Name: 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
Uploaded: 2017-10-03T14:00:00
Description: Watch this Scientific Journal Video about 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-fu

Vi takker M. Buechner (University of Kansas, Kansas, USA), K. Nehrke (University of Rochester Medical Center, Rochester, New York, USA) og byens Caenorhabditis genetik, finansieret af National Institutes of Health, kontoret for forskningsinfrastruktur Programmer (P40 OD010440). Dette arbejde blev støttet af tilskud NIH GM078653, MGH er 224570 og SAA 223809 at V.G.

1. mærkning af C. elegans Excretory kanalen af fluorescerende Fusion proteiner 14 Note: Se den ledsagende papir på tarm tubulogenesis 3 for mærkning af i situ antistof farvning procedurer kan tilpasses til ekskretionsorganerne kanalen. Se tabel 1 eksempler på molekyler vist sig nyttige til at visualisere C. elegans ekskretionsorganerne canal endo- og plasma membraner, tabel 2 eks...

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C. elegans genetiske alsidighed, gennemsigtighed, enkel krop plan og invariante celle lineage gør det en fremragende model for analyse af morfogenese. Denne protokol beskriver hvordan man kombinerer standard genetiske manipulationer og billedbehandling undersøgelser for at drage fordel af 2 micron tynd C. elegans ekskretionsorganerne kanaler for at studere polariseret membran og intracellulære lumen Biogenese i en enkelt celle tube.
Mærkning C. ele...

Alle indre organer er sammensat af rør, afgørende for deres mange forskellige funktioner, såsom transport og udveksling af gasser, væsker og næringsstoffer og udskillelsen af metaboliske affald. Deres polariseret karakter, med særskilte apikale og lumenal membraner, er tilpasset til disse bestemte funktioner, og mangler i Biogenese aspekt(er) endo- og plasma membran er en hyppig årsag til sygdom hos mennesker1,2. Fleste af rør Vaskulaturen og indre organer er flercellede og danne en lumen intercellularly; dog kan, encellede rør, der udgør lumen intracellulært, for eksempel repræsenterer så meget som 30 – 50% af menneskelige kapillær senge2. Polariseret membraner i multi- og encellede rør er ens i sammensætning, selv om deres microdomains kan variere baseret på tube's specifikke funktion (fx, ekskretionsorganerne canal canaliculi versus intestinal microvilli i Caenorhabditis elegans; Se ledsager papir på C. elegans intestinale tubulogenesis)3. Principperne for polariseret membran Biogenese og tubulogenesis er bevaret blandt metazoans, og en lignende molekylære maskineri dirigerer dem1,2,4.
C. elegans ekskretionsorganerne system består af fem celler: ekskretionsorganerne cellen (EF), kanal celle (DC), pore celle (PC) og to kirtel celler. Ablation af EF, DC eller PC forårsager væskeophobning i kroppen hulrum og dyr dø på en tidlig larve fase5. Intriguingly, disse tre encellede rør oprette deres lumen på tre forskellige måder: ved celle udhuling (EF); celle indpakning kombineret med autocellular junction dannelse (PC); og af celle indpakning kombineret med autofusion (DC); forskellige mekanismer af lumen morfogenese, der alle Fylogenetisk bevarede6,7. EF, beliggende på den venstre laterale side af den bageste svælg pære, sender ud to laterale udvidelser fra som de fire kanaler forgrene sig for at udvide Anteriort og posteriort (i både højre og venstre side) til spidsen af den orm næse og hale , henholdsvis (figur 1)5,6,8. EF strækker sig fra ca 1 µm til 2 x 1.000 µm, hvilket gør det til den største celle i dyret. På en subcellulært niveau er den ekskretionsorganerne kanalen en enkel tube, genereret fra en basal membran rettet mod pseudocoelom, og tunneled af en lumenal membran (endotube). Canal lumenal membranen forbinder til kanalen lumenal membran på sin kun intercellulære junction; kanalerne er ellers junctionless langs deres længder (figur 1). Ekskretionsorganerne canal lumenal membranen og dens submembraneous cytoskelet er apikale, defineret ved deres molekylære sammensætning, der minder om sammensætningen af den apikale membran og submembraneous cytoskelet flercellede rør, såsom tarmen, og af andre (fxflade) epitheler. Cytoplasmatisk organeller, inklusive endosomal vesikulær og andre (fxGolgi) endomembranes er fordelt langs længden af kanalen. Derudover er flere kanalikulær vesikler - enten tilsluttet lumenal membranen, og/eller indbyrdes forbundne eller isoleret - gevind gennem kanalen cytoplasma7,8,9,10 . Denne dynamiske plasma-membran/kanalikulær forbindelse yderligere udvider kanalens membran system og bidrager til begge lumen morfogenese og osmoregulering10. Ekskretionsorganerne kanalen består således næsten udelukkende af endo- og plasma membraner, give en fremragende model for analyse af polariseret membran Biogenese og regulering af endo-plasma membran interface. En dramatisk udvidelse af den apikale membran under kanalen morfogenese - i denne single-celle systemet sammenfaldende med lumen udvidelse – giver også mulighed for at analysere de arkitektoniske problemer som følge af behovet for at stabilisere og centrere en intracellulær lumenal membran . Denne protokol fokuserer på analyse af kanalen røret og lumen's strukturelle morfogenese og intracellulære membran dynamics kræves for denne proces og ikke på de signaler, der direkte celle bevægelser, der genererer EFS holdning i de ekskretionsorganerne system og konstruere dets indviklede forbindelser til andre cellulære elementer (gennemgik i6).
En yderligere fordel af C. elegans encellede canal system til analyse af polariseret membran og intracellulære lumen Biogenese er dens evne til at adskille, gennem udviklingsmæssige tid, generation af forskellige komponenter af dets membraner og knudepunkter. EF er født på tidspunktet for ventrale lukning og afregner ventro sideværts i svælget under midten af embryogenese5,6,8, hvor tid laterale canal udvidelse og forgrening forekomme. Dette er efterfulgt af anterior-posterior canal udvidelse under sent embryogenese, en proces, som fortsætter ind i stadiet L1-larver (figur 1). I en nyklækkede larve, den bageste kanalen tip når frem til cirka midten af ormen, fuldt udvide til halen slutningen af L1 scenen, hvorefter kanalen elongates sammen med orm8. Aktive canal vækst på en hastighed på over for dyrets vækst dermed ender på den første larve stadie, men yderligere vækst sker sideløbende med en vækst på hele dyret under de ekstra larve faser (L2-4). Denne indstilling giver mulighed for at analysere forskellige trin i de novo polariseret membran Biogenese uafhængigt af polariseret celledeling eller migration. Desuden, det tillader adskillelse af denne proces fra samling af knudepunkter (som forekommer i embryoet før lumen indledning); deres nøjagtige krav i membranen polarisering er stadig et åbent spørgsmål i feltet polaritet. Endelig adskiller det entydigt apikale fra basale membran ekspansion, sidstnævnte processen forud for tidligere i ekskretionsorganerne kanaler10. C. elegans ekskretionsorganerne kanalen model er derfor en særlig informativ supplement til den intestinale model, som deler en række af disse fordele til analyse af polariseret membran Biogenese men udfører det i flercellede omgivelser (jf. den ledsagende papir på tarm tubulogenesis3).
Selv om wild-type kanaler ultratynde tubuli i denne lille orm, kan deres lumen være visualized direkte af Nomarski optik i dette gennemsigtige dyr. Mutant cystisk kanalen morfologier kan faktisk karakteriseres i umærkede dyr ved hjælp af lav forstørrelse dissekere mikroskopi, som har været brugt med stor effekt i fremad genetiske skærme til at identificere gener involveret i tubulogenesis11. Forbedret visualisering af morfologi af kanaler og skelnen mellem deres polariseret membraner, cytoskeletal komponenter, forskellige intracellulære organeller og andre subcellulært strukturer, men kræver mærkning og højere magt fluorescerende dissekere og konfokal mikroskopi. Selv om kanaler fine struktur udgør en række vanskeligheder for mærkning og mikroskopi, membraner og subcellulært komponenter kan skelnes via de specifikke molekyler unikke for hvert rum, og dyr kan være sikkert monteret til mikroskopi, hvis visse forholdsregler er truffet for at undgå at indføre artefakter (Se protokollen og diskussion). Mærkning kan gøres af Immunhistokemi i faste enheder, eller ved at generere transgene orme at udtrykke fluorescerende fusion proteiner under deres egen kontrol eller ekskretionsorganerne canal-specifikke initiativtagere til i vivo billeddannelse. Denne protokol beskriver den sidstnævnte mærkning teknik (se den ledsagende papir på tarm tubulogenesis for antistof farvning3).
Evnen til at kombinere i vivo tab eller gevinst-af-funktion undersøgelser med i vivo billeddannelse analyse på det enkelt celle niveau i hele udvikling gør C. elegans ekskretionsorganerne kanalen en særlig stærk model for den molekylære og cellulære analyse af encellede tubulogenesis. Frem eller omvendt genetiske skærme kan udføres starter med en wild-type eller mærket transgene dyr til at identificere canal morfogenese fænotyper (for eksempel, cyster) og deres underliggende gendefekter. Alternativt, sådanne skærme kan starte med en mutant fænotype (fx, en cystisk kanalen) og identificere overspændingsbeskyttere eller smagsforstærkere af denne fænotype for at identificere gener, der funktionelt interagerer med genet forårsager mutant fænotype. Den genetiske defekt forårsager mutant fænotype kan fremkalde et tab (f.eks., via gen sletning) eller en gevinst (f.eks. via en aktiverende mutation eller via indførelse af overskydende gen kopier) af de undersøgte funktion. Videresende mutagenese eller systematisk RNAi skærme er uden forudfattede meninger om genfunktioner og til upartisk identificering af gener involveret i funktionen af interesse. Betragtning af tilgængeligheden af genome-wide RNAi fodring biblioteker, kan næsten hvert gen være let slået ned af RNAi i C. elegans, således at enhver enkelt gen af interesse eller enhver gruppe af gener (fxi målrettede skærme) kan også være hurtigt aftestede for deres virkning i en omvendt genetik tilgang. For at påvise en eventuel kombination af tilgange, vi her beskrive en målrettet RNAi interaktion skærm, begyndende med en gevinst af funktion cystisk ekskretionsorganerne canal mutant, mærket med cytoplasmatisk canal grøn fluorescerende proteiner (NGL). Mutant fænotype blev genereret af overekspression af erm-1, en yderst velbevarede C. elegans ortholog af familien membran-actin linker Ezrin-Radixin-Moesin (ERM), som har været impliceret i lumen morfogenese og membran organisationen i mange arter12. C. elegans ERM-1 lokaliserer til lumenal membraner af indre organer, såsom ekskretionsorganerne canal og tarmen, og der kræves til lumen dannelse i begge13. ERM-1 overekspression rekrutter overskydende aktin og vesikler til canal lumenal membran, stigende flux i lumen og generere en kort cystisk kanalen og en krympede lumenal membran med fortykket actin underuld9. Protokollen beskriver hvordan man kan generere transgene stammer med ekskretionsorganerne udtrykt canal mærket fusion proteiner (eller andre proteiner); Hvordan til at udføre målrettede RNAi skærme starter med disse stammer til at identificere modifikatorer for en canal fænotype; og hvordan man visuelt analysere resultaterne af sådanne skærme af fluorescens dissekere og konfokal mikroskopi, herunder enkle måder at kvantificere informative tubulogenesis fænotyper. Alternativ mærkning teknikker og detaljer af RNAi, justeret til de ofte dødbringende tubulogenesis gener, kan findes i det ledsagende dokument på tarm tubulogenesis3. Alle metoder kan anvendes i forskellige kombinationer for undersøgelse af andre spørgsmål på kanalen tubulogenesis.

<table border="0" cellpadding="0" cellspacing="0" width="983">
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			<td height="20" style="height:20px;width:811px;">Cloning</td>
			<td style="width:173px;"></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Plasmid pPD95.75</td>
			<td>Addgene</td>
			<td>Cat. No. 37464</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">PCR Kit</td>
			<td>Qiagen</td>
			<td>Cat. No. 27106</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Ligation kit</td>
			<td>New England Biolabs</td>
			<td>Cat. No. E2611L</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">DNA marker</td>
			<td>Thermo Scientific</td>
			<td>Cat. No. SM1331</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Agarose DNA grade</td>
			<td>Fisher Scientific</td>
			<td>Cat. No. BP164-100</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Competent cells</td>
			<td>New England Biolabs</td>
			<td>Cat. No. C2987H</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Tris</td>
			<td>Fisher Scientific</td>
			<td>Cat. No. BP154-1</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">EDTA</td>
			<td>Sigma</td>
			<td>Cat. No. ED-1KG</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Acetic acid</td>
			<td>Fisher Scientific</td>
			<td>Cat. No. A38S-500</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Ethidium bromide</td>
			<td>Fisher Scientific</td>
			<td>Cat. No. BP1302-10</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Equipments</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">PCR machine&#160;</td>
			<td>MJ Research</td>
			<td>Cat. No. PTG-200&#160;</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Centrifuge</td>
			<td>Eppendorf&#160;</td>
			<td>Cat. No. 5415C</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Water Bath</td>
			<td>Precision Scientific&#160;</td>
			<td>Cat. No. 666A3&#160;</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Gel running instrument</td>
			<td>Fisher Scientific</td>
			<td>Cat. No. 09-528-165</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Gel running power supply</td>
			<td>Fisher Scientific</td>
			<td>Cat. No. 45-000-465</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:216px;">Molecular Imager&#160;Gel Doc XR System</td>
			<td>Bio-Rad</td>
			<td>Cat. No. 1708195EDU</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Nanodrop Spectrophotometer</td>
			<td>Thermo Scientific</td>
			<td>Cat. No. ND1000</td>
			<td></td>
		</tr>
		<tr height="23">
			<td height="23" style="height:23px;">C. elegans related1</td>
			<td></td>
			<td></td>
			<td>1see reference27 for standard C. elegans culture and maintenance procedures.</td>
		</tr>
		<tr height="23">
			<td height="23" style="height:23px;">LB Medium and plates2</td>
			<td></td>
			<td></td>
			<td>2see reference24 for protocols.</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Tryptone&#160;</td>
			<td>Acros Organics</td>
			<td>Cat. no. 611845000</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Yeast Extract</td>
			<td>BD Biosciences</td>
			<td>Cat. no. 212750</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">NaCl</td>
			<td>Sigma</td>
			<td>Cat. no. S7653</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Bacto Agar&#160;</td>
			<td>BD Biosciences</td>
			<td>Cat. no. 214040</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Ampicillin</td>
			<td>Sigma</td>
			<td>Cat. no. A0116</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Tetracycline</td>
			<td>Fisher Scientific</td>
			<td>Cat. no. BP912</td>
			<td></td>
		</tr>
		<tr height="23">
			<td colspan="3" height="23" style="height:23px;">M9 Medium2</td>
			<td></td>
			<td></td>
			<td>2see reference24 for protocols.</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">NaCl</td>
			<td>Sigma</td>
			<td>Cat. no. S7653</td>
			<td></td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;">KH2PO4</td>
			<td>Sigma</td>
			<td>Cat. no. P0662</td>
			<td></td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;">Na2HPO4</td>
			<td>Sigma</td>
			<td>Cat. no. S7907</td>
			<td></td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;">MgSO4</td>
			<td>Sigma</td>
			<td>Cat. no. M2773</td>
			<td></td>
		</tr>
		<tr height="23">
			<td height="23" style="height:23px;">NGM plates 2</td>
			<td></td>
			<td></td>
			<td>2see reference24 for protocols.</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">NaCl</td>
			<td>Sigma</td>
			<td>Cat. no. S7653</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Peptone&#160;</td>
			<td>BD Biosciences</td>
			<td>Cat. no. 211677</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Tryptone&#160;</td>
			<td>Acros Organics</td>
			<td>Cat. no. 611845000</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Bacto Agar&#160;</td>
			<td>BD Biosciences</td>
			<td>Cat. no. 214040</td>
			<td></td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;">MgSO4</td>
			<td>Sigma</td>
			<td>Cat. no. M2773</td>
			<td></td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;">CaCl2</td>
			<td>Sigma</td>
			<td>Cat. no. C3881</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Cholesterol&#160;</td>
			<td>Sigma</td>
			<td>Cat. no. C8667</td>
			<td></td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;">K2HPO4&#160;</td>
			<td>Sigma</td>
			<td>Cat. no. P3786</td>
			<td></td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;">KH2PO4</td>
			<td>Sigma</td>
			<td>Cat. no. P0662</td>
			<td></td>
		</tr>
		<tr height="23">
			<td height="23" style="height:23px;">RNAi plates3</td>
			<td></td>
			<td></td>
			<td>3see reference60 for protocols.</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">NaCl</td>
			<td>Sigma</td>
			<td>Cat. no. S7653</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Peptone&#160;</td>
			<td>BD Biosciences</td>
			<td>Cat. no. 211677</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Tryptone&#160;</td>
			<td>Acros Organics</td>
			<td>Cat. no. 611845000</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Bacto Agar&#160;</td>
			<td>BD Biosciences</td>
			<td>Cat. no. 214040</td>
			<td></td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;">MgSO4</td>
			<td>Sigma</td>
			<td>Cat. no. M2773</td>
			<td></td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;">CaCl2</td>
			<td>Sigma</td>
			<td>Cat. no. C3881</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Cholesterol&#160;</td>
			<td>Sigma</td>
			<td>Cat. no. C8667</td>
			<td></td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;">K2HPO4&#160;</td>
			<td>Sigma</td>
			<td>Cat. no. P3786</td>
			<td></td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;">KH2PO4</td>
			<td>Sigma</td>
			<td>Cat. no. P0662</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">IPTG&#160;</td>
			<td>US Biological</td>
			<td>Cat. no. I8500</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Carbenicillin</td>
			<td>Fisher Scientific</td>
			<td>Cat. no. BP2648</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">NaOH</td>
			<td>Fisher Scientific</td>
			<td>Cat. no. SS266-1</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Sodium hypochlorite</td>
			<td>Fisher Scientific</td>
			<td>Cat. no. 50371500</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Bacteria</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">OP50 bacteria</td>
			<td colspan="2">CGC</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">HT115 bacteria</td>
			<td colspan="2">CGC</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Genome-wide RNAi libraries</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="23">
			<td height="23" style="height:23px;">Ahringer genome-wide RNAi feeding library (ref29,49)</td>
			<td>Source BioScience</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="23">
			<td height="23" style="height:23px;">C. elegans ORF-RNAi feeding library (ref50)</td>
			<td>Source BioScience</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Imaging related</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Lidocaine</td>
			<td>MP Biomedicals,LLG</td>
			<td>Cat. no. 193917</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Materials</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Vacuum Grease Silicone</td>
			<td>Beckman</td>
			<td>Cat. no. 335148</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Microscope slides&#160;</td>
			<td>Fisher Scientific</td>
			<td>Cat. no. 4448</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Microscope coverslips (22&#215;22-1&#65289;</td>
			<td>Fisher Scientific</td>
			<td>Cat. no. 12-542-B</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Tissue culture plate, 6 well&#160;</td>
			<td>Corning Inc.</td>
			<td>Cat. no. 08-772-33</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Equipment</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">SMZ-U dissecting microscope (Nikon)</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:811px;">SZX12 dissecting microscope (Olympus), equipped with a high-power stereo fluorescence attachment (Kramer Scientific), CCD camera with Q capture software and X-Cite fluorescent lamp (Photonic Solutions).</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">TCS SL Laser-scanning confocal microscope (Leica Microsystem)</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">C2 laser-scanning confocal mounted on an ECLIPSE Ti-E inverted microscope (Nikon)</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
	</tbody>
</table>

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

De fire C. elegans ekskretionsorganerne kanaler er smalle rør udvidet gennem længden af dyret fra en enkelt celle, med næsten lige så langt udvidede intracellulære endotubes, der opbygger og stabilisere lumen med en membran og submembraneous cytoskelettet apikale karakter. Cellen ekskretionsorganerne udvider sin længde ca 2.000 gange til at generere disse kanaler, hvilket gør denne model enestående i vivo -vurdering af de novo polariseret membran Biogenese, intracellulære lumen morfogenese og encellede tubulogenesis. Protokollen præsenteres her viser, hvordan man kombinerer standard mærkning, gevinst og tab-af-funktion genetiske eller RNA-interferens (RNAi)-, og mikroskopisk tilgange til at bruge denne model til visuelt dissekere og funktionelt analyserer disse processer på molekylært niveau. Som et eksempel på en mærkning tilgang protokollen skitserer generation af transgene dyr med fluorescerende fusion proteiner til live analyse af tubulogenesis. Som et eksempel på en genetisk tilgang fremhæver det hovedpunkterne i en visuel RNAi-baserede interaktion skærm designet til at ændre en gevinst af funktion cystisk kanalen fænotype. De specifikke metoder beskrevet sådan: etiket og visualisere kanalerne ved at udtrykke fluorescerende proteiner; konstruere en målrettet RNAi bibliotek og strategize RNAi screening for Molekylær analyse af canal morfogenese; visuelt vurdere ændringer af canal fænotyper; score dem ved at dissekere Fluorescens mikroskopi; karakterisere subcellulært canal komponenter på højere opløsning af konfokalmikroskopi; og kvantificere visuelle parametre. Fremgangsmåde er nyttig for den efterforsker, der er interesseret i at drage fordel af C. elegans ekskretionsorganerne kanalen for at identificere og karakterisere gener involveret i de Fylogenetisk bevaret intracellulære lumen og encellede Tube morfogenese.

Denne protokol beskriver hvordan man bruger C. elegans ekskretionsorganerne kanaler til visuelt og molekylært analysere encellede tubulogenesis og intracellulære lumen morfogenese i en enkelt celle. Under deres udvidelse fra tidspunktet for midten embryogenese til voksenalderen, de fire ekskretionsorganerne kanaler fortsætter med at udvide deres basolateral og apikale/lumenal membraner sammen med deres kanalikulær og endosomal endomembrane system, der giver en enestående mod...

Watch this Scientific Journal Video about 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-fu

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

C. elegans Excretory kanalen som en Model for intracellulære Lumen morfogenese og In Vivo polariseret membran Biogenese i en enkelt celle: mærkning af normal god landbrugspraksis-fusioner, RNAi interaktion skærm og billedbehandling

C. elegans ekskretionsorganerne kanalen er en unik én celle model for den visuelle i vivo analyse af de novo polariseret membran Biogenese. Denne protokol beskriver en kombination af standard genetiske/RNAi og imaging tilgange, kan tilpasses til identifikation og karakterisering af molekyler lede encellede tubulogenesis og apikale membran og lumen Biogenese.

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 ...

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JoVE Journal

Developmental Biology

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

Analyzing In Vivo Cell Migration using Cell Transplantations and Time-lapse Imaging in Zebrafish Embryos

Analyzing In Vivo Cell Migration using Cell Transplantations and Time-lapse Imaging in Zebrafish Embryos

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