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Martin Chalfie

Publications and source records attributed to Martin Chalfie.

15 recordsLinked to original sources
PubMed2006

WormBook: the online review of Caenorhabditis elegans biology.

WormBook (www.wormbook.org) is an open-access, online collection of original, peer-reviewed chapters on the biology of Caenorhabditis elegans and related nematodes. Since WormBook was launched in June 2005 with 12 chapters, it has grown to over 100 chapters, covering nearly every aspect of C.elegans research, from Cell Biology and Neurobiology to Evolution and Ecology. WormBook also serves as the text companion to WormBase, the C.elegans model organism database. Objects such as genes, proteins and cells are linked to the relevant pages in WormBase, providing easily accessible background information. Additionally, WormBook chapters contain links to other relevant topics in WormBook, and the in-text citations are linked to their abstracts in PubMed and full-text references, if available. Since WormBook is online, its chapters are able to contain movies and complex images that would not be possible in a print version. WormBook is designed to keep up with the rapid pace of discovery in the field of C.elegans research and continues to grow. WormBook represents a generic publishing infrastructure that is easily adaptable to other research communities to facilitate the dissemination of knowledge in the field.

Animals↗
PubMed2006

Podocin and MEC-2 bind cholesterol to regulate the activity of associated ion channels.

The prohibitin (PHB)-domain proteins are membrane proteins that regulate a variety of biological activities, including mechanosensation, osmotic homeostasis, and cell signaling, although the mechanism of this regulation is unknown. We have studied two members of this large protein family, MEC-2, which is needed for touch sensitivity in Caenorhabditis elegans, and Podocin, a protein involved in the function of the filtration barrier in the mammalian kidney, and find that both proteins bind cholesterol. This binding requires the PHB domain (including palmitoylation sites within it) and part of the N-terminally adjacent hydrophobic domain that attaches the proteins to the inner leaflet of the plasma membrane. By binding to MEC-2 and Podocin, cholesterol associates with ion-channel complexes to which these proteins bind: DEG/ENaC channels for MEC-2 and TRPC channels for Podocin. Both the MEC-2-dependent activation of mechanosensation and the Podocin-dependent activation of TRPC channels require cholesterol. Thus, MEC-2, Podocin, and probably many other PHB-domain proteins by binding to themselves, cholesterol, and target proteins regulate the formation and function of large protein-cholesterol supercomplexes in the plasma membrane.

Amino Acid Sequence↗
PubMed2006

Loss of LIN-35, the Caenorhabditis elegans ortholog of the tumor suppressor p105Rb, results in enhanced RNA interference.

BACKGROUND: Genome-wide RNA interference (RNAi) screening is a very powerful tool for analyzing gene function in vivo in Caenorhabditis elegans. The effectiveness of RNAi varies from gene to gene, however, and neuronally expressed genes are largely refractive to RNAi in wild-type worms. RESULTS: We found that C. elegans strains carrying mutations in lin-35, the worm ortholog of the tumor suppressor gene p105Rb, or a subset of the genetically related synMuv B family of chromatin-modifying genes, show increased strength and penetrance for many germline, embryonic, and post-embryonic RNAi phenotypes, including neuronal RNAi phenotypes. Mutations in these same genes also enhance somatic transgene silencing via an RNAi-dependent mechanism. Two genes, mes-4 and zfp-1, are required both for the vulval lineage defects resulting from mutations in synMuv B genes and for RNAi, suggesting a common mechanism for the function of synMuv B genes in vulval development and in regulating RNAi. Enhanced RNAi in the germline of lin-35 worms suggests that misexpression of germline genes in somatic cells cannot alone account for the enhanced RNAi observed in this strain. CONCLUSION: A worm strain with a null mutation in lin-35 is more sensitive to RNAi than any other previously described single mutant strain, and so will prove very useful for future genome-wide RNAi screens, particularly for identifying genes with neuronal functions. As lin-35 is the worm ortholog of the mammalian tumor suppressor gene p105Rb, misregulation of RNAi may be important during human oncogenesis.

Animals↗
PubMed2004

The MEC-4 DEG/ENaC channel of Caenorhabditis elegans touch receptor neurons transduces mechanical signals.

Transformation of mechanical energy into ionic currents is essential for touch, hearing and nociception. Although DEG/ENaC proteins are believed to form sensory mechanotransduction channels, the evidence for this role remains indirect. By recording from C. elegans touch receptor neurons in vivo, we found that external force evokes rapidly activating mechanoreceptor currents (MRCs) carried mostly by Na(+) and blocked by amiloride-characteristics consistent with direct mechanical gating of a DEG/ENaC channel. Like mammalian Pacinian corpuscles, these neurons depolarized with both positive and negative changes in external force but not with sustained force. Null mutations in the DEG/ENaC gene mec-4 and in the accessory ion channel subunit genes mec-2 and mec-6 eliminated MRCs. In contrast, the genetic elimination of touch neuron-specific microtubules reduced, but did not abolish, MRCs. Our findings link the application of external force to the activation of a molecularly defined metazoan sensory transduction channel.

Amiloride↗
PubMed2004

Extracellular proteins organize the mechanosensory channel complex in C. elegans touch receptor neurons.

Specialized extracellular matrix (ECM) is associated with virtually every mechanosensory system studied. C. elegans touch receptor neurons have specialized ECM and attach to the surrounding epidermis. The mec-1 gene encodes an ECM protein with multiple EGF and Kunitz domains. MEC-1 is needed for the accumulation of the collagen MEC-5 and other ECM components, attachment, and, separately, for touch sensitivity. MEC-1 and MEC-5 bind to touch processes uniformly and in puncta. These puncta colocalize with and localize the mechanosensory channel complex in the touch neurons. In turn, the production of the MEC-1 and MEC-5 puncta appears to rely on interactions with the neighboring epidermal tissue. These and other observations lead us to propose that extracellular, but not cytoskeletal, tethering of the degenerin channel is needed for mechanosensory transduction. Additionally, our experiments demonstrate an important role of the ECM in organizing the placement of the channel complex.

Alleles↗
PubMed2004

MEC-2 is recruited to the putative mechanosensory complex in C. elegans touch receptor neurons through its stomatin-like domain.

BACKGROUND: The response to gentle body touch in C. elegans requires a degenerin channel complex containing four proteins (MEC-2, MEC-4, MEC-6, and MEC-10). The central portion of the integral membrane protein MEC-2 contains a stomatin-like region that is highly conserved from bacteria to mammals. The molecular function of this domain in MEC-2, however, is unknown. RESULTS: Here, we show that MEC-2 colocalizes with the degenerin MEC-4 in regular puncta along touch receptor neuron processes. This punctate localization requires the other channel complex proteins. The stomatin-like region of MEC-2 interacts with the intracellular cytoplasmic portion of MEC-4. Missense mutations in this region that destroy the interaction also disrupt the punctate localization and degenerin-regulating function of MEC-2. Missense mutations outside this region apparently have no effect on the punctate localization but significantly reduce the regulatory effect of MEC-2 on the MEC-4 degenerin channel. A second stomatin-like protein, UNC-24, colocalizes with MEC-2 in vivo and coimmunoprecipitates with MEC-2 and MEC-4 in Xenopus oocytes; unc-24 enhances the touch insensitivity of temperature-sensitive alleles of mec-4 and mec-6. CONCLUSION: Two stomatin homologs, MEC-2 and UNC-24, interact with the MEC-4 degenerin through their stomatin-like regions, which act as protein binding domains. At least in the case of MEC-2, this binding allows its nonstomatin domains to regulate channel activity. Stomatin-like regions in other proteins may serve a similar protein binding function.

Alleles↗
PubMed2004

Combinatorial marking of cells and organelles with reconstituted fluorescent proteins.

Expression of GFP and other fluorescent proteins depends on cis-regulatory elements. Because these elements rarely direct expression to specific cell types, GFP production cannot always be sufficiently limited. Here we show that reconstitution of GFP, YFP, and CFP previously split into two polypeptides yields fluorescent products when coexpressed in C. elegans. Because this reconstitution involves two components, it can confirm cellular coexpression and identify cells expressing a previously uncharacterized promoter. By choosing promoters whose expression patterns overlap for a single cell type, we can produce animals with fluorescence only in those cells. Furthermore, when one partial GFP polypeptide is fused with a subcellularly localized protein or peptide, this restricted expression leads to the fluorescent marking of cellular components in a subset of cells.

Animals↗
PubMed2003

Nucleotide binding by the Mdm2 RING domain facilitates Arf-independent Mdm2 nucleolar localization.

The RING domain of Mdm2 contains a conserved Walker A or P loop motif that is a characteristic of nucleotide binding proteins. We found that Mdm2 binds adenine-containing nucleotides preferentially and that nucleotide binding leads to a conformational change in the Mdm2 C terminus. Although nucleotide binding is not required for Mdm2 E3 ubiquitin ligase activity, we show that nucleotide binding-defective P loop mutants are impaired in p14(ARF)-independent nucleolar localization both in vivo and in vitro. Consistent with this, ATP-bound Mdm2 is preferentially localized to the nucleolus. Indeed, we identify a unique amino acid substitution in the P loop motif (K454A) that uncouples nucleolar localization and E3 ubiquitin ligase activity of Mdm2 and leads to upregulation of the E3 activity both in human cells and in Caenorhabditis elegans. We propose that nucleotide binding-facilitated nucleolar localization of Mdm2 is an evolutionarily conserved regulator of Mdm2 activity.

Active Transport, Cell Nucleus↗
PubMed2003

The Caenorhabditis elegans spalt-like gene sem-4 restricts touch cell fate by repressing the selector Hox gene egl-5 and the effector gene mec-3.

Members of the spalt (sal) gene family encode zinc-finger proteins that are putative tumor suppressors and regulate anteroposterior (AP) patterning, cellular identity, and, possibly, cell cycle progression. The mechanism through which sal genes carry out these functions is unclear. The Caenorhabditis elegans sal gene sem-4 controls the fate of several different cell types, including neurons, muscle and hypodermis. Mutation of sem-4 transforms particular tail neurons into touch-neuron-like cells. In wild-type C. elegans, six touch receptor neurons mediate the response of the worm to gentle touch. All six touch neurons normally express the LIM homeobox gene mec-3. A subset, the two PLM cells, also express the Hox gene egl-5, an Abdominal-B homolog, which we find is required for correct mec-3 expression in these cells. The abnormal touch-neuron-like-cells in sem-4 animals express mec-3; we show that a subset also express egl-5. We report: (1) that ectopic expression of sem-4 in normal touch cells represses mec-3 expression and reduces touch cell function; (2) that egl-5 expression is required for both the fate of normal PLM touch neurons in wild-type animals and the fate of a subset of abnormal touch neurons in sem-4 animals, and (3) that SEM-4 specifically binds a shared motif in the mec-3 and egl-5 promoters that mediates repression of these genes in cells in the tail. We conclude that sem-4 represses egl-5 and mec-3 through direct interaction with regulatory sequences in the promoters of these genes, that sem-4 indirectly modulates mec-3 expression through its repression of egl-5 and that this negative regulation is required for proper determination of neuronal fates. We suggest that the mechanism and targets of regulation by sem-4 are conserved throughout the sal gene family: other sal genes might regulate patterning and cellular identity through direct repression of Hox selector genes and effector genes.

Animals↗
PubMed2002

The mechanosensory protein MEC-6 is a subunit of the C. elegans touch-cell degenerin channel.

Mechanosensory transduction in touch receptor neurons is believed to be mediated by DEG/ENaC (degenerin/epithelial Na+ channel) proteins in nematodes and mammals. In the nematode Caenorhabditis elegans, gain-of-function mutations in the degenerin genes mec-4 and mec-10 (denoted mec-4(d) and mec-10(d), respectively) cause degeneration of the touch cells. This phenotype is completely suppressed by mutation in a third gene, mec-6 (refs 3, 4), that is needed for touch sensitivity. This last gene is also required for the function of other degenerins. Here we show that mec-6 encodes a single-pass membrane-spanning protein with limited similarity to paraoxonases, which are implicated in human coronary heart disease. This gene is expressed in muscle cells and in many neurons, including the six touch receptor neurons. MEC-6 increases amiloride-sensitive Na+ currents produced by MEC-4(d)/MEC-10(d) by approximately 30-fold, and functions synergistically with MEC-2 (a stomatin-like protein that regulates MEC-4(d)/MEC-10(d) channel activity) to increase the currents by 200-fold. MEC-6 physically interacts with all three channel proteins. In vivo, MEC-6 co-localizes with MEC-4, and is required for punctate MEC-4 expression along touch-neuron processes. We propose that MEC-6 is a part of the degenerin channel complex that may mediate mechanotransduction in touch cells.

Animals↗
PubMed2002

Identification of genes expressed in C. elegans touch receptor neurons.

The extent of gene regulation in cell differentiation is poorly understood. We previously used saturation mutagenesis to identify 18 genes that are needed for the development and function of a single type of sensory neuron--the touch receptor neuron for gentle touch in Caenorhabditis elegans. One of these genes, mec-3, encodes a transcription factor that controls touch receptor differentiation. By culturing and isolating wild-type and mec-3 mutant cells from embryos and applying their amplified RNA to DNA microarrays, here we have identified genes that are known to be expressed in touch receptors, a previously uncloned gene (mec-17) that is needed for maintaining touch receptor differentiation, and more than 50 previously unknown mec-3-dependent genes. These genes are randomly distributed in the genome and under-represented both for genes that are co-expressed in operons and for multiple members of gene families. Using regions 5' of the start codon of the first 20 genes, we have also identified an over-represented heptanucleotide, AATGCAT, that is needed for the expression of touch receptor genes.

Amino Acid Sequence↗
PubMed2002

MEC-2 regulates C. elegans DEG/ENaC channels needed for mechanosensation.

Touch sensitivity in animals relies on nerve endings in the skin that convert mechanical force into electrical signals. In the nematode Caenorhabditis elegans, gentle touch to the body wall is sensed by six mechanosensory neurons that express two amiloride-sensitive Na+ channel proteins (DEG/ENaC). These proteins, MEC-4 and MEC-10, are required for touch sensation and can mutate to cause neuronal degeneration. Here we show that these mutant or 'd' forms of MEC-4 and MEC-10 produce a constitutively active, amiloride-sensitive ionic current when co-expressed in Xenopus oocytes, but not on their own. MEC-2, a stomatin-related protein needed for touch sensitivity, increased the activity of mutant channels about 40-fold and allowed currents to be detected with wild-type MEC-4 and MEC-10. Whereas neither the central, stomatin-like domain of MEC-2 nor human stomatin retained the activity of full-length MEC-2, both produced amiloride-sensitive currents with MEC-4d. Our findings indicate that MEC-2 regulates MEC-4/MEC-10 ion channels and raise the possibility that similar ion channels may be formed by stomatin-like proteins and DEG/ENaC proteins that are co-expressed in both vertebrates and invertebrates. Some of these channels may mediate mechanosensory responses.

Amiloride↗
PubMed2002

MTD-1, a touch-cell-specific membrane protein with a subtle effect on touch sensitivity.

We have used representational difference analysis (RDA) applied to cDNA to isolate transcripts regulated by MEC-3, a transcription factor needed for the differentiation of the six touch receptor neurons in Caenorhabditis elegans. Six percent of 595 cDNAs isolated by cDNA RDA were mec-3-dependent. These cDNAs represented mRNA from two previously known genes, mec-18 and mec-7, and one new gene, mtd-1 (mec-three-dependent). mtd-1 encodes a novel transmembrane protein that is exclusively expressed in the six touch cells throughout development. mtd-1 loss results in a subtle defect in the touch receptor neurons. Neither mtd-1 RNAi nor a putative mtd-1 loss-of-function mutation resulted in touch insensitivity, but both enhanced the touch insensitivity of mec-6(u247), a temperature sensitive allele, at the permissive temperature.

Alleles↗
PubMed2002

Genetics of sensory mechanotransduction.

The molecular mechanisms for the transduction of light and chemical signals in animals are fairly well understood. In contrast, the processes by which the senses of touch, balance, hearing, and proprioception are transduced are still largely unknown. Biochemical approaches to identify transduction components are difficult to use with mechanosensory systems, but genetic approaches are proving more successful. Genetic research in several organisms has demonstrated the importance of cytoskeletal, extracellular, and membrane components for sensory mechanotransduction. In particular, researchers have identified channel proteins in the DEG/ENaC and TRP families that are necessary for signaling in a variety of mechanosensory cells. Proof that these proteins are components of the transduction channel, however, is incomplete.

Animals↗