Abstract. We have been investigating a set of genes, collectively called mups, that are essential to striated body wall mu...__scle 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(upl), 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(upl) 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 intraceUular Ca 2÷. 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. STUDIES of many organisms, including vertebrates, indicate that the stable attachment of muscle to the skeleton requires muscle sarcomere assembly, muscle contraction and extracellular matrix formation. Caenorhabditis elegans offers unique advantages for investigations of the cellular mechanisms influencing the establishment and maintenance of muscle attachment. C. elegans has a simple cellular anatomy of only 959 adult somatic ceils and contains a small number of muscle types (for review see Waterston, 1988). The cell divisions and migrations giving rise to these muscle types have been fully described at the cellular level (Sulston et al., 1983). Since the spatial relationships of individual muscle cells are easily visualized and are invariant, the attachments of muscle cells can be precisely determined in wild-type and mutant worms. Given these attributes, it is possible to study specific gene mutations to

Phytochelatins (PCs), (␥-Glu-Cys) n Gly polymers that were formerly considered to be restricted to plants and some fungal systems, are now known to play a critical role in heavy metal (notably Cd 2؉ ) detoxification in Caenorhabditis elegans. In view of the functional equivalence of the gene encoding C. elegans PC synthase 1, ce-pcs-1, to its homologs from plant and fungal sources, we have gone on to explore processes downstream of PC fabrication in this organism. Here we describe the identification of a half-molecule ATP-binding cassette transporter, CeHMT-1, from C. elegans with an equivalent topology to that of the putative PC transporter SpHMT-1 from Schizosaccharomyces pombe. At one level, Ce-HMT-1 satisfies the requirements of a Cd . These results and those from our previous investigations of the requirement for PC synthase for heavy metal tolerance in C. elegans demonstrate PC-dependent, HMT-1-mediated heavy metal detoxification not only in S. pombe but also in some invertebrates while at the same time indicating that the action of Ce-HMT-1 does not depend exclusively on PC synthesis.The toxicity of supraoptimal levels of the essential heavy metals copper and zinc and of trace or higher levels of the nonessential heavy metals cadmium, arsenic, mercury, and lead is thought to result from the displacement of endogenous co-factors from their cellular binding sites, thiol-capping of essential proteins and peptides, and promotion of the formation of reactive oxygen species (1). In humans, the repercussions of heavy metal exposure can range from acute poisoning to progressive kidney, liver, and lung dysfunction and, in some instances, cancer. Although its true clinical and epidemiological significance remains to be determined, chronic exposure to heavy metals is often associated with muscular and neurological degenerative conditions reminiscent of muscular dystrophy, multiple sclerosis, Alzheimer disease, and Parkinson disease (2-4).Three classes of heavy metal-binding peptides are known to participate in the homeostasis and detoxification of heavy metals in most animals. These are the ubiquitous thiol tripeptide glutathione (GSH), a family of small (4 -8 kDa) cysteine-rich proteins termed metallothioneins, and several higher molecular mass albeit sequence-unrelated metal-binding proteins. In all of the plants studied and in some fungi (as exemplified by Schizosaccharomyces pombe and Candida glabrata) and some animals (as exemplified by the model nematode Caenorhabditis elegans), a third class of cysteine-rich peptides termed phytochelatins (PCs) 1 has also been shown to be involved in heavy metal detoxification.PCs, which constitute a family of short-chain heavy metalbinding peptides with the general structure (␥-EC) n Xaa, where n ϭ 2-11, are derived from GSH and related thiols in a ␥-glutamylcysteinyl transpeptidation reaction catalyzed by phytochelatin synthases (EC 2.3.2.15) (1,5,6). It is now almost 15 years since the partial purification of the enzyme capable of catalyzing PC synthesis (5), yet it is onl...

Increasing emissions of heavy metals such as cadmium, mercury, and arsenic into the environment pose an acute problem for all organisms. Considerations of the biochemical basis of heavy metal detoxification in animals have focused exclusively on two classes of peptides, the thiol tripeptide, glutathione (GSH, ␥-Glu-CysGly), and a diverse family of cysteine-rich low molecular weight proteins, the metallothioneins. Plants and some fungi, however, not only deploy GSH and metallothioneins for metal detoxification but also synthesize another class of heavy metal binding peptides termed phytochelatins (PCs) from GSH. Here we show that PC-mediated heavy metal detoxification is not restricted to plants and some fungi but extends to animals by demonstrating that the ce-pcs-1 gene of the nematode worm Caenorhabditis elegans encodes a functional PC synthase whose activity is critical for heavy metal tolerance in the intact organism.Plants and some fungi post-translationally synthesize novel peptides termed phytochelatins (PCs) 1 when exposed to heavy metals. Fabricated from the ubiquitous thiol tripeptide GSH and related thiols in a novel transpeptidation reaction catalyzed by PC synthases (␥-glutamylcysteinyltransferase; EC 2.3.2.15), PCs have the general structure (␥-Glu-Cys) n -Xaa, contain 2-11 ␥-Glu-Cys repeats, chelate heavy metals at high affinity, and facilitate the vacuolar sequestration of heavy metals, most notably Cd 2ϩ (1-3). Although it is more than a decade since the first report of the partial purification of a heavy metal-, primarily Cd 2ϩ -activated PC synthase from plant extracts (1), it is only recently that the small family of genes encoding these enzymes has been identified in plants and the fission yeast Schizosaccharomyces pombe (4 -6). As exemplified by the clone from Arabidopsis thaliana (AtPCS1), these genes encode 45-55-kDa proteins that are sufficient for heavy metalactivated PC synthesis from GSH both in vivo and in vitro (6, 7).An unexpected outcome of the cloning of AtPCS1 and its equivalents from other plants and S. pombe was the identification of a single-copy gene homolog (accession number Z66513) in the nematode worm Caenorhabditis elegans (4 -6). Designated ce-pcs-1, this gene encodes a hypothetical 40.8-kDa protein (CePCS1) bearing 32% identity (45% similarity) to AtPCS1 in an overlap of 367 amino acid residues (6). Disclosure of a PCS1 homolog in the genome of C. elegans was surprising in that it raised for the first time the possibility that not only GSH and metallothioneins (8) but also PCs might participate in metal homeostasis in at least some animals.In the report that follows we demonstrate unequivocally that ce-pcs-1 encodes a bona fide PC synthase whose activity is necessary for the detoxification of heavy metals in the intact organism. Discovery of the PC synthase-dependent pathway in the model organism C. elegans establishes a firm basis for determining the ubiquity of this pathway in other animals and for elucidation of the identity and organization of the cellular machiner...

Tissue functions and mechanical coupling of cells must be integrated throughout development. A striking example of this coupling is the interactions of body wall muscle and hypodermal cells in Caenorhabditis elegans. These tissues are intimately associated in development and their interactions generate structures that provide a continuous mechanical link to transmit muscle forces across the hypodermis to the cuticle. Previously, we established that mup-4 is essential in embryonic epithelial (hypodermal) morphogenesis and maintenance of muscle position. Here, we report that mup-4 encodes a novel transmembrane protein that is required for attachments between the apical epithelial surface and the cuticular matrix. Its extracellular domain includes epidermal growth factor-like repeats, a von Willebrand factor A domain, and two sea urchin enterokinase modules. Its intracellular domain is homologous to filaggrin, an intermediate filament (IF)-associated protein that regulates IF compaction and that has not previously been reported as part of a junctional complex. MUP-4 colocalizes with epithelial hemidesmosomes overlying body wall muscles, beginning at the time of embryonic cuticle maturation, as well as with other sites of mechanical coupling. These findings support that MUP-4 is a junctional protein that functions in IF tethering, cell–matrix adherence, and mechanical coupling of tissues.

We compared the developmental regulation of the three troponin genes that encode the proteins of the Ca2+ regulatory complex in striated muscles of the Japanese quail. Nuclear run-on transcription and RNA protection analyses showed that the fast skeletal troponin I, the fast skeletal troponin T, and the slow skeletal-cardiac troponin C genes were transcriptionally coactivated and that transcripts rapidly accumulated within 6 to 12 h after the initiation of myoblast differentiation. The fast-isoform mRNAs of troponin I and troponin T were coexpressed at similar levels in different skeletal muscles, whereas the slow-cardiac troponin C mRNA varied independently and was the only one of these genes expressed in embryonic and adult heart. We conclude that these troponin genes are transcriptionally coactivated during skeletal myoblast differentiation, indicating that their transcription is under precise temporal control. However, this troponin C gene is regulated independently in specialized striated muscles.During embryonic development, the differential expression of genes in specific embryonic cell lineages generates a diversity of functionally differentiated tissues. We are interested in the mechanisms that coordinate the expression of muscle-specific genes during cellular differentiation. Muscle structure and function require the simultaneous expression of a large array of genes encoding proteins forming the contractile apparatus. When embryonic myoblasts differentiate, many muscle-specific mRNAs are accumulated, and muscle-specific proteins are synthesized (13,19,25,39,53). It is important to distinguish whether transcriptional or posttranscriptional processes regulate the accumulation of these muscle-specific mRNAs to determine whether a common molecular mechanism regulates any group of musclespecific genes (6,12,13,27). To address this question requires the comparison of nuclear transcription and mRNA accumulation to determine whether contractile protein genes are transcriptionally coactivated during muscle differentiation and during the formation of functionally specialized muscle types later in development.The particular genes we have selected for investigation are the three contractile protein genes, troponins C, I, and T (TnC, TnI, and TnT, respectively) (2,26,34), that encode the calcium regulatory complex of vertebrate striated muscles. The expression of these three troponin genes is particularly interesting for studies of coordinate gene regulation because they encode the functionally related subunits of the troponin complex. Muscle contraction is initiated by an increase in intracellular Ca2+. TnI inhibits the interaction of actin-myosin required for muscle contraction. Binding of Ca2+ by TnC is thought to result in a conformational change,