Functional genomic analysis of C. elegans molting.

Although the molting cycle is a hallmark of insects and nematodes, neither the endocrine control of molting via size, stage, and nutritional inputs nor the enzymatic mechanism for synthesis and release of the exoskeleton is well understood. Here, we identify endocrine and enzymatic regulators of molting in C. elegans through a genome-wide RNA-interference screen. Products of the 159 genes discovered include annotated transcription factors, secreted peptides, transmembrane proteins, and extracellular matrix enzymes essential for molting. Fusions between several genes and green fluorescent protein show a pulse of expression before each molt in epithelial cells that synthesize the exoskeleton, indicating that the corresponding proteins are made in the correct time and place to regulate molting. We show further that inactivation of particular genes abrogates expression of the green fluorescent protein reporter genes, revealing regulatory networks that might couple the expression of genes essential for molting to endocrine cues. Many molting genes are conserved in parasitic nematodes responsible for human disease, and thus represent attractive targets for pesticide and pharmaceutical development

Regulation of the C. elegans molt by pqn-47

AbstractC. elegans molts at the end of each of its four larval stages but this cycle ceases at the reproductive adult stage. We have identified a regulator of molting, pqn-47. Null mutations in pqn-47 cause a developmental arrest at the first larval molt, showing that this gene activity is required to transit the molt. Mutants with weak alleles of pqn-47 complete the larval molts but fail to exit the molting cycle at the adult stage. These phenotypes suggest that pqn-47 executes key aspects of the molting program including the cessation of molting cycles. The pqn-47 gene encodes a protein that is highly conserved in animal phylogeny but probably misannotated in genome sequences due to much less significant homology to a yeast transcription factor. A PQN-47::GFP fusion gene is expressed in many neurons, vulval precursor cells, the distal tip cell (DTC), intestine, and the lateral hypodermal seam cells but not in the main body hypodermal syncytium (hyp7) that underlies, synthesizes, and releases most of the collagenous cuticle. A functional PQN-47::GFP fusion protein localizes to the cytoplasm rather than the nucleus at all developmental stages, including the periods preceding and during ecdysis when genetic analysis suggests that pqn-47 functions. The cytoplasmic localization of PQN-47::GFP partially overlaps with the endoplasmic reticulum, suggesting that PQN-47 is involved in the extensive secretion of cuticle components or hormones that occurs during molts. The mammalian and insect homologues of pqn-47 may serve similar roles in regulated secretion

Functional Genomic Analysis of C. elegans Molting

Although the molting cycle is a hallmark of insects and nematodes, neither the endocrine control of molting via size, stage, and nutritional inputs nor the enzymatic mechanism for synthesis and release of the exoskeleton is well understood. Here, we identify endocrine and enzymatic regulators of molting in C. elegans through a genome-wide RNA-interference screen. Products of the 159 genes discovered include annotated transcription factors, secreted peptides, transmembrane proteins, and extracellular matrix enzymes essential for molting. Fusions between several genes and green fluorescent protein show a pulse of expression before each molt in epithelial cells that synthesize the exoskeleton, indicating that the corresponding proteins are made in the correct time and place to regulate molting. We show further that inactivation of particular genes abrogates expression of the green fluorescent protein reporter genes, revealing regulatory networks that might couple the expression of genes essential for molting to endocrine cues. Many molting genes are conserved in parasitic nematodes responsible for human disease, and thus represent attractive targets for pesticide and pharmaceutical development

The role of ERO1 in oxidative protein folding in the endoplasmic reticulum

Thesis (Ph.D.)--Massachusetts Institute of Technology, Dept. of Biology, 1999.Includes bibliographical references.The formation of native disulfide bonds is critical for the folding and stability of many secreted proteins. We describe an essential S. cerevisiae gene, ER01, which encodes a conserved ER membrane protein required for disulfide bond formation in the er .doplasmic reticulum (ER). In a conditional ero 1-1 mutant, secretory proteins that would normally contain disulfide bonds, such as carboxypeptidase Y (CPY), are retained in the ER in reduced form, as shown by thiol modification with AMS. ER01 levels determine cellular oxidizing capacity, since mutation of ER01 causes hypersensitivil/ to the reductant OTT, whereas overexpression of ER01 confers resistance to OTT. Moreover, the thiol oxidant diamide can restore growth and secretion to ero1 mutants. These results suggest that Ero1p provides the oxidizing equivalents utilized for disulfide bond formation in the ER. Oxidizing equivalents are transferred directly from Ero1p to the abundant ER oxidoreductase PDI (protein disulfide isomerase) and its homolog Mpd2p. PDI is oxidized in wild-type cells, but reduced in the ero 1-1 mutant. Thiol-disulfide exchange between POI and Ero1p is indicated by the capture of PD1-Ero1p mixed-disulfides. PDI oxidizes secretory proteins, since newly-synthesized CPY remains fully reduced in POI-depleted cells. Mixed-disulfides between PDI and p1 CPY are also detected, indicating that PDI engages directly in thiol-disulfide exchange with this substrate. Together, these results define a pathway for protein disulfide bond formation in the ER wherein oxidizing equivalents flow from Ero1p to POI (and Mpd2p) and then to substrate proteins through direct thiol-disulfide exchange reactions. Oxidized glutathione (GSSG) does not serve as an obligate intermediate In this pathway, since oxidative protein folding proceeds normally in a gsh 1.1 mutant devoid of intracellular glutathione. Mutational analysis of ER01 identifies two pairs of conserved, vlclnal cystelnes essential for Ero1p function. Mutation of Cys100, Cys105, Cys352, or Cys355 of Ero1 p disrupts cell viability, CPY folding, and thiol-disulfide exchange between POI and Ero1p. Cys100 of Ero1p may be preferentially attacked by POI, while the Cys352- Cys355 disulfide may re-oxidize the Cys 100-Cys 105 cystelne pair. The properties of yeast Ero1 p resemble those of E. coli DsbB.by Alison R. Frand.Ph.D

Toward a unified model of developmental timing: A "molting" approach.

Animal development requires temporal coordination between recurrent processes and sequential events, but the underlying timing mechanisms are not yet understood. The molting cycle of C. elegans provides an ideal system to study this basic problem. We recently characterized LIN-42, which is related to the circadian clock protein PERIOD, as a key component of the developmental timer underlying rhythmic molting cycles. In this context, LIN-42 coordinates epithelial stem cell dynamics with progression of the molting cycle. Repeated actions of LIN-42 may enable the reprogramming of seam cell temporal fates, while stage-specific actions of LIN-42 and other heterochronic genes select fates appropriate for upcoming, rather than passing, life stages. Here, we discuss the possible configuration of the molting timer, which may include interconnected positive and negative regulatory loops among lin-42, conserved nuclear hormone receptors such as NHR-23 and -25, and the let-7 family of microRNAs. Physiological and environmental conditions may modulate the activities of particular components of this molting timer. Finding that LIN-42 regulates both a sleep-like behavioral state and epidermal stem cell dynamics further supports the model of functional conservation between LIN-42 and mammalian PERIOD proteins. The molting timer may therefore represent a primitive form of a central biological clock and provide a general paradigm for the integration of rhythmic and developmental processes

Functional genomic analysis of C. elegans molting.

Although the molting cycle is a hallmark of insects and nematodes, neither the endocrine control of molting via size, stage, and nutritional inputs nor the enzymatic mechanism for synthesis and release of the exoskeleton is well understood. Here, we identify endocrine and enzymatic regulators of molting in C. elegans through a genome-wide RNA-interference screen. Products of the 159 genes discovered include annotated transcription factors, secreted peptides, transmembrane proteins, and extracellular matrix enzymes essential for molting. Fusions between several genes and green fluorescent protein show a pulse of expression before each molt in epithelial cells that synthesize the exoskeleton, indicating that the corresponding proteins are made in the correct time and place to regulate molting. We show further that inactivation of particular genes abrogates expression of the green fluorescent protein reporter genes, revealing regulatory networks that might couple the expression of genes essential for molting to endocrine cues. Many molting genes are conserved in parasitic nematodes responsible for human disease, and thus represent attractive targets for pesticide and pharmaceutical development

Functional genomic analysis of C. elegans molting.

Although the molting cycle is a hallmark of insects and nematodes, neither the endocrine control of molting via size, stage, and nutritional inputs nor the enzymatic mechanism for synthesis and release of the exoskeleton is well understood. Here, we identify endocrine and enzymatic regulators of molting in C. elegans through a genome-wide RNA-interference screen. Products of the 159 genes discovered include annotated transcription factors, secreted peptides, transmembrane proteins, and extracellular matrix enzymes essential for molting. Fusions between several genes and green fluorescent protein show a pulse of expression before each molt in epithelial cells that synthesize the exoskeleton, indicating that the corresponding proteins are made in the correct time and place to regulate molting. We show further that inactivation of particular genes abrogates expression of the green fluorescent protein reporter genes, revealing regulatory networks that might couple the expression of genes essential for molting to endocrine cues. Many molting genes are conserved in parasitic nematodes responsible for human disease, and thus represent attractive targets for pesticide and pharmaceutical development