the-c-elegans-intestine-as-model-for-intercellular-lumen

The C. elegans Intestine As a Model for Intercellular Lumen Morphogenesis and In Vivo Polarized Membrane Biogenesis at the Single-cell Level: Labeling by Antibody Staining, RNAi Loss-of-function Analysis and Imaging

Multicellular tubes, fundamental units of all internal organs, are composed of polarized epithelial or endothelial cells, with apical membranes lining the ...

the-c-elegans-excretory-canal-as-model-for-intracellular-lumen

The C. elegans Excretory Canal as a Model for Intracellular Lumen Morphogenesis and In Vivo Polarized Membrane Biogenesis in a Single Cell: labeling by GFP-fusions, RNAi Interaction Screen and Imaging

The four C. elegans excretory canals are narrow tubes extended through the length of the animal from a single cell, with almost equally far extended ...

Harvard Medical School

Epithelial tubes are basic building blocks of complex organs, but their architectural requirements are not well understood. Here we show that erm-1 is a unique C. elegans ortholog of the ERM family of cytoskeleton-membrane linkers, with an essential role in lumen morphogenesis. ERM-1 localizes to the luminal membranes of those tubular organ epithelia which lack stabilization by cuticle. RNA interference (RNAi), a germline deletion, and overexpression of erm-1 cause cystic luminal phenotypes in these epithelia. Confocal and ultrastructural analyses indicate that erm-1 functions directly in apical membrane morphogenesis, rather than in epithelial polarity and junction assembly as has been previously proposed for ERMs. We also show that act-5/cytoplasmic actin and sma-1/beta-H-spectrin are required for lumen formation and functionally interact with erm-1. Our findings suggest that there are common structural constraints on the architecture of diverse organ lumina.

Lumen morphogenesis in C. elegans requires the membrane-cytoskeleton linker erm-1.

Now that some genomes have been completely sequenced, the ability to direct specific mutations into genomes is particularly desirable. Here we present a method to create mutations in the Caenorhabditis elegans genome efficiently through transgene-directed, transposon-mediated gene conversion. Engineered deletions targeted into two genes show that the frequency of obtaining the desired mutation was higher using this approach than using standard transposon insertion-deletion approaches. We also targeted an engineered green fluorescent protein insertion-replacement cassette to one of these genes, thereby confirming that custom alleles of different types can be created in vitro to make the corresponding mutations in vivo. This approach should also be applicable to heterologous transposons in C. elegans and other organisms, including vertebrates.

Targeted gene alteration in Caenorhabditis elegans by gene conversion.

Case records of the Massachusetts General Hospital. Case 30-2007. A newborn girl with skin lesions.

Metazoan internal organs are assembled from polarized tubular epithelia that must set aside an apical membrane domain as a lumenal surface. In a global Caenorhabditis elegans tubulogenesis screen, interference with several distinct fatty-acid-biosynthetic enzymes transformed a contiguous central intestinal lumen into multiple ectopic lumens. We show that multiple-lumen formation is caused by apicobasal polarity conversion, and demonstrate that in situ modulation of lipid biosynthesis is sufficient to reversibly switch apical domain identities on growing membranes of single post-mitotic cells, shifting lumen positions. Follow-on targeted lipid-biosynthesis pathway screens and functional genetic assays were designed to identify a putative single causative lipid species. They demonstrate that fatty-acid biosynthesis affects polarity through sphingolipid synthesis, and reveal ceramide glucosyltransferases (CGTs) as end-point biosynthetic enzymes in this pathway. Our findings identify glycosphingolipids, CGT products and obligate membrane lipids, as critical determinants of in vivo polarity and indicate that they sort new components to the expanding apical membrane.

Apicobasal domain identities of expanding tubular membranes depend on glycosphingolipid biosynthesis.

Clathrin coats vesicles in all eukaryotic cells and has a well-defined role in endocytosis, moving molecules away from the plasma membrane. Its function on routes towards the plasma membrane was only recently appreciated and is thought to be limited to basolateral transport. Here, an unbiased RNAi-based tubulogenesis screen identifies a role of clathrin (CHC-1) and its AP-1 adaptor in apical polarity during de novo lumenal membrane biogenesis in the C. elegans intestine. We show that CHC-1/AP-1-mediated polarized transport intersects with a sphingolipid-dependent apical sorting process. Depleting each presumed trafficking component mislocalizes the same set of apical membrane molecules basolaterally, including the polarity regulator PAR-6, and generates ectopic lateral lumens. GFP::CHC-1 and BODIPY-ceramide vesicles associate perinuclearly and assemble asymmetrically at polarized plasma membrane domains in a co-dependent and AP-1-dependent manner. Based on these findings, we propose a trafficking pathway for apical membrane polarity and lumen morphogenesis that implies: (1) a clathrin/AP-1 function on an apically directed transport route; and (2) the convergence of this route with a sphingolipid-dependent apical trafficking path.

Clathrin and AP-1 regulate apical polarity and lumen formation during C. elegans tubulogenesis.

&#x8BB8;&#x591A;&#x5355;&#x7EC6;&#x80DE;&#x7BA1;&#x5982;&#x6BDB;&#x7EC6;&#x8840;&#x7BA1;&#x5F62;&#x6210;&#x6D41;&#x660E;&#x819C;&#x5C42;&#xFF0C;&#x4E00;&#x4E2A;&#x4E0D;&#x5F88;&#x4E86;&#x89E3;&#x7684;&#x8FC7;&#x7A0B;&#x3002;&#x5728;&#x8FD9;&#x91CC;&#x6211;&#x4EEC;&#x5C55;&#x793A; ERM-1 &#x76AE;&#x8D28;&#x819C;&#x7EC4;&#x7EC7;&#x8005;&#x9700;&#x5728;&#x5355;&#x4E2A;&#x5355;&#x5143;&#x683C;&#x79C0;&#x4E3D;&#x9690;&#x6746;&#x7EBF;&#x866B;&#x6392;&#x6CC4;&#x8FD0;&#x6CB3;&#x5F62;&#x6001;&#x53D1;&#x751F;&#x671F;&#x95F4;&#x5C55;&#x5F00;&#x7684;&#x80DE;&#x5185;&#x7684;&#x5FC3;&#x5C16;/&#x80A0;&#x58C1;&#x819C;&#x548C;&#x5176;&#x808C;&#x52A8;&#x86CB;&#x767D;&#x5185;&#x6D82;&#x5C42;&#x3002;&#x6211;&#x4EEC;&#x7684;&#x7279;&#x70B9;&#x6C34;&#x901A;&#x9053;&#x86CB;&#x767D;-8&#xFF0C;&#x786E;&#x5B9A;&#x7684; ERM 1 &#x8868;&#x8FBE; (ERM-1[++]) &#x6291;&#x5236;&#x5668;&#x5C4F;&#x5E55;&#x4E0A;&#xFF0C;&#x4F5C;&#x4E3A;&#x6CEA;&#x9053;&#x7684;&#x6C34;&#x901A;&#x9053;&#x86CB;&#x767D;&#x8FDB;&#x884C;&#x4EA4;&#x4E92;&#x4EE5; ERM-1 &#x6D41;&#x660E;&#x6269;&#x5C55;&#x4E2D;&#x6C5E;&#x654F;&#x611F;&#x7684;&#x65B9;&#x5F0F;&#xFF0C;&#x6C34;&#x901A;&#x9053;&#x6D3B;&#x52A8;&#x6709;&#x7275;&#x8FDE;&#x3002;&#x6C34;&#x901A;&#x9053;&#x86CB;&#x767D;-8 &#x662F;&#x77AC;&#x6001;&#x62DB;&#x52DF;&#x7531; ERM-1&#xFF0C;&#x5728;&#x56F4;&#x80A0;&#x58C1;&#x8896;&#x53E3;&#x6CBF;&#x6269;&#x5927;&#x8FD0;&#x6CB3; interspaced co-localizing &#x6D41;&#x660E;&#x3002;ERM-1 + [+]-&#x4ECB;&#x5BFC;&#x7684;&#x589E;&#x52A0;&#x6D41;&#x660E;&#x5173;&#x8054;&#x5C0F;&#x7BA1;&#x65AD;&#x88C2;&#x7684;&#x6570;&#x76EE;&#x626D;&#x8F6C;&#x7684;&#x6C34;&#x901A;&#x9053;&#x86CB;&#x767D; 8 &#x67AF;&#x7AED;&#x3002;&#x6211;&#x4EEC;&#x5EFA;&#x8BAE;&#x7531; translumenal &#x901A;&#x91CF;&#xFF0C;ERM-1/&#x6C34;&#x901A;&#x9053;&#x86CB;&#x767D;-8 &#x4E92;&#x52A8;&#x63A8;&#x8FDB;&#x6D41;&#x660E;&#x6269;&#x5C55;&#x540D;&#x6697;&#x793A;&#x7684;&#x6C34;&#x901A;&#x9053;&#x53D7;&#x89C4;&#x7BA1;&#x7684;&#x6D41;&#x4F53;&#x538B;&#x529B;&#x76F4;&#x63A5;&#x5F62;&#x6001;&#x53D1;&#x751F;&#x6548;&#x5E94;&#x3002;

&#x80DE;&#x8154;&#x6269;&#x5C55;&#x9700;&#x8981; ERM-1-&#x4F9D;&#x8D56;&#x9876;&#x7AEF;&#x819C;&#x6269;&#x5F20;&#x548C;&#x6C34;&#x901A;&#x9053;&#x86CB;&#x767D;&#x4ECB;&#x5BFC; 8 &#x901A;&#x91CF;&#x3002;

Many unicellular tubes such as capillaries form lumens intracellularly, a process that is not well understood. Here we show that the cortical membrane organizer ERM-1 is required to expand the intracellular apical/lumenal membrane and its actin undercoat during single-cell Caenorhabditis elegans excretory canal morphogenesis. We characterize AQP-8, identified in an ERM-1-overexpression (ERM-1[++]) suppressor screen, as a canalicular aquaporin that interacts with ERM-1 in lumen extension in a mercury-sensitive manner, implicating water-channel activity. AQP-8 is transiently recruited to the lumen by ERM-1, co-localizing in peri-lumenal cuffs interspaced along expanding canals. An ERM-1[++]-mediated increase in the number of lumen-associated canaliculi is reversed by AQP-8 depletion. We propose that the ERM-1/AQP-8 interaction propels lumen extension by translumenal flux, suggesting a direct morphogenetic effect of water-channel-regulated fluid pressure.

Intracellular lumen extension requires ERM-1-dependent apical membrane expansion and AQP-8-mediated flux.

Nombreux tubes unicellulaires comme les capillaires forment lumens intracellulaire, un processus qui n&#39;est pas bien compris. Ici, nous montrons que l&#39;organisateur de la membrane corticale ERM-1 est n&eacute;cessaire pour &eacute;largir la membrane apicale/lumi&egrave;re intracellulaire et son sous-poil actine au cours de la seule cellule Caenorhabditis elegans canal excr&eacute;teur morphogen&egrave;se. Nous caract&eacute;risons AQP-8, identifi&eacute; dans une MCE-1-surexpression (&eacute;cran de suppresseur ERM-1[++]), comme une aquaporine canaliculaire qui interagit avec MCE-1 en prolongation de lumen de mani&egrave;re sensible au mercure, impliquant l&#39;activit&eacute; de l&#39;eau-canal. AQP-8 est transitoirement recrut&eacute;s &agrave; la lumi&egrave;re de MCE-1, co-localizing en peri-lumi&egrave;re poignets espac&eacute;es le long de l&#39;expansion des canaux. Un MTC-1 [++]-m&eacute;di&eacute;e par augmentation du nombre des canalicules associ&eacute;s &agrave; lumen est renvers&eacute;e par l&#39;appauvrissement AQP-8. Nous proposons que l&#39;interaction de l&#39;ERM-1/AQP-8 propulse extension lumen par flux translumenal, sugg&eacute;rant un effet morphog&eacute;n&eacute;tique direct de pression du fluide eau-canal-r&eacute;gl&eacute;es.

Lumen intracellulaire extension n&eacute;cessite expansion membrane apicale MCE-1-d&eacute;pendante et le flux induite par l&#39;AQP-8.

Molti tubi unicellulari come capillari formano lumen intracellularly, un processo che non &egrave; ben compreso. Qui ci mostra che l&#39;organizzatore della membrana corticale ERM-1 &egrave; necessario espandere la membrana apicale/lumenal intracellulare e il suo sottopelo actina durante la cella singola Caenorhabditis elegans canale escretore morfogenesi. Ci caratterizzano AQP-8, identificato in una sovraespressione ERM-1 (ERM-1[++]) schermo soppressore, come un aquaporina canalicolare che interagisce con ERM-1 nell&#39;estensione del lume in maniera sensibile al mercurio, implicando attivit&agrave; di acqua-canale. AQP-8 &egrave; transitoriamente reclutati per il lume di ERM-1, co-localizing in peri-lumenal polsini intervallate lungo canali in espansione. Un ERM-1 [+ +]-mediata aumento del numero di associati al lume canaliculi &egrave; invertito da deplezione di AQP-8. Proponiamo che l&#39;interazione di ERM-1/AQP-8 aziona estensione lumen di flusso translumenal, suggerendo un effetto morfogenetico diretto della pressione del fluido acqua-canale-regolato.

Lume intracellulare estensione richiede espansione membrana apicale ERM-1-dipendente e flux AQP-8-mediata.

&#x6BDB;&#x7D30;&#x8840;&#x7BA1;&#x306A;&#x3069;&#x591A;&#x304F;&#x306E;&#x5358;&#x7D30;&#x80DE;&#x7BA1;&#x5185;&#x8154;&#x7D30;&#x80DE;&#x5185;&#x3001;&#x30D5;&#x30A9;&#x30FC;&#x30E0;&#x3088;&#x304F;&#x7406;&#x89E3;&#x3055;&#x308C;&#x3066;&#x3044;&#x306A;&#x3044;&#x30D7;&#x30ED;&#x30BB;&#x30B9;&#x3002;&#x3053;&#x3053;&#x3067;&#x76AE;&#x8CEA;&#x819C;&#x30AA;&#x30FC;&#x30AC;&#x30CA;&#x30A4;&#x30B6;&#x30FC; ERM 1 &#x7D30;&#x80DE;&#x9802;/&#x306A;&#x3063;&#x305F;&#x5185;&#x8154;&#x5074;&#x819C;&#x3068;&#x305D;&#x306E;&#x30A2;&#x30AF;&#x30C1;&#x30F3;&#x4E0B;&#x5730;&#x5358;&#x4E00;&#x30BB;&#x30EB;&#x7DDA;&#x866B;&#x7DDA;&#x866B;&#x6392;&#x6CC4;&#x904B;&#x6CB3;&#x306E;&#x5F62;&#x614B;&#x5F62;&#x6210;&#x4E2D;&#x306B;&#x5C55;&#x958B;&#x3059;&#x308B;&#x5FC5;&#x8981;&#x304C;&#x3042;&#x308B;&#x3053;&#x3068;&#x3092;&#x793A;&#x3057;&#x307E;&#x3059;&#x3002;&#x30A2;&#x30AF;&#x30A2;&#x30DD;&#x30EA;&#x30F3;-8&#x3001;ERM-1 &#x767A;&#x73FE; (ERM-1[++]) &#x6291;&#x5236;&#x753B;&#x9762;&#x3001;ERM-1 &#x3068;&#x306E;&#x5185;&#x8154;&#x62E1;&#x5F35; implicating &#x6C34;&#x30C1;&#x30E3;&#x30CD;&#x30EB;&#x306E;&#x5229;&#x7528;&#x72B6;&#x6CC1;&#x3001;&#x6C34;&#x9280;&#x306B;&#x654F;&#x611F;&#x306A;&#x65B9;&#x6CD5;&#x3067;&#x5BFE;&#x8A71;&#x6BDB;&#x7D30;&#x30A2;&#x30AF;&#x30A2;&#x30DD;&#x30EA;&#x30F3;&#x3068;&#x3057;&#x3066;&#x3002; &#x8B58;&#x5225;&#x3092;&#x7279;&#x5FB4;&#x4ED8;&#x3051;&#x308B;&#x30A2;&#x30AF;&#x30A2;&#x30DD;&#x30EA;&#x30F3; 8 &#x306F;&#x4E00;&#x904E;&#x6027;&#x306B;&#x5185;&#x8154; ERM - 1 co-localizing &#x904B;&#x6CB3;&#x3092;&#x62E1;&#x5927;&#x306B;&#x6CBF;&#x3063;&#x3066; interspaced &#x30DA;&#x30EA;&#x306A;&#x3063;&#x305F;&#x5185;&#x8154;&#x5074;&#x306E;&#x8896;&#x53E3;&#x3067;&#x52DF;&#x96C6;&#x3057;&#x3066;&#x3044;&#x307E;&#x3059;&#x3002;ERM 1 &#x306E; [+ +]-&#x7D30;&#x7BA1;&#x306E;&#x5185;&#x8154;&#x306B;&#x95A2;&#x9023;&#x4ED8;&#x3051;&#x3089;&#x308C;&#x305F;&#x6570;&#x306E;&#x5A92;&#x4ECB;&#x5897;&#x52A0;&#x304C;&#x9006;&#x30A2;&#x30AF;&#x30A2;&#x30DD;&#x30EA;&#x30F3; 8 &#x6E1B;&#x5C11;&#x306B;&#x3088;&#x3063;&#x3066;&#x3002;&#x6211;&#x3005; &#x306F;&#x6C34;&#x30C1;&#x30E3;&#x30CD;&#x30EB;&#x8ABF;&#x6574;&#x5727;&#x529B;&#x306E;&#x76F4;&#x63A5;&#x5F62;&#x6210;&#x52B9;&#x679C;&#x3092;&#x793A;&#x5506;&#x3001;ERM 1/&#x30A2;&#x30AF;&#x30A2;&#x30DD;&#x30EA;&#x30F3; 8 &#x76F8;&#x4E92;&#x4F5C;&#x7528;&#x304C;&#x8D8A;&#x7D4C;&#x7BA1;&#x8154;&#x30D5;&#x30E9;&#x30C3;&#x30AF;&#x30B9;&#x306B;&#x3088;&#x308B;&#x5185;&#x8154;&#x62E1;&#x5F35;&#x3092;&#x63A8;&#x9032;&#x3059;&#x308B;&#x3053;&#x3068;&#x3092;&#x63D0;&#x6848;&#x3057;&#x307E;&#x3059;&#x3002;

&#x7D30;&#x80DE;&#x5185;&#x306E;&#x5185;&#x8154;&#x62E1;&#x5F35;&#x6A5F;&#x80FD;&#x3067;&#x306F;&#x3001;ERM-1 &#x4F9D;&#x5B58;&#x30A2;&#x30D4;&#x30AB;&#x30EB;&#x819C;&#x306E;&#x62E1;&#x5F35;&#x30A2;&#x30AF;&#x30A2;&#x30DD;&#x30EA;&#x30F3; 8 &#x3092;&#x4ECB;&#x3057;&#x305F;&#x30D5;&#x30E9;&#x30C3;&#x30AF;&#x30B9;&#x3092;&#x5FC5;&#x8981;&#x304C;&#x3042;&#x308A;&#x307E;&#x3059;&#x3002;

&#xBAA8;&#xC138; &#xD608;&#xAD00; &#xB4F1; &#xB9CE;&#xC740; &#xB2E8; &#xC138;&#xD3EC; &#xAD00; &#xCE68;, &#xB8E8;&#xBA58;&#xC744; &#xD615;&#xC131; &#xD504;&#xB85C;&#xC138;&#xC2A4;&#xB97C; &#xC798; &#xC774;&#xD574; &#xB418;&#xC9C0; &#xC54A;&#xC2B5;&#xB2C8;&#xB2E4;. &#xC5EC;&#xAE30;&#xC5D0; &#xC6B0;&#xB9AC;&#xAC00; &#xD45C;&#xC2DC; &#xC678;&#xD53C; &#xB9C9; organizer &#xC74C; 1 &#xB2E8;&#xC77C; &#xC140; &#xAF2C;&#xB9C8; &#xC120; &#xCDA9; &#xBC30;&#xC124; &#xC6B4;&#xD558; morphogenesis &#xB3D9;&#xC548; &#xC138;&#xD3EC;&#xB0B4; &#xAF2D;&#xB300;&#xAE30;/lumenal &#xB9C9;&#xACFC; &#xADF8;&#xAC83;&#xC758; &#xAC78; &#xC18D; &#xD138;&#xC744; &#xD655;&#xC7A5; &#xD558;&#xB294; &#xB370; &#xD544;&#xC694;&#xD55C;&#xC785;&#xB2C8;&#xB2E4;. &#xC6B0;&#xB9AC;&#xB294; &#xC74C;-1-overexpression (ERM-1[++]) &#xC5B5;&#xC555; &#xD654;&#xBA74;, &#xC74C; 1 &#xB8E8;&#xBA58; &#xD655;&#xC7A5;&#xC5D0;&#xC11C; &#xBB3C; &#xCC44;&#xB110; &#xD65C;&#xB3D9;&#xC744; &#xB0B4;&#xD3EC; &#xC218;&#xC740; &#xBBFC;&#xAC10;&#xD55C; &#xBC29;&#xC2DD;&#xC73C;&#xB85C; &#xC0C1;&#xD638;&#xC791;&#xC6A9; canalicular aquaporin&#xB85C; &#xC2DD;&#xBCC4; AQP 8 &#xD2B9;&#xC9D5;. &#xC815;&#xB3C4; &#xC74C;-1, peri lumenal &#xC218; &#xAC11; &#xC6B4;&#xD558; &#xD655;&#xC7A5; &#xD568;&#xAED8; interspaced&#xC5D0; co-localizing&#xC5D0; &#xC758;&#xD574; AQP 8 &#xB8E8;&#xBA58;&#xC744; &#xBAA8;&#xC9D1;. &#xC74C; 1 + [+]-&#xB8E8;&#xBA58; &#xAD00;&#xB828; canaliculi &#xC218; &#xC788;&#xB294; &#xC911;&#xC7AC; &#xC99D;&#xAC00; AQP 8 &#xACE0;&#xAC08;&#xC5D0; &#xC758;&#xD574; &#xBC18;&#xB300;&#xC785;&#xB2C8;&#xB2E4;. &#xC6B0;&#xB9AC; &#xC74C; 1/AQP 8 &#xC0C1;&#xD638; &#xC791;&#xC6A9; translumenal &#xD50C;&#xB7ED;&#xC2A4;, &#xB8E8;&#xBA58; &#xD655;&#xC7A5; &#xCD94;&#xC9C4; &#xBB3C; &#xCC44;&#xB110; &#xADDC;&#xC81C; &#xC720;&#xCCB4; &#xC555;&#xB825;&#xC758; &#xC9C1;&#xC811;&#xC801;&#xC778; &#xC804;&#xCCB4;&#xC801; &#xD6A8;&#xACFC; &#xC81C;&#xC548; &#xC81C;&#xC548; &#xD569;&#xB2C8;&#xB2E4;.

&#xC138;&#xD3EC;&#xB0B4; &#xB8E8;&#xBA58; &#xD655;&#xC7A5; &#xC74C; 1 &#xC885;&#xC18D; &#xAF2D;&#xB300;&#xAE30; &#xB9C9; &#xD655;&#xC7A5; AQP 8 &#xC911;&#xC7AC; &#xD558;&#xB294; &#xD50C;&#xB7ED;&#xC2A4;&#xB97C; &#xD544;&#xC694;&#xD569;&#xB2C8;&#xB2E4;.

Muchos tubos unicelulares como capilares forman l&uacute;menes intracelular, un proceso que no se comprende bien. Aqu&iacute; mostramos que el organizador de la membrana cortical ERM-1 es necesaria para ampliar la membrana apical/lumenal intracelular y su capa de actina durante la morfog&eacute;nesis de canal excretor unicelular Caenorhabditis elegans. Caracterizamos AQP-8, identificado en una sobreexpresi&oacute;n ERM-1 (pantalla del supresor ERM-1[++]), como un aquaporin con canal&iacute;culos que interact&uacute;a con ERM-1 extensi&oacute;n de luz de manera sensible al mercurio, implicando la actividad del canal de agua. AQP-8 es reclutado transitoriamente a la luz por ERM-1, Junta en pu&ntilde;os peri lumenal otro a lo largo de la expansi&oacute;n de canales. Un ERM-1 [++]-mediada por aumento en el n&uacute;mero de asociados de lumen canal&iacute;culos se invierte por el agotamiento de AQP-8. Proponemos que la interacci&oacute;n de ERM-1/AQP-8 impulsa la extensi&oacute;n de lumen por flujo translumenal, sugiriendo un efecto morfogen&eacute;tico directo de presi&oacute;n regulada por el canal de agua.

Lumen intracelular extensi&oacute;n requiere expansi&oacute;n de membrana apical de ERM-1-dependiente y AQP-8-mediada por flujo.

Biological tubes consist of polarized epithelial cells with apical membranes building the central lumen and basolateral membranes contacting adjacent cells or the extracellular matrix. Cellular polarity requires distinct inputs from outside the cell, e.g., the matrix, inside the cell, e.g., vesicular trafficking and the plasma membrane and its junctions.(1) Many highly conserved polarity cues have been identified, but their integration during the complex process of polarized tissue and organ morphogenesis is not well understood. It is assumed that plasma-membrane-associated polarity determinants, such as the partitioning-defective (PAR) complex, define plasma membrane domain identities, whereas vesicular trafficking delivers membrane components to these domains, but lacks the ability to define them. In vitro studies on lumenal membrane biogenesis in mammalian cell lines now indicate that trafficking could contribute to defining membrane domains by targeting the polarity determinants, e.g., the PARs, themselves.(2) This possibility suggests a mechanism for PARs' asymmetric distribution on membranes and places vesicle-associated polarity cues upstream of membrane-associated polarity determinants. In such an upstream position, trafficking might even direct multiple membrane components, not only polarity determinants, an original concept of polarized plasma membrane biogenesis(3) (,) (4)that was largely abandoned due to the failure to identify a molecularly defined intrinsic vesicular sorting mechanism. Our two recent studies on C. elegans intestinal tubulogenesis reveal that glycosphingolipids (GSLs) and the well-recognized vesicle components clathrin and its AP-1 adaptor are required for targeting multiple apical molecules, including polarity regulators, to the expanding apical/lumenal membrane.(5) (,) (6) These findings support GSLs' long-proposed role in in vivo polarized epithelial membrane biogenesis and development and identify a novel function in apical polarity for classical post-Golgi vesicle components. They are also compatible with a vesicle-intrinsic sorting mechanism during membrane biogenesis and suggest a model for how vesicles could acquire apical directionality during the assembly of the functionally critical polarized lumenal surfaces of epithelial tubes.

Vesicular sorting controls the polarity of expanding membranes in the C. elegans intestine.

The field of metabolomics continues to catalog new compounds, but their functional analysis remains technically challenging, and roles beyond metabolism are largely unknown. Unbiased genetic/RNAi screens are powerful tools to identify the in vivo functions of protein-encoding genes, but not of nonproteinaceous compounds such as lipids. They can, however, identify the biosynthetic enzymes of these compounds-findings that are usually dismissed, as these typically synthesize multiple products. Here, we provide a method using follow-on biosynthetic pathway screens to identify the endpoint biosynthetic enzyme and thus the compound through which they act. The approach is based on the principle that all subsequently identified downstream biosynthetic enzymes contribute to the synthesis of at least this one end product. We describe how to systematically target lipid biosynthetic pathways; optimize targeting conditions; take advantage of pathway branchpoints; and validate results by genetic assays and biochemical analyses. This approach extends the power of unbiased genetic/RNAi screens to identify in vivo functions of non-nucleic acid-based metabolites beyond their metabolic roles. It will typically require several months to identify a metabolic end product by biosynthetic pathway screens, but this time will vary widely depending, among other factors, on the end product's location in the pathway, which determines the number of screens required for its identification.

RNAi-based biosynthetic pathway screens to identify in vivo functions of non-nucleic acid-based metabolites such as lipids.

Corrigendum: RNAi-based biosynthetic pathway screens to identify in vivo functions of non-nucleic acid-based metabolites such as lipids.

Determination of luminal diameter is critical to the function of small single-celled tubes. A series of EXC proteins, including EXC-1, prevent swelling of the tubular excretory canals in Caenorhabditis elegans In this study, cloning of exc-1 reveals it to encode a homolog of mammalian IRG proteins, which play roles in immune response and autophagy and are associated with Crohn's disease. Mutants in exc-1 accumulate early endosomes, lack recycling endosomes, and exhibit abnormal apical cytoskeletal structure in regions of enlarged tubules. EXC-1 interacts genetically with two other EXC proteins that also affect endosomal trafficking. In yeast two-hybrid assays, wild-type and putative constitutively active EXC-1 binds to the LIM-domain protein EXC-9, whose homolog, cysteine-rich intestinal protein, is enriched in mammalian intestine. These results suggest a model for IRG function in forming and maintaining apical tubule structure via regulation of endosomal recycling.

ArticleTotal : 2	Year
Publication title Cited by 13	2017
Publication title Cited by 6	2017

Article	Year
Lumen morphogenesis in C. elegans requires the membrane-cytoskeleton linker erm-1. Developmental cell\| PubMed ID: 15177034	2004
Targeted gene alteration in Caenorhabditis elegans by gene conversion. Nature genetics\| PubMed ID: 15502826	2004
Case records of the Massachusetts General Hospital. Case 30-2007. A newborn girl with skin lesions. The New England journal of medicine\| PubMed ID: 17898103	2007
Apicobasal domain identities of expanding tubular membranes depend on glycosphingolipid biosynthesis. Nature cell biology\| PubMed ID: 21926990	2011
Clathrin and AP-1 regulate apical polarity and lumen formation during C. elegans tubulogenesis. Development (Cambridge, England)\| PubMed ID: 22535410	2012
Intracellular lumen extension requires ERM-1-dependent apical membrane expansion and AQP-8-mediated flux. Nature cell biology\| PubMed ID: 23334498	2013
Vesicular sorting controls the polarity of expanding membranes in the C. elegans intestine. Worm\| PubMed ID: 24058862	2013
RNAi-based biosynthetic pathway screens to identify in vivo functions of non-nucleic acid-based metabolites such as lipids. Nature protocols\| PubMed ID: 25837419	2015
Corrigendum: RNAi-based biosynthetic pathway screens to identify in vivo functions of non-nucleic acid-based metabolites such as lipids. Nature protocols\| PubMed ID: 26020617	2015
Facilitation of Endosomal Recycling by an IRG Protein Homolog Maintains Apical Tubule Structure in Caenorhabditis elegans. Genetics\| PubMed ID: 27334269	2016

Verena Gobel

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