Long-Term Culture and Monitoring of Isolated <em>Caenorhabditis elegans</em> on Solid Media in Multi-Well Devices

The nematode Caenorhabditis elegans is among the most common model systems used in aging research owing to its simple and inexpensive culture techniques, ...

University of Arizona

The nuclear vitamin D receptor (VDR) mediates the actions of its 1,25-dihydroxyvitamin D(3) ligand to control gene expression in terrestrial vertebrates. Prominent functions of VDR-regulated genes are to promote intestinal absorption of calcium and phosphate for bone mineralization and to potentiate the hair cycle in mammals. We report the cloning of VDR from Petromyzon marinus, an unexpected finding because lampreys lack mineralized tissues and hair. Lamprey VDR (lampVDR) clones were obtained via RT-PCR from larval protospleen tissue and skin and mouth of juveniles. LampVDR expressed in transfected mammalian COS-7 cells bound 1,25-dihydroxyvitamin D(3) with high affinity, and transactivated a reporter gene linked to a vitamin D-responsive element from the human CYP3A4 gene, which encodes a P450 enzyme involved in xenobiotic detoxification. In tests with other vitamin D responsive elements, such as that from the rat osteocalcin gene, lampVDR showed little or no activity. Phylogenetic comparisons with nuclear receptors from other vertebrates revealed that lampVDR is a basal member of the VDR grouping, also closely related to the pregnane X receptors and constitutive androstane receptors. We propose that, in this evolutionarily ancient vertebrate, VDR may function in part, like pregnane X receptors and constitutive androstane receptors, to induce P450 enzymes for xenobiotic detoxification.

Cloning of a functional vitamin D receptor from the lamprey (Petromyzon marinus), an ancient vertebrate lacking a calcified skeleton and teeth.

Nuclear hormone receptor-responsive element binding specificity has been reported to reside predominantly in the proximal box (P-box), three amino acids located in a DNA-recognition alpha-helix situated on the C-terminal side of the first zinc finger. To further define the residues in the vitamin D receptor (VDR) DNA binding domain (DBD) that mediate its interaction as a retinoid X receptor (RXR) heterodimer with the rat osteocalcin vitamin D-responsive element (VDRE), chimeric receptors were created in which the core DBD of VDR was replaced with that of the homodimerizing glucocorticoid receptor (GR). Systematic alteration of GR DBD amino acids in these chimeras to VDR DBD residues identified arg-49 and lys-53, just C-terminal of the P-box within the base recognition alpha-helix of human VDR (hVDR), as the only two amino acids among 36 differences required to convert the GR core zinc finger domain to that of the VDR. Gel mobility shift and 1,25-dihydroxyvitamin D3-stimulated transcription assays verified that an hVDR-GR DBD chimera is functional on the rat osteocalcin VDRE with only the conservative change of lys-49 to arg, and of the negatively charged glu-53 to a basic amino acid (lys or arg). Thus, for RXR heterodimerizing receptors like VDR, the P-box requires redefinition and expansion to include a DNA specificity element corresponding to arg-49 and lys-53 of hVDR. Examination of DNA specificity element amino acids in other nuclear receptors in terms of conservation and base contact in cocrystal structures supports the conclusion that these residues are crucial for selective DNA recognition.

Two basic amino acids C-terminal of the proximal box specify functional binding of the vitamin D receptor to its rat osteocalcin deoxyribonucleic acid-responsive element.

MTM1, MTMR2, and SBF2 belong to a family of proteins called the myotubularins. X-linked myotubular myopathy, a severe congenital disorder characterized by hypotonia and generalized muscle weakness in newborn males, is caused by mutations in MTM1 (Laporte et al., 1996). Charcot-Marie-Tooth types 4B1 and 4B2 are severe demyelinating neuropathies caused by mutations in MTMR2 (Bolino et al., 2000) and SBF2/MTMR13 (Senderek et al., 2003), respectively. Although several myotubularins are known to regulate phosphoinositide-phosphate levels in cells, little is known about the actual cellular process that is defective in patients with these diseases. Mutations in worm MTM-6 and MTM-9, myotubularins belonging to two subgroups, disorganize phosphoinositide 3-phosphate localization and block endocytosis in the coelomocytes of Caenorhabditis elegans. We demonstrate that MTM-6 and MTM-9 function as part of a complex to regulate an endocytic pathway that involves the Arf6 GTPase, and we define protein domains required for MTM-6 activity.

Disease-related myotubularins function in endocytic traffic in Caenorhabditis elegans.

The human vitamin D receptor (hVDR), which is a substrate for several protein kinases, mediates the actions of its 1,25-dihydroxyvitamin D3 (1,25(OH)2D3) ligand to regulate gene expression. To determine the site, and functional impact, of cAMP-dependent protein kinase (PKA)-catalyzed phosphorylation of hVDR, we generated a series of C-terminally truncated and point mutant receptors. Incubation of mutant hVDRs with PKA and [gamma-32P]ATP, in vitro, or overexpressing them in COS-7 kidney cells labeled with [32P]orthophosphate, revealed that serine-182 is the predominant residue in hVDR phosphorylated by PKA. An aspartate substituted mutant (S182D), incorporating a negative charge to mimic phosphorylation, displayed only 50% of the transactivation capacity in response to 1,25(OH)2D3 of either wild-type or an S182A-altered hVDR. When the catalytic subunit of PKA was overexpressed, a similar reduction in wild-type but not S182D hVDR transactivity was observed. In a mammalian two-hybrid system, S182D bound less avidly than wild-type or S182A hVDR to the retinoid X receptor (RXR) heterodimeric partner that co-mediates vitamin D responsive element recognition and transactivation. These data suggest that hVDR serine-182 is a primary site for PKA phosphorylation, an event that leads to an attenuation of both RXR heterodimerization and resultant transactivation of 1,25(OH)2D3 target genes.

Phosphorylation of human vitamin D receptor serine-182 by PKA suppresses 1,25(OH)2D3-dependent transactivation.

Ligand-gated ion channels are transmembrane proteins that respond to a variety of transmitters, including acetylcholine, gamma-aminobutyric acid (GABA), glycine, and glutamate [1 and 2]. These proteins play key roles in neurotransmission and are typically found in the nervous system and at neuromuscular junctions [3]. Recently, acetylcholine receptor family members also have been found in nonneuronal cells, including macrophages [4], keratinocytes [5], bronchial epithelial cells [5], and endothelial cells of arteries [6]. The function of these channels in nonneuronal cells in mammals remains to be elucidated, though it has been shown that the acetylcholine receptor alpha7 subunit is required for acetylcholine-mediated inhibition of tumor necrosis factor release by activated macrophages [4]. We show that cup-4, a gene required for efficient endocytosis of fluids by C. elegans coelomocytes, encodes a protein that is homologous to ligand-gated ion channels, with the highest degree of similarity to nicotinic acetylcholine receptors. Worms lacking CUP-4 have reduced phosphatidylinositol 4,5-bisphosphate levels at the plasma membrane, suggesting that CUP-4 regulates endocytosis through modulation of phospholipase C activity.

Endocytosis function of a ligand-gated ion channel homolog in Caenorhabditis elegans.

Mutations in MCOLN1, which encodes the protein h-mucolipin-1, result in the lysosomal storage disease Mucolipidosis Type IV. Studies on CUP-5, the human orthologue of h-mucolipin-1 in Caenorhabditis elegans, have shown that these proteins are required for lysosome biogenesis. We show here that the lethality in cup-5 mutant worms is due to two defects, starvation of embryonic cells and general developmental defects. Starvation leads to apoptosis through a CED-3-mediated pathway. We also show that providing worms with a lipid-soluble metabolite partially rescues the embryonic lethality but has no effect on the developmental defects, the major cause of the lethality. These results indicate that supplementing the metabolic deficiency of Mucolipidosis Type IV patients mat not be sufficient to alleviate the symptoms due to tissue degeneration.

Basis of lethality in C. elegans lacking CUP-5, the Mucolipidosis Type IV orthologue.

Mucolipidosis Type IV is currently characterized as a lysosomal storage disorder with defects that include corneal clouding, achlorhydria and psychomotor retardation. MCOLN1, the gene responsible for this disease, encodes the protein mucolipin-1 that belongs to the "Transient Receptor Potential" family of proteins and has been shown to function as a non-selective cation channel whose activity is modulated by pH. Two cell biological defects that have been described in MLIV fibroblasts are a hyperacidification of lysosomes and a delay in the exit of lipids from lysosomes.

Lysosomal trafficking functions of mucolipin-1 in murine macrophages.

Quality-control mechanisms of protein folding of transmembrane and secreted proteins is mediated by endoplasmic-reticulum-associated degradation (ERAD), which is used to detect and to degrade misfolded proteins in the ER. The ERAD machinery consists of chaperones, transmembrane proteins and ubiquitin-associated enzymes that detect, modify, and retro-translocate the misfolded proteins to the cytoplasm for degradation by the proteasome. In contrast to ERAD, little is known about the fates of integral membrane and secreted proteins that become misfolded at the plasma membrane or in the extracellular space. Derlin proteins are a family of proteins that are conserved in all eukaryotes, where they function in ERAD. Here, we show that loss of Derlin function in Caenorhabditis elegans and in mouse macrophages results in the accumulation of integral membrane proteins at the plasma membrane. Induction of LDL receptor misfolding at the plasma membrane results in a sharp decrease in its half-life, which can be rescued by proteasomal inhibitors or by reduction of Derlin-1 levels. We also show that Derlin proteins localize to endosomes as well as to the ER. Our data are consistent with a model where Derlin proteins function in a spatially segregated quality control pathway that is used for the recognition and degradation of transmembrane proteins that become misfolded at the plasma membrane and/or in endosomes.

Derlin-dependent accumulation of integral membrane proteins at cell surfaces.

Developing methods for protecting organisms in metal-polluted environments is contingent upon our understanding of cellular detoxification mechanisms. In this regard, half-molecule ATP-binding cassette (ABC) transporters of the HMT-1 subfamily are required for cadmium (Cd) detoxification. HMTs have conserved structural architecture that distinguishes them from other ABC transporters and allows the identification of homologs in genomes of different species including humans. We recently discovered that HMT-1 from the simple, unicellular organism, Schizosaccharomyces pombe, SpHMT1, acts independently of phytochelatin synthase (PCS) and detoxifies Cd, but not other heavy metals. Whether HMTs from multicellular organisms confer tolerance only to Cd or also to other heavy metals is not known.

Detoxification of multiple heavy metals by a half-molecule ABC transporter, HMT-1, and coelomocytes of Caenorhabditis elegans.

D&#xE9;velopper des m&#xE9;thodes pour prot&#xE9;ger les organismes en m&#xE9;tal pollu&#xE9;es environnements d&#xE9;pend de notre compr&#xE9;hension des m&#xE9;canismes de d&#xE9;toxification cellulaire. &#xC0; cet &#xE9;gard, une demi-mol&#xE9;cule d&#x26;#39;ATP-binding cassette (ABC), les transporteurs de la sous-famille HMT-1 sont n&#xE9;cessaires pour le cadmium (Cd) de d&#xE9;sintoxication. HMTS ont conserv&#xE9; l&#x26;#39;architecture structurelle qui les distingue des autres transporteurs ABC et permet l&#x26;#39;identification des homologues dans les g&#xE9;nomes de diff&#xE9;rentes esp&#xE8;ces, y compris les humains. Nous avons r&#xE9;cemment d&#xE9;couvert que HMT-1 du simple, organisme unicellulaire, Schizosaccharomyces pombe, SpHMT1, agit ind&#xE9;pendamment de la synthase phytoch&#xE9;latine (PCS) et d&#xE9;toxifie Cd, mais pas d&#x26;#39;autres m&#xE9;taux lourds. Que HMTS d&#x26;#39;organismes multicellulaires ne leur conf&#xE8;rent une tol&#xE9;rance au Cd ou &#xE9;galement &#xE0; d&#x26;#39;autres m&#xE9;taux lourds n&#x26;#39;est pas connue.

D&#xE9;sintoxication de multiples m&#xE9;taux lourds par une demi-mol&#xE9;cule Transporter ABC, HMT-1, et coelomocytes de Caenorhabditis elegans

&#x53D1;&#x5C55;&#x4E2D;&#x56FD;&#x5BB6;&#x4FDD;&#x62A4;&#x91D1;&#x5C5E;&#x6C61;&#x67D3;&#x73AF;&#x5883;&#x4E2D;&#x7684;&#x751F;&#x7269;&#x7684;&#x65B9;&#x6CD5;&#x5C06;&#x53D6;&#x51B3;&#x4E8E;&#x6211;&#x4EEC;&#x5BF9;&#x7EC6;&#x80DE;&#x89E3;&#x6BD2;&#x673A;&#x5236;&#x7684;&#x7406;&#x89E3;&#x3002;&#x5728;&#x8FD9;&#x65B9;&#x9762;&#xFF0C;&#x9700;&#x8981;&#x9549; (Cd) &#x6212;&#x6BD2;&#x534A;&#x5206;&#x5B50; ATP &#x7ED3;&#x5408;&#x76D2; &#xFF08;ABC&#xFF09; &#x8F6C;&#x8FD0;&#x86CB;&#x767D; HMT 1 &#x4E9A;&#x79D1;&#x3002;HMTs &#x6709;&#x4FDD;&#x5B88;&#x7ED3;&#x6784;&#x7684;&#x4F53;&#x7CFB;&#x7ED3;&#x6784;&#xFF0C;&#x4F7F;&#x5B83;&#x4EEC;&#x533A;&#x522B;&#x4E8E;&#x5176;&#x4ED6; ABC &#x8F6C;&#x8FD0;&#x86CB;&#x767D;&#x548C;&#x5141;&#x8BB8;&#x540C;&#x7CFB;&#x7269;&#x7684;&#x9274;&#x5B9A;&#xFF0C;&#x5305;&#x62EC;&#x4EBA;&#x7C7B;&#x7684;&#x4E0D;&#x540C;&#x7269;&#x79CD;&#x7684;&#x57FA;&#x56E0;&#x7EC4;&#x4E2D;&#x3002;&#x6211;&#x4EEC;&#x6700;&#x8FD1;&#x53D1;&#x73B0; HMT 1 &#x4ECE;&#x7B80;&#x5355;&#x7684;&#x5355;&#x7EC6;&#x80DE;&#x6709;&#x673A;&#x4F53;&#xFF0C;&#x7C9F;&#xFF0C;SpHMT1&#xFF0C;&#x72EC;&#x7ACB;&#x4E8E;&#x7EDC;&#x5408;&#x9176; (PC) &#x7684;&#x884C;&#x4E3A;&#x548C;&#x51CF;&#x5C11; Cd&#xFF0C;&#x4F46;&#x6CA1;&#x6709;&#x5176;&#x4ED6;&#x91CD;&#x91D1;&#x5C5E;&#x3002;&#x4E0D;&#x77E5;&#x9053;&#x662F;&#x5426;&#x4ECE;&#x591A;&#x7EC6;&#x80DE;&#x6709;&#x673A;&#x4F53; HMTs &#x8D4B;&#x4E88;&#x5BB9;&#x5FCD;&#x53EA;&#x5411; Cd &#x6216;&#x5176;&#x4ED6;&#x91CD;&#x91D1;&#x5C5E;&#x4E5F;&#x3002;

&#x5BF9;&#x591A;&#x4E2A;&#x91CD;&#x91D1;&#x5C5E;&#x7684;&#x534A;&#x5206;&#x5B50; ABC &#x8F6C;&#x8FD0;&#x86CB;&#x767D;&#x3001; HMT-1 &#x548C;&#x4F53;&#x8154;&#x7684;&#x79C0;&#x4E3D;&#x9690;&#x6746;&#x7EBF;&#x866B;&#x7684;&#x6392;&#x6BD2;&#x3002;

Entwicklung von Methoden zum Schutz von Organismen in Metall-verschmutzten Umgebungen ist von unserem Verst&auml;ndnis der Mechanismen der zellul&auml;ren Entgiftung abh&auml;ngig. In diesem Zusammenhang sind halb-Molek&uuml;l ATP verbindliche Kassette (ABC) Transporter der HMT-1-Unterfamilie f&uuml;r Kadmium (Cd) Entgiftung erforderlich. HMTs haben strukturelle Architektur konserviert, das unterscheidet sie von anderen ABC-Transporter und erm&ouml;glicht die Identifizierung von UCP1 in Genome verschiedener Arten, einschlie&szlig;lich des Menschen. Vor kurzem entdeckten wir, dass HMT-1 aus einfachen, einzelligen Organismus, Schizosaccharomyces Pombe, SpHMT1, unabh&auml;ngig von der Phytochelatine-Synthase (PCS agiert) und Cd, aber nicht andere Schwermetalle entgiftet. Ob HMTs aus vielzelligen Organismen Toleranz nur auf Cd oder auch andere Schwermetalle verleihen, ist nicht bekannt.

Entgiftung von mehreren Schwermetallen durch eine halb-Molek&uuml;l ABC-Transporter, HMT-1 und Coelomocytes von Caenorhabditis Elegans.

Sviluppo di metodi per proteggere gli organismi in ambienti inquinati in metallo &egrave; subordinato la nostra comprensione dei meccanismi di disintossicazione cellulare. A questo proposito, met&agrave;-molecola ATP-binding cassette (ABC) della sottofamiglia HMT-1 sono necessari per la disintossicazione del cadmio (Cd). HMTs hanno conservato di architettura strutturale che li distingue da altri trasportatori ABC e permette l&#39;identificazione di omologhi nel genoma di specie diverse, tra cui gli esseri umani. Abbiamo recentemente scoperto che HMT-1 dall&#39;organismo semplice, unicellulare, Schizosaccharomyces pombe, SpHMT1, agisce indipendentemente da fitochelatina sintasi (pz) e disintossica il Cd, ma non altri metalli pesanti. Non &egrave; noto se HMTs da organismi pluricellulari conferisce tolleranza solo su Cd o anche di altri metalli pesanti.

Disintossicazione dei metalli pesanti pi&ugrave; di una molecola di mezzo trasportatore ABC HMT-1 e coelomocytes di Caenorhabditis elegans.

&#x91D1;&#x5C5E;&#x6C5A;&#x67D3;&#x3055;&#x308C;&#x305F;&#x74B0;&#x5883;&#x3067;&#x751F;&#x7269;&#x3092;&#x4FDD;&#x8B77;&#x3059;&#x308B;&#x305F;&#x3081;&#x306E;&#x30E1;&#x30BD;&#x30C3;&#x30C9;&#x3092;&#x958B;&#x767A;&#x3059;&#x308B;&#x306B;&#x306F;&#x3001;&#x643A;&#x5E2F;&#x96FB;&#x8A71;&#x306E;&#x89E3;&#x6BD2;&#x30E1;&#x30AB;&#x30CB;&#x30BA;&#x30E0;&#x306E;&#x7406;&#x89E3;&#x304C;&#x6761;&#x4EF6;&#x3068;&#x306A;&#x308A;&#x307E;&#x3059;&#x3002;&#x3053;&#x306E;&#x70B9;&#x3067;&#x306F;&#x3001;HMT-1&#x30D5;&#x30A1;&#x30DF;&#x30EA;&#x30FC;&#x306E;&#x534A;&#x5206;&#x5B50;&#x306E;ATP&#x7D50;&#x5408;&#x30AB;&#x30BB;&#x30C3;&#x30C8;&#xFF08;ABC&#xFF09;&#x30C8;&#x30E9;&#x30F3;&#x30B9;&#x30DD;&#x30FC;&#x30BF;&#x30FC;&#x306F;&#x3001;&#x30AB;&#x30C9;&#x30DF;&#x30A6;&#x30E0;&#xFF08;Cd&#xFF09;&#x89E3;&#x6BD2;&#x306B;&#x5FC5;&#x8981;&#x3068;&#x3055;&#x308C;&#x308B;&#x3002; HMTs&#x306F;&#x3001;&#x4ED6;&#x306E;ABC&#x30C8;&#x30E9;&#x30F3;&#x30B9;&#x30DD;&#x30FC;&#x30BF;&#x30FC;&#x304B;&#x3089;&#x305D;&#x308C;&#x3089;&#x3092;&#x533A;&#x5225;&#x3057;&#x3001;&#x30D2;&#x30C8;&#x3092;&#x542B;&#x3080;&#x69D8;&#x3005;&#x306A;&#x7A2E;&#x306E;&#x30B2;&#x30CE;&#x30E0;&#x306B;&#x76F8;&#x540C;&#x4F53;&#x306E;&#x540C;&#x5B9A;&#x3092;&#x53EF;&#x80FD;&#x306B;&#x3059;&#x308B;&#x69CB;&#x9020;&#x306E;&#x30A2;&#x30FC;&#x30AD;&#x30C6;&#x30AF;&#x30C1;&#x30E3;&#x3092;&#x4FDD;&#x5B58;&#x3057;&#x3066;&#x3044;&#x307E;&#x3059;&#x3002;&#x6211;&#x3005;&#x306F;&#x6700;&#x8FD1;&#x3001;&#x5358;&#x7D14;&#x306A;&#x5358;&#x7D30;&#x80DE;&#x751F;&#x7269;&#x3001;&#x30B7;&#x30BE;&#x30B5;&#x30C3;&#x30AB;&#x30ED;&#x30DF;&#x30BB;&#x30B9;&#xB7;&#x30DD;&#x30F3;&#x30D9;&#x3001;SpHMT1&#x304B;&#x3089;&#x3001;HMT-1&#x306F;&#x30D5;&#x30A3;&#x30C8;&#x30B1;&#x30E9;&#x30C1;&#x30F3;&#x5408;&#x6210;&#x9175;&#x7D20;&#xFF08;PCS&#xFF09;&#x304B;&#x3089;&#x72EC;&#x7ACB;&#x3057;&#x3066;&#x52D5;&#x4F5C;&#x3057;&#x3001;&#x30AB;&#x30C9;&#x30DF;&#x30A6;&#x30E0;&#x3092;&#x89E3;&#x6BD2;&#x3059;&#x308B;&#x3053;&#x3068;&#x3092;&#x767A;&#x898B;&#x3057;&#x305F;&#x304C;&#x3001;&#x4ED6;&#x306E;&#x306A;&#x3044;&#x91CD;&#x91D1;&#x5C5E;&#x3002;&#x591A;&#x7D30;&#x80DE;&#x751F;&#x7269;&#x304B;&#x3089;HMTs CD&#x306B;&#x306E;&#x307F;&#x8010;&#x6027;&#x3092;&#x4ED8;&#x4E0E;&#x3059;&#x308B;&#x304B;&#x3001;&#x4ED6;&#x306E;&#x91CD;&#x91D1;&#x5C5E;&#x306B;&#x77E5;&#x3089;&#x308C;&#x3066;&#x3044;&#x306A;&#x3044;&#x304B;&#x3069;&#x3046;&#x304B;&#x3092;&#x6307;&#x5B9A;&#x3057;&#x307E;&#x3059;&#x3002;

&#x30CF;&#x30FC;&#x30D5;&#x5206;&#x5B50;ABC&#x30C8;&#x30E9;&#x30F3;&#x30B9;&#x30DD;&#x30FC;&#x30BF;&#x30FC;&#x3001;HMT-1&#x3001;&#x7DDA;&#x866B;Caenorhabditis elegans&#x306E;Coelomocytes&#x306B;&#x3088;&#x308B;&#x8907;&#x6570;&#x306E;&#x91CD;&#x91D1;&#x5C5E;&#x306E;&#x89E3;&#x6BD2;

&#xAE08;&#xC18D; &#xC624;&#xC5FC; &#xD658;&#xACBD;&#xC5D0;&#xC11C; &#xC720;&#xAE30; &#xCCB4;&#xB97C; &#xBCF4;&#xD638; &#xD558;&#xAE30; &#xC704;&#xD55C; &#xBC29;&#xBC95;&#xC744; &#xAC1C;&#xBC1C; &#xD558;&#xB294; &#xAC83;&#xC740; &#xC138;&#xD3EC; &#xD574;&#xB3C5; &#xBA54;&#xCEE4;&#xB2C8;&#xC998;&#xC5D0; &#xB300; &#xD55C; &#xC6B0;&#xB9AC;&#xC758; &#xC774;&#xD574;&#xC5D0; &#xB530;&#xB77C; &#xD30C;&#xACAC;&#xC785;&#xB2C8;&#xB2E4;. &#xC774;&#xC640; &#xAD00;&#xB828;, HMT 1 &#xC544;&#xC758; &#xC808;&#xBC18;-&#xBD84;&#xC790; ATP &#xBC14;&#xC778;&#xB529; &#xCE74;&#xC138;&#xD2B8; (ABC) &#xC804;&#xC1A1;&#xAE30;&#xB294; &#xCE74;&#xB4DC;&#xBBB4; (Cd) &#xD574;&#xB3C5;&#xC744; &#xC704;&#xD55C; &#xD544;&#xC694; &#xD569;&#xB2C8;&#xB2E4;. HMTs &#xB2E4;&#xB978; ABC &#xC804;&#xC1A1;&#xAE30;&#xC5D0;&#xC11C; &#xADF8;&#xB4E4;&#xC744; &#xAD6C;&#xBCC4; &#xD558; &#xACE0; &#xC778;&#xAC04;&#xC744; &#xD3EC;&#xD568; &#xD55C; &#xB2E4;&#xB978; &#xC885;&#xC758; &#xAC8C;&#xB188;&#xC5D0;&#xC11C; homologs &#xC2DD;&#xBCC4; &#xAD6C;&#xC870; &#xAC74;&#xCD95;&#xC744; &#xBCF4;&#xC874; &#xD588;&#xC2B5;&#xB2C8;&#xB2E4;. &#xC6B0;&#xB9AC;&#xB294; &#xCD5C;&#xADFC;&#xC5D0; HMT-1 &#xAC04;&#xB2E8; &#xD558; &#xACE0;, &#xB2E8; &#xC138;&#xD3EC; &#xC0DD;&#xBB3C;, Schizosaccharomyces pombe, SpHMT1, phytochelatin synthase (PC) &#xB3C5;&#xB9BD;&#xC801;&#xC73C;&#xB85C; &#xC791;&#xB3D9; &#xD558; &#xACE0; Cd&#xB9CC; &#xD558;&#xC9C0; &#xB2E4;&#xB978; &#xC911; &#xAE08;&#xC18D;&#xC744; &#xD574;&#xB3C5;&#xC744; &#xBC1C;&#xACAC; &#xD588;&#xB2E4;. HMTs &#xB2E4;&#xC138;&#xD3EC; &#xC0DD;&#xBB3C;&#xC5D0;&#xC11C; Cd&#xC5D0;&#xB9CC; &#xB610;&#xB294; &#xB2E4;&#xB978; &#xC911; &#xAE08;&#xC18D;&#xC5D0; &#xB0B4;&#xC131;&#xC744; &#xBD80;&#xC5EC; &#xD558;&#xB294; &#xC5EC;&#xBD80;&#xB97C; &#xC54C; &#xC218; &#xC5C6;&#xC2B5;&#xB2C8;&#xB2E4;.

&#xBC18; &#xBD84;&#xC790; ABC transporter, HMT 1 &#xBC0F; Caenorhabditis elegans&#xC758; coelomocytes&#xC5D0; &#xC758;&#xD574; &#xC5EC;&#xB7EC; &#xC911; &#xAE08;&#xC18D; &#xD574;&#xB3C5;.

El desarrollo de m&#xE9;todos para la protecci&#xF3;n de los organismos en ambientes contaminados de metales depende de nuestra comprensi&#xF3;n de los mecanismos de destoxificaci&#xF3;n celular. A este respecto, la mol&#xE9;cula media-ATP vinculante cassette (ABC) transportadores de la subfamilia de HMT-1 se requieren para el cadmio (Cd) desintoxicaci&#xF3;n. HMTs han conservado la arquitectura estructural que los distingue de los otros transportadores ABC y permite la identificaci&#xF3;n de los hom&#xF3;logos en los genomas de diferentes especies, incluyendo seres humanos. Recientemente hemos descubierto que HMT-1 desde el organismo simple, unicelular, Schizosaccharomyces pombe, SpHMT1, act&#xFA;a de forma independiente de la sintasa de fitoquelatina (PCS) y desintoxica CD, pero no a otros metales pesados. Si HMTs de los organismos multicelulares confieren tolerancia s&#xF3;lo en un CD o tambi&#xE9;n a otros metales pesados &#x200B;&#x200B;no se conoce.

La desintoxicaci&#xF3;n de varios metales pesados &#x200B;&#x200B;por un transportador de media mol&#xE9;cula de ABC, HMT-1, y Coelomocytes de Caenorhabditis elegans

Cells have to maintain stable plasma membrane protein and lipid compositions under normal conditions and to remodel their plasma membranes in response to stimuli. This maintenance and remodeling require that integral membrane proteins at the plasma membrane that become misfolded, because of the relatively harsher extracellular milieu or carbohydrate and amino acid sequence changes, are degraded. We had previously shown that Derlin proteins, required for quality control mechanisms in the endoplasmic reticulum, also localize to endosomes and function in the degradation of misfolded integral membrane proteins at the plasma membrane. In this study, we show that Derlin proteins physically associate with sorting nexins that function in retrograde membrane transport from endosomes to the Golgi apparatus. Using genetic studies in Caenorhabditis elegans and ricin pulse-chase analyses in murine RAW264.7 macrophages, we show that the Derlin-sorting nexin interaction is physiologically relevant. Our studies suggest that at least some integral membrane proteins that are misfolded at the plasma membrane are retrogradely transported to the Golgi apparatus and ultimately to the endoplasmic reticulum for degradation via resident quality control mechanisms.

Derlin-dependent retrograde transport from endosomes to the Golgi apparatus.

Mutations in MCOLN1, which encodes the cation channel protein TRPML1, result in the neurodegenerative lysosomal storage disorder Mucolipidosis type IV. Mucolipidosis type IV patients show lysosomal dysfunction in many tissues and neuronal cell death. The ortholog of TRPML1 in Caenorhabditis elegans is CUP-5; loss of CUP-5 results in lysosomal dysfunction in many tissues and death of developing intestinal cells that results in embryonic lethality. We previously showed that a null mutation in the ATP-Binding Cassette transporter MRP-4 rescues the lysosomal defect and embryonic lethality of cup-5(null) worms. Here we show that reducing levels of the Endosomal Sorting Complex Required for Transport (ESCRT)-associated proteins DID-2, USP-50, and ALX-1/EGO-2, which mediate the final de-ubiquitination step of integral membrane proteins being sequestered into late endosomes, also almost fully suppresses cup-5(null) mutant lysosomal defects and embryonic lethality. Indeed, we show that MRP-4 protein is hypo-ubiquitinated in the absence of CUP-5 and that reducing levels of ESCRT-associated proteins suppresses this hypo-ubiquitination. Thus, increased ESCRT-associated de-ubiquitinating activity mediates the lysosomal defects and corresponding cell death phenotypes in the absence of CUP-5.

ESCRT-Dependent Cell Death in a Caenorhabditis elegans Model of the Lysosomal Storage Disorder Mucolipidosis Type IV.

Lysosomes, the major membrane-bound degradative organelles, have a multitude of functions in eukaryotic cells. Lysosomes are the terminal compartments in the endocytic pathway, though they display highly dynamic behaviors, fusing with each other and with late endosomes in the endocytic pathway, and with the plasma membrane during regulated exocytosis and for wound repair. After fusing with late endosomes, lysosomes are reformed from the resulting hybrid organelles through a process that involves budding of a nascent lysosome, extension of the nascent lysosome from the hybrid organelle, while remaining connected by a membrane bridge, and scission of the membrane bridge to release the newly formed lysosome. The newly formed lysosomes undergo cycles of homotypic fusion and fission reactions to form mature lysosomes. In this study, we used a forward genetic screen in  to identify six regulators of lysosome biology. We show that these proteins function in different steps of lysosome biology, regulating lysosome formation, lysosome fusion, and lysosome degradation.

Regulators of Lysosome Function and Dynamics in .

Assays have been developed to study trafficking in various tissues of  Adult  intestinal cells are large and have extensive endocytic networks, thus making them a good system for deciphering the endocytic pathway using live imaging techniques. However, the presence of auto-fluorescent gut granules in adult intestine can interfere with the signals of endocytic compartment reporters, like GFP. Here we demonstrate a protocol adapted from the original method developed by the Grant laboratory to identify signals from reporters in adult intestinal cells. The goal of this protocol is to identify endocytic compartments tagged with fluorescent markers without any confounding effects of background autofluorescent gut granules in adult intestinal cells of .

Hope Dang

Department of Molecular & Cellular Biology

University of Arizona

Long-Term Culture and Monitoring of Isolated Caenorhabditis elegans on Solid Media in Multi-Well Devices

Hope Dang

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University of Arizona

Long-Term Culture and Monitoring of Isolated Caenorhabditis elegans on Solid Media in Multi-Well Devices

Department of Molecular & Cellular Biology