Journal of Molecular Biology
unc-94 Encodes a Tropomodulin in Caenorhabditis elegans
Introduction
Sarcomeres are specialized actin cytoskeletal structures that perform the work of muscle contraction. Sarcomeres are molecular or “nano”-machines consisting of a highly ordered assemblage of many proteins. Despite ever-increasing knowledge of the components and functions of sarcomeric proteins, we still do not have a clear picture about how sarcomeres are assembled and how sarcomeres are maintained in the face of repeated muscle activity. The ability to analyze mutants in the nematode Caenorhabditis elegans is being exploited to obtain insights into these questions.1., 2., [3] Molecular genetic experiments using C. elegans complement the typical biochemical analyses that are carried out in mammalian systems. The major striated muscle in the worm lies in the body wall and is used for locomotion. In adults, there are 95 spindle-shaped cells that are divided among four quadrants, which lie just underneath a basement membrane, hypodermis and cuticle. Due to the optical transparency of the worm, the myofibrils can be viewed by polarized light which reveals obvious striations. Bright A-bands alternate with dark I-bands, and each I-band contains a row of dense bodies, which are the analogs of Z-disks of vertebrate striated muscle. Because the striations lie at a slightly oblique angle with respect to the long axis of the worm, this muscle is “obliquely striated.” Rather than filling the entire cell, as is the case for vertebrate striated muscle, in C. elegans body wall muscle, the myofibrils are restricted to a narrow zone of ∼ 1.5 μm along one side of the cell. All the M-lines and Z-disks are attached to the muscle cell membrane, making these structures good models for studying muscle costameres4 and focal adhesions of non-muscle cells.3
C. elegans has been used profitably to obtain mutants defective in the formation, function and/or structure of muscle. There are two major classes of muscle-affecting mutations. In one class, the uncoordinated or “Unc” class, the worms develop into adults but are slow moving or paralyzed.5., 6., [7] The second class, the paralyzed arrested at twofold (“Pat”) class of mutants, displays a characteristic embryonic lethality in which embryos do not move within the eggshell and stop development at the twofold stage.8 A few genes have both hypomorphic Unc and null Pat phenotypes; examples include unc-1129 and unc-45.10 To date, nearly all the muscle Unc and many of the Pat genes have been cloned and studied at the molecular level. The encoded proteins include both previously known and novel components of the thick and thin filaments and their organizing and membrane attachment structures (M-lines and dense bodies). The cloning of M-line and dense body components has revealed not only a number of familiar components of focal adhesions (perlecan, integrins, vinculin, integrin-linked kinase, PINCH), but also new components of these structures (UNC-112, UNC-98, UNC-96 and UNC-89).[3], [11] This analysis is consistent with a model in which myofibril assembly is directed by signals first laid down in the extracellular matrix and the muscle cell membrane. In addition, most of the components of dense bodies and M-lines are shared, except for the proteins involved in the later stages of assembly, for example, for the dense bodies, vinculin and α-actinin, and for the M-lines, UNC-89.
For thick filaments, the expected genes for the myosins and paramyosin were among the first worm muscle genes identified. New and evolutionarily conserved components of thick filaments were first revealed by this genetic analysis. One example is twitchin, the founding member of the giant kinases that include mammalian titin and insect projectin.12., [13], 14. Another example is UNC-45, which is a conserved chaperone for myosin head folding and for the assembly of myosin into thick filaments.10., 15. For thin filaments, genes encoding the actins and the troponin–tropomyosin complex have been found. In addition, novel components have been identified (e.g., UNC-87),16 and roles in myofibril assembly were first revealed for several previously known proteins. There are many actin-binding proteins, many of which regulate actin filament dynamics in many eukaryotic cells. This includes proteins that promote actin polymerization (e.g., profilin), severing (e.g., gelsolins, ADF/cofilin), stability (e.g., tropomyosin), depolymerization (e.g., ADF/cofilin), barbed end capping (e.g., capZ) or pointed end capping (e.g., tropomodulin). Molecular genetic analysis of UNC-60B (an ADF/cofilin protein),17., [18] tropomyosin19 and UNC-78,20., 21. has clearly demonstrated the requirement for regulating actin filament dynamics to ensure proper assembly and maintenance of muscle thin filaments.
In 1980, Zengel and Epstein reported results of a screen for mutants with altered body wall muscle structure.7 Their screen involved enrichment for slow-moving worms, followed by assessment by polarized light. Mutants represented new alleles of 10 previously identified genes5., 6. and 7 new genes. Among them was a single mutant allele for a new gene, unc-94. unc-94 (su177) was described as slow moving and by polarized light microscopy to have “irregular birefringent areas.” Electron microscopy (EM) showed large collections of thin filaments interspersed with possible intermediate filaments and patches of thick filaments. Here, we report that unc-94 encodes a tropomodulin, an F-actin pointed-end capping protein. Our results show the expected localization of a tropomodulin in the sarcomere of another animal and its in vivo importance, and point to a new role for tropomodulin in regulating F-actin at muscle cell–to-cell boundaries.
Section snippets
Results
As is the case for most muscle Unc mutants, unc-94(su177) displays a less organized myofilament lattice as compared to wild type (Fig. 1a). There is alternation between normal and increased width of individual birefringent bands. To gain further understanding about the unc-94 mutant phenotype, we sought to identify additional unc-94 mutant alleles. By an F1 noncomplementation screen, we recovered a new allele, sf20. As shown in Fig. 1a, sf20 has the same polarized light phenotype as su177.
Discussion
We have shown here that the mutationally identified gene unc-94 corresponds with the sequence-predicted gene tmd-1, which encodes a tropomodulin. This discovery demonstrates yet another level of conservation in sarcomere components between C. elegans and mammals. Here we show that mutation in or RNAi against unc-94 results in reduced organization of myofibrils, abnormal accumulation of F-actin near muscle cell-to-cell boundaries, and reduced motility and brood size. In cultured chick
Strains and genetics
Two alleles of unc-94 were employed in this study. The first allele, su177, was isolated and described by Zengel and Epstein using a motility and polarized light screen.7 When we obtained unc-94 (su177) from the Caenorhabditis Genetics Center, it had only been outcrossed three times. These three times outcrossed animals were used for polarized light, motility, and immunofluorescence experiments. We noticed the same polarized light phenotype in these animals as was reported by Zengel and
Acknowledgements
We thank Hiroshi Qadota for valuable advice on the project and help with the microscopy, Jeannette Taylor for EM images, Andy Fire for the promoterless GFP vector, Alan Coulson for cosmid clones, and Yuji Kohara for cDNA clones. Some strains used in this work were provided by the Caenorhabditis Genetics Center, which is supported by the National Center for Research Resources of the National Institutes of Health. These studies were supported by grant AR052133 from the National Institutes of
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Mutual dependence between tropomodulin and tropomyosin in the regulation of sarcomeric actin assembly in Caenorhabditis elegans striated muscle
2022, European Journal of Cell BiologyCitation Excerpt :Tpm binds along the side of actin filaments and also directly binds to Tmod to enhance its capping activity (Fowler, 1990; Kostyukova et al., 2006; Kostyukova and Hitchcock-DeGregori, 2004; Weber et al., 1994). Tmod mutations cause disorganized sarcomeric actin in the mouse heart (Fritz-Six et al., 2003), zebrafish (Berger et al., 2014), and Caenorhabditis elegans (Stevenson et al., 2007; Yamashiro et al., 2008). Tmod has been proposed as a regulator of thin filament lengths (Littlefield and Fowler, 2008), because inhibition of Tmod capping activity causes elongation of thin filaments (Gregorio et al., 1995), and overexpression of Tmod causes shortening of thin filaments (Mardahl-Dumesnil and Fowler, 2001; Sussman et al., 1998).
Tropomodulins and Leiomodins: Actin Pointed End Caps and Nucleators in Muscles
2017, Biophysical JournalCitation Excerpt :Thus, loss of Tmod1 in the mouse heart results in severe disorganization of the thin filaments, and accumulation of abnormal actin filament bundles, resulting in defective contractility, aborted cardiac development, and lethality at E9.5 (29,30,78) (Fig. 6 A; Table 1). Similarly, unc94/tmd-1 loss of function alleles in C. elegans and morpholino knockdown of Tmod4 in developing X. laevis skeletal muscle result in aberrant myofibril assembly and defective movement (15,79,80). Due to isoform compensation during muscle development, Tmod1 is dispensable for myofibril assembly and specification of thin filament lengths in mouse skeletal muscle, as Tmod4 (endogenously present) and additionally recruited Tmod3 substitute for Tmod1 at pointed ends during myofibril assembly (Table 1) (24,81).
Cadherins and their partners in the nematode worm Caenorhabditis elegans
2013, Progress in Molecular Biology and Translational ScienceLevamisole and ryanodine receptors (I): A contraction study in Ascaris suum
2010, Molecular and Biochemical Parasitology
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Present address: E. A. Cox, Department of Biology, SUNY College at Geneseo, Geneseo, NY 14454, USA.