scholarly journals Ca2+-dependent Muscle Dysfunction Caused by Mutation of the Caenorhabditis elegans Troponin T-1 Gene

1998 ◽  
Vol 143 (5) ◽  
pp. 1201-1213 ◽  
Author(s):  
Kristen McArdle ◽  
Taylor StC. Allen ◽  
Elizabeth A. Bucher
Keyword(s):  
Caenorhabditis Elegans ◽  
Thin Filament ◽  
Body Wall ◽  
Troponin I ◽  
Troponin T ◽  
Muscle Dysfunction ◽  
Excessive Force ◽  
Thin Filaments ◽  
Attachment Sites

We have investigated the functions of troponin T (CeTnT-1) in Caenorhabditis elegans embryonic body wall muscle. TnT tethers troponin I (TnI) and troponin C (TnC) to the thin filament via tropomyosin (Tm), and TnT/Tm regulates the activation and inhibition of myosin-actin interaction in response to changes in intracellular [Ca2+]. Loss of CeTnT-1 function causes aberrant muscle trembling and tearing of muscle cells from their exoskeletal attachment sites (Myers, C.D., P.-Y. Goh, T. StC. Allen, E.A. Bucher, and T. Bogaert. 1996. J. Cell Biol. 132:1061–1077). We hypothesized that muscle tearing is a consequence of excessive force generation resulting from defective tethering of Tn complex proteins. Biochemical studies suggest that such defective tethering would result in either (a) Ca2+-independent activation, due to lack of Tn complex binding and consequent lack of inhibition, or (b) delayed reestablishment of TnI/TnC binding to the thin filament after Ca2+ activation and consequent abnormal duration of force. Analyses of animals doubly mutant for CeTnT-1 and for genes required for Ca2+ signaling support that CeTnT-1 phenotypes are dependent on Ca2+ signaling, thus supporting the second model and providing new in vivo evidence that full inhibition of thin filaments in low [Ca2+] does not require TnT.

scholarly journals Developmental genetic analysis of troponin T mutations in striated and nonstriated muscle cells of Caenorhabditis elegans.

1996 ◽  
Vol 132 (6) ◽  
pp. 1061-1077 ◽  
Author(s):  
C D Myers ◽  
P Y Goh ◽  
T S Allen ◽  
E A Bucher ◽  
T Bogaert
Keyword(s):  
Caenorhabditis Elegans ◽  
Muscle Cell ◽  
Body Wall ◽  
Troponin I ◽  
Troponin T ◽  
Body Wall Muscle ◽  
Termination Codon ◽  
Cell Positioning ◽  
Sensitive Allele

We have been investigating a set of genes, collectively called mups, that are essential to striated body wall muscle cell positioning in Caenorhabditis elegans. Here we report our detailed characterization of the mup-2 locus, which encodes troponin T (TnT). Mutants for a heat-sensitive allele, called mup-2(e2346ts), and for a putative null, called mup-2(up1), are defective for embryonic body wall muscle cell contraction, sarcomere organization, and cell positioning. Characterizations of the heat-sensitive allele demonstrate that mutants are also defective for regulated muscle contraction in larval and adult body wall muscle, defective for function of the nonstriated oviduct myoepithelial sheath, and defective for epidermal morphogenesis. We cloned the mup-2 locus and its corresponding cDNA. The cDNA encodes a predicted 405-amino acid protein homologous to vertebrate and invertebrate TnT and includes an invertebrate-specific COOH-terminal tail. The mup-2 mutations lie within these cDNA sequences: mup-2(up1) is a termination codon near NH2 terminus (Glu94) and mup-2(e2346ts) is a termination codon in the COOH-terminal invertebrate-specific tail (Trp342). TnT is a muscle contractile protein that, in association with the thin filament proteins tropomyosin, troponin I and troponin C, regulates myosin-actin interaction in response to a rise in intracellular Ca2+. Our findings demonstrate multiple essential functions for TnT and provide a basis to investigate the in vivo functions and protein interactions of TnT in striated and nonstriated muscles.

scholarly journals Atomic resolution probe for allostery in the regulatory thin filament

2016 ◽  
Vol 113 (12) ◽  
pp. 3257-3262 ◽  
Author(s):  
Michael R. Williams ◽  
Sarah J. Lehman ◽  
Jil C. Tardiff ◽  
Steven D. Schwartz
Keyword(s):  
Binding Site ◽  
Human Disease ◽  
Calcium Binding ◽  
Cardiac Troponin ◽  
Thin Filament ◽  
Troponin I ◽  
Troponin T ◽  
Calcium Binding Site

Calcium binding and dissociation within the cardiac thin filament (CTF) is a fundamental regulator of normal contraction and relaxation. Although the disruption of this complex, allosterically mediated process has long been implicated in human disease, the precise atomic-level mechanisms remain opaque, greatly hampering the development of novel targeted therapies. To address this question, we used a fully atomistic CTF model to test both Ca2+ binding strength and the energy required to remove Ca2+ from the N-lobe binding site in WT and mutant troponin complexes that have been linked to genetic cardiomyopathies. This computational approach is combined with measurements of in vitro Ca2+ dissociation rates in fully reconstituted WT and cardiac troponin T R92L and R92W thin filaments. These human disease mutations represent known substitutions at the same residue, reside at a significant distance from the calcium binding site in cardiac troponin C, and do not affect either the binding pocket affinity or EF-hand structure of the binding domain. Both have been shown to have significantly different effects on cardiac function in vivo. We now show that these mutations independently alter the interaction between the Ca2+ ion and cardiac troponin I subunit. This interaction is a previously unidentified mechanism, in which mutations in one protein of a complex indirectly affect a third via structural and dynamic changes in a second to yield a pathogenic change in thin filament function that results in mutation-specific disease states. We can now provide atom-level insight that is potentially highly actionable in drug design.

scholarly journals Muscle organization in Caenorhabditis elegans: localization of proteins implicated in thin filament attachment and I-band organization.

1985 ◽  
Vol 101 (4) ◽  
pp. 1532-1549 ◽  
Author(s):  
G R Francis ◽  
R H Waterston
Keyword(s):  
Caenorhabditis Elegans ◽  
Cell Membrane ◽  
Cell Surface ◽  
Thin Filament ◽  
Body Wall ◽  
Dense Body ◽  
The Body ◽  
Major Constituent ◽  
Thin Filaments ◽  
A Cell

The body wall muscle cells of Caenorhabditis elegans contain an obliquely striated myofibrillar lattice that is associated with the cell membrane through two structures: an M-line analogue in the A-band and a Z-disc analogue, or dense-body, in the I-band. By using a fraction enriched in these structures as an immunogen for hybridoma production, we prepared monoclonal antibodies that identify four components of the I-band as determined by immunofluorescence and Western transfer analysis. A major constituent of the dense-body is a 107,000-D polypeptide that shares determinants with vertebrate alpha-actinin. A second dense-body constituent is a more basic and antigenically distinct 107,000-D polypeptide that is localized to a narrow domain of the dense-body at or subjacent to the plasma membrane. This basic dense-body polypeptide is also found at certain cell boundaries where thin filaments in half-bands terminate at membrane-associated structures termed attachment plaques. A third, unidentified antigen is also found closely apposed to the cell membrane in regions of not only the dense-body and attachment plaque, but also the M-line analogue. Finally, a fourth high molecular weight antigen, composed of two polypeptides of approximately 400,000-D, is localized to the I-band regions surrounding the dense-body. The attachment of the dense-body to the cell surface and the differential localization of the dense-body-associated antigens suggest a model for their organization in which the unidentified antigen is a cell surface component, and the two 107,000-D polypeptides define different cytoplasmic domains of the dense-body.

scholarly journals Overexpression of troponin T in Drosophila muscles causes a decrease in the levels of thin-filament proteins

2005 ◽  
Vol 386 (1) ◽  
pp. 145-152 ◽  
Author(s):  
Raquel MARCO-FERRERES ◽  
Juan J. ARREDONDO ◽  
Benito FRAILE ◽  
Margarita CERVERA
Keyword(s):  
Thin Filament ◽  
Troponin I ◽  
Troponin T ◽  
Thin Filaments ◽  
Muscle Formation ◽  
Thin Filament Proteins ◽  
Flight Muscles ◽  
Transgenic Drosophila ◽  
Regulated Thin Filament

Formation of the contractile apparatus in muscle cells requires co-ordinated activation of several genes and the proper assembly of their products. To investigate the role of TnT (troponin T) in the mechanisms that control and co-ordinate thin-filament formation, we generated transgenic Drosophila lines that overexpress TnT in their indirect flight muscles. All flies that overexpress TnT were unable to fly, and the loss of thin filaments themselves was coupled with ultrastructural perturbations of the sarcomere. In contrast, thick filaments remained largely unaffected. Biochemical analysis of these lines revealed that the increase in TnT levels could be detected only during the early stages of adult muscle formation and was followed by a profound decrease in the amount of this protein as well as that of other thin-filament proteins such as tropomyosin, troponin I and actin. The decrease in thin-filament proteins is not only due to degradation but also due to a decrease in their synthesis, since accumulation of their mRNA transcripts was also severely diminished. This decrease in expression levels of the distinct thin-filament components led us to postulate that any change in the amount of TnT transcripts might trigger the down-regulation of other co-regulated thin-filament components. Taken together, these results suggest the existence of a mechanism that tightly co-ordinates the expression of thin-filament genes and controls the correct stoichiometry of these proteins. We propose that the high levels of unassembled protein might act as a sensor in this process.

Functional and evolutionary relationships of troponin C

2007 ◽  
Vol 32 (1) ◽  
pp. 16-27 ◽  
Author(s):  
Todd E. Gillis ◽  
Christian R. Marshall ◽  
Glen F. Tibbits
Keyword(s):  
Functional Properties ◽  
Thin Filament ◽  
Troponin I ◽  
Striated Muscle ◽  
Troponin T ◽  
Extensive Study ◽  
Troponin C ◽  
High Conservation ◽  
Cross Bridges ◽  
And Function

Striated muscle contraction is initiated when, following membrane depolarization, Ca2+ binds to the low-affinity Ca2+ binding sites of troponin C (TnC). The Ca2+ activation of this protein results in a rearrangement of the components (troponin I, troponin T, and tropomyosin) of the thin filament, resulting in increased interaction between actin and myosin and the formation of cross bridges. The functional properties of this protein are therefore critical in determining the active properties of striated muscle. To date there are 61 known TnCs that have been cloned from 41 vertebrate and invertebrate species. In vertebrate species there are also distinct fast skeletal muscle and cardiac TnC proteins. While there is relatively high conservation of the amino acid sequence of TnC homologs between species and tissue types, there is wide variation in the functional properties of these proteins. To date there has been extensive study of the structure and function of this protein and how differences in these translate into the functional properties of muscles. The purpose of this work is to integrate these studies of TnC with phylogenetic analysis to investigate how changes in the sequence and function of this protein, integrate with the evolution of striated muscle.

scholarly journals Specific Myosin Heavy Chain Mutations Suppress Troponin I Defects in Drosophila Muscles

1999 ◽  
Vol 144 (5) ◽  
pp. 989-1000 ◽  
Author(s):  
William A. Kronert ◽  
Angel Acebes ◽  
Alberto Ferrús ◽  
Sanford I. Bernstein
Keyword(s):  
Myosin Heavy Chain ◽  
Heavy Chain ◽  
Thin Filament ◽  
Troponin I ◽  
Point Mutations ◽  
Thick Filament ◽  
Actin Binding ◽  
Filament Protein ◽  
Phenotypic Rescue

We show that specific mutations in the head of the thick filament molecule myosin heavy chain prevent a degenerative muscle syndrome resulting from the hdp2 mutation in the thin filament protein troponin I. One mutation deletes eight residues from the actin binding loop of myosin, while a second affects a residue at the base of this loop. Two other mutations affect amino acids near the site of nucleotide entry and exit in the motor domain. We document the degree of phenotypic rescue each suppressor permits and show that other point mutations in myosin, as well as null mutations, fail to suppress the hdp2 phenotype. We discuss mechanisms by which the hdp2 phenotypes are suppressed and conclude that the specific residues we identified in myosin are important in regulating thick and thin filament interactions. This in vivo approach to dissecting the contractile cycle defines novel molecular processes that may be difficult to uncover by biochemical and structural analysis. Our study illustrates how expression of genetic defects are dependent upon genetic background, and therefore could have implications for understanding gene interactions in human disease.

scholarly journals Cardiac muscle thin filament structures reveal calcium regulatory mechanism

2020 ◽  
Vol 11 (1) ◽  
Author(s):  
Yurika Yamada ◽  
Keiichi Namba ◽  
Takashi Fujii
Keyword(s):  
Cardiac Muscle ◽  
Actin Filament ◽  
Conformational Changes ◽  
Thin Filament ◽  
Structural Changes ◽  
Troponin I ◽  
Regulatory Mechanism ◽  
Troponin T ◽  
Terminal Region ◽  
Muscle Thin Filament

AbstractContraction of striated muscles is driven by cyclic interactions of myosin head projecting from the thick filament with actin filament and is regulated by Ca2+ released from sarcoplasmic reticulum. Muscle thin filament consists of actin, tropomyosin and troponin, and Ca2+ binding to troponin triggers conformational changes of troponin and tropomyosin to allow actin-myosin interactions. However, the structural changes involved in this regulatory mechanism remain unknown. Here we report the structures of human cardiac muscle thin filament in the absence and presence of Ca2+ by electron cryomicroscopy. Molecular models in the two states built based on available crystal structures reveal the structures of a C-terminal region of troponin I and an N-terminal region of troponin T in complex with the head-to-tail junction of tropomyosin together with the troponin core on actin filament. Structural changes of the thin filament upon Ca2+ binding now reveal the mechanism of Ca2+ regulation of muscle contraction.

scholarly journals Genomic Organization, Expression, and Analysis of the Troponin C Gene pat-10 of Caenorhabditis elegans

1999 ◽  
Vol 146 (1) ◽  
pp. 193-202 ◽  
Author(s):  
Hiromi Terami ◽  
Benjamin D. Williams ◽  
Shin-ichi Kitamura ◽  
Yasuji Sakube ◽  
Shinji Matsumoto ◽  
...  
Keyword(s):  
Caenorhabditis Elegans ◽  
Calcium Binding ◽  
Troponin I ◽  
Amino Acid Level ◽  
Troponin C ◽  
In Vivo Studies ◽  
Calcium Binding Site ◽  
Expression Studies ◽  
Missense Alteration

We have cloned and characterized the troponin C gene, pat-10 of the nematode Caenorhabditis elegans. At the amino acid level nematode troponin C is most similar to troponin C of Drosophila (45% identity) and cardiac troponin C of vertebrates. Expression studies demonstrate that this troponin is expressed in body wall muscle throughout the life of the animal. Later, vulval muscles and anal muscles also express this troponin C isoform. The structural gene for this troponin is pat-10 and mutations in this gene lead to animals that arrest as twofold paralyzed embryos late in development. We have sequenced two of the mutations in pat-10 and both had identical two mutations in the gene; one changes D64 to N and the other changes W153 to a termination site. The missense alteration affects a calcium-binding site and eliminates calcium binding, whereas the second mutation eliminates binding to troponin I. These combined biochemical and in vivo studies of mutant animals demonstrate that this troponin is essential for proper muscle function during development.

scholarly journals Interaction between Troponin C and Troponin I of body wall in Caenorhabditis elegans

2003 ◽  
Vol 43 (supplement) ◽  
pp. S122
Author(s):  
M. Soda ◽  
T. Takaya ◽  
H. Kagawa ◽  
T. Iio
Keyword(s):  
Caenorhabditis Elegans ◽  
Body Wall ◽  
Troponin I ◽  
Troponin C

scholarly journals The Caenorhabditis elegans muscle-affecting gene unc-87 encodes a novel thin filament-associated protein.

1994 ◽  
Vol 127 (1) ◽  
pp. 79-93 ◽  
Author(s):  
S Goetinck ◽  
R H Waterston
Keyword(s):  
Caenorhabditis Elegans ◽  
Gene Product ◽  
Thin Filament ◽  
Genomic Sequence ◽  
Muscle Protein ◽  
Thin Filaments ◽  
C Elegans ◽  
Neuronal Protein ◽  
And Function ◽  
Filament Protein

Mutations in the unc-87 gene of Caenorhabditis elegans affect the structure and function of bodywall muscle, resulting in variable paralysis. We cloned the unc-87 gene by taking advantage of a transposon-induced allele of unc-87 and the correspondence of the genetic and physical maps in C. elegans. A genomic clone was isolated that alleviates the mutant phenotype when introduced into unc-87 mutants. Sequence analysis of a corresponding cDNA clone predicts a 357-amino acid, 40-kD protein that is similar to portions of the vertebrate smooth muscle proteins calponin and SM22 alpha, the Drosophila muscle protein mp20, the deduced product of the C. elegans cDNA cm7g3, and the rat neuronal protein np25. Analysis of the genomic sequence and of various transcripts represented in a cDNA library suggest that unc-87 mRNAs are subject to alternative splicing. Immunohistochemistry of wildtype and mutant animals with antibodies to an unc-87 fusion protein indicates that the gene product is localized to the I-band of bodywall muscle. Studies of the UNC-87 protein in other muscle mutants suggest that the unc-87 gene product associates with thin filaments, in a manner that does not depend on the presence of the thin filament protein tropomyosin.

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