin Dynamics ◽

Muscle Physiology ◽

Skeletal Muscle Function ◽

Capping Proteins ◽

Locomotor Deficits ◽

End Capping

During myofibril assembly, thin filament lengths are precisely specified to optimize skeletal muscle function. Tropomodulins (Tmods) are capping proteins that specify thin filament lengths by controlling actin dynamics at pointed ends. In this study, we use a genetic targeting approach to explore the effects of deleting Tmod1 from skeletal muscle. Myofibril assembly, skeletal muscle structure, and thin filament lengths are normal in the absence of Tmod1. Tmod4 localizes to thin filament pointed ends in Tmod1-null embryonic muscle, whereas both Tmod3 and -4 localize to pointed ends in Tmod1-null adult muscle. Substitution by Tmod3 and -4 occurs despite their weaker interactions with striated muscle tropomyosins. However, the absence of Tmod1 results in depressed isometric stress production during muscle contraction, systemic locomotor deficits, and a shift to a faster fiber type distribution. Thus, Tmod3 and -4 compensate for the absence of Tmod1 structurally but not functionally. We conclude that Tmod1 is a novel regulator of skeletal muscle physiology.

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Tropomodulin caps the pointed ends of actin filaments.

The Journal of Cell Biology ◽

10.1083/jcb.127.6.1627 ◽

1994 ◽

Vol 127 (6) ◽

pp. 1627-1635 ◽

Cited By ~ 198

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.

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Effects of cardiomyopathy-linked mutations K15N and R21H in tropomyosin on thin-filament regulation and pointed-end dynamics

Molecular Biology of the Cell ◽

10.1091/mbc.e18-06-0406 ◽

2019 ◽

Vol 30 (2) ◽

pp. 268-281 ◽

Cited By ~ 5

Author(s):

Thu Ly ◽

Christopher T. Pappas ◽

Dylan Johnson ◽

William Schlecht ◽

Mert Colpan ◽

...

Keyword(s):

Thin Filament ◽

Structural Changes ◽

Striated Muscle ◽

In Vitro Assays ◽

Missense Mutations ◽

Thin Filaments ◽

Thin Filament Regulation ◽

Pointed End ◽

Kinetics Of

Missense mutations K15N and R21H in striated muscle tropomyosin are linked to dilated cardiomyopathy (DCM) and hypertrophic cardiomyopathy (HCM), respectively. Tropomyosin, together with the troponin complex, regulates muscle contraction and, along with tropomodulin and leiomodin, controls the uniform thin-filament lengths crucial for normal sarcomere structure and function. We used Förster resonance energy transfer to study effects of the tropomyosin mutations on the structure and kinetics of the cardiac troponin core domain associated with the Ca2+-dependent regulation of cardiac thin filaments. We found that the K15N mutation desensitizes thin filaments to Ca2+ and slows the kinetics of structural changes in troponin induced by Ca2+ dissociation from troponin, while the R21H mutation has almost no effect on these parameters. Expression of the K15N mutant in cardiomyocytes decreases leiomodin’s thin-filament pointed-end assembly but does not affect tropomodulin’s assembly at the pointed end. Our in vitro assays show that the R21H mutation causes a twofold decrease in tropomyosin’s affinity for F-actin and affects leiomodin’s function. We suggest that the K15N mutation causes DCM by altering Ca2+-dependent thin-filament regulation and that one of the possible HCM-causing mechanisms by the R21H mutation is through alteration of leiomodin’s function.

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Tropomodulin Capping of Actin Filaments in Striated Muscle Development and Physiology

Journal of Biomedicine and Biotechnology ◽

10.1155/2011/103069 ◽

2011 ◽

Vol 2011 ◽

pp. 1-16 ◽

Cited By ~ 26

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.

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A method for determining the periodicity of a troponin component in isolated insect flight muscle thin filaments by gold/Fab labelling

Journal of Cell Science ◽

10.1242/jcs.101.3.503 ◽

1992 ◽

Vol 101 (3) ◽

pp. 503-508

Author(s):

R. Newman ◽

G.W. Butcher ◽

B. Bullard ◽

K.R. Leonard

Keyword(s):

Gold Particle ◽

Colloidal Gold ◽

Striated Muscle ◽

Flight Muscle ◽

Insect Flight ◽

Insect Flight Muscle ◽

Fab Fragment ◽

Thin Filaments ◽

Gold Particles ◽

Almost All

Insect flight muscle has a large component (Tn-H) in the tropomyosin-troponin complex that is not present in vertebrate striated muscle thin filaments. Tn-H is shown by gold/Fab labelling to be present at regular intervals in insect flight muscle thin filaments. The Fab fragment of a monoclonal antibody to Tn-H was conjugated directly with colloidal gold and this probe used to label isolated thin filaments from the flight muscle of Lethocerus indicus (water bug). The distribution of gold particles seen in electron microscope images of negatively stained thin filaments was analysed to show that the probe bound to sites having a periodicity of approximately 40 nm, which is the expected value for the tropomyosin-troponin repeat. Conjugates of Fab with colloidal gold particles of 3 nm diameter labelled almost all sites. Conjugates with gold particles of 5 nm and 10 nm diameter labelled less efficiently (70% and 30%, respectively) but analysis of the distribution of inter-particle intervals among a number of filaments again gave the same fundamental spacing of 40 nm. The error in the measurements (standard deviation approximately +/− 4.2 for 5 nm gold/Fab) is less than earlier estimates for the size of the gold/Fab complex. Measurements on gold/Fab in negative stain suggest that the bound Fab contributes a shell about 2 nm in thickness around the gold particle. The radius of the probe (about 4.5 nm for 5 nm gold/Fab) would then be consistent with the value of error found. The size of the probe suggests that the gold particle binds to the side of the Fab molecule, rather close to the antibody combining site. The potential resolution of the technique may thus be better than originally expected.

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EGF stimulates an increase in actin nucleation and filament number at the leading edge of the lamellipod in mammary adenocarcinoma cells

Journal of Cell Science ◽

10.1242/jcs.111.2.199 ◽

1998 ◽

Vol 111 (2) ◽

pp. 199-211 ◽

Cited By ~ 16

Author(s):

A.Y. Chan ◽

S. Raft ◽

M. Bailly ◽

J.B. Wyckoff ◽

J.E. Segall ◽

...

Keyword(s):

Actin Polymerization ◽

De Novo ◽

Leading Edge ◽

Mammary Adenocarcinoma ◽

Actin Dynamics ◽

Actin Nucleation ◽

Nucleation Sites ◽

Nucleation Activity ◽

Pointed End ◽

Stimulation Of

Stimulation of metastatic MTLn3 cells with EGF causes the rapid extension of lamellipods, which contain a zone of F-actin at the leading edge. In order to establish the mechanism for accumulation of F-actin at the leading edge and its relationship to lamellipod extension in response to EGF, we have studied the kinetics and location of EGF-induced actin nucleation activity in MTLn3 cells and characterized the actin dynamics at the leading edge by measuring the changes at the pointed and barbed ends of actin filaments upon EGF stimulation of MTLn3 cells. The major result of this study is that stimulation of MTLn3 cells with EGF causes a transient increase in actin nucleation activity resulting from the appearance of free barbed ends very close to the leading edge of extending lamellipods. In addition, cytochalasin D causes a significant decrease in the total F-actin content in EGF-stimulated cells, indicating that both actin polymerization and depolymerization are stimulated by EGF. Pointed end incorporation of rhodamine-labeled actin by the EGF stimulated cells is 2.12+/−0.47 times higher than that of control cells. Since EGF stimulation causes an increase in both barbed and pointed end incorporation of rhodamine-labeled actin in the same location, the EGF-stimulated nucleation sites are more likely due either to severing of pre-existing filaments or de novo nucleation of filaments at the leading edge thereby creating new barbed and pointed ends. The timing and location of EGF-induced actin nucleation activity in MTLn3 cells can account for the observed accumulation of F-actin at the leading edge and demonstrate that this F-actin rich zone is the primary actin polymerization zone after stimulation.

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Tropomodulin isoforms regulate thin filament pointed-end capping and skeletal muscle physiology

The Journal of General Physiology ◽

10.1085/jgp1355oia4 ◽

2010 ◽

Vol 135 (5) ◽

pp. i4-i4

Author(s):

David S. Gokhin ◽

Raymond A. Lewis ◽

Caroline R. McKeown ◽

Roberta B. Nowak ◽

Nancy E. Kim ◽

...

Keyword(s):

Skeletal Muscle ◽

Thin Filament ◽

Muscle Physiology ◽

End Capping ◽

Pointed End

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Molecular-scale visualization of sarcomere contraction within native cardiomyocytes

10.1101/2020.09.09.288977 ◽

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.

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The development of indirect flight muscle innervation in Drosophila melanogaster

Development ◽

10.1242/dev.118.1.215 ◽

1993 ◽

Vol 118 (1) ◽

pp. 215-227 ◽

Cited By ~ 5

Author(s):

J. Fernandes ◽

K. VijayRaghavan

Keyword(s):

Muscle Development ◽

Flight Muscle ◽

Neuromuscular Junctions ◽

Indirect Flight Muscle ◽

Larval Life ◽

Spatial And Temporal Patterns ◽

Muscle Innervation ◽

Flight Muscles ◽

Nerve Muscle ◽

Longitudinal Muscles

We have examined the development of innervation to the indirect flight muscles of Drosophila. During metamorphosis, the larval intersegmental nerve of the mesothorax is remodelled to innervate the dorsal longitudinal muscles and two of the dorsoventral muscles. Another modified larval nerve innervates the remaining dorsoventral muscle. The dorsal longitudinal muscles develop using modified larval muscles as templates while dorsoventral muscles develop without the use of such templates. The development of innervation to the two groups of indirect flight muscles differs in spatial and temporal patterns, which may reflect the different ways in which these muscles develop. The identification of myoblasts associated with thoracic nerves during larval life and the association of migrating myoblasts with nerves during metamorphosis indicate the existence of nerve-muscle interactions during indirect flight muscle development. In addition, the developing pattern of axonal branching suggests a role for the target muscles in respecifying neuromuscular junctions during metamorphosis.

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