Name: nanogold labeling of the yeast endosomal system for ultrastructural analyses
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Description: Watch this Scientific Journal Video about Nanogold Labeling of the Yeast Endosomal System for Ultrastructural Analyses at JoVE.com

Forfatterne takker René Scriwanek for assistance med udarbejdelse af figuren. FR er støttet af ECHO (700.59.003), ALW Open Program (821.02.017 og 822.02.014) DFG-NWO samarbejde (DN82-303) og ZonMw VICI (016.130.606) tilskud.

1.. Sfæroblast Forberedelse <ol><li> Inkuber gær natten over ved 30 ° C i 10 ml af det passende medium bestemmes af udformningen af ​​forsøget. </li><li> Dagen efter, at måle den optiske densitet af kulturen ved 600 nm (OD600) ved anvendelse af et fotometer, Fortynd celler i det samme kulturmedium til en OD600 på 0,2-0,4, og dyrke dem i en eksponentiel vækstfase, medmindre udformningen af eksperiment kræver en anden tilstand. Celler er i den eksponentielle fase, når kulturen har en <...

<ol>
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Immuno-elektron-mikroskopi er en teknik, der gør det muligt at kombinere lokalisering af proteiner med ultrastrukturelle opløsning af luftfartsselskaberne og organeller, hvor disse proteiner bor. Dette er især vigtigt, når man studerer gær endosomale system, fordi dens rum vises som pointere strukturer i fluorescens mikroskopi. Det er derfor vanskeligt at skelne dem. Af denne grund er anvendelsen af ​​en probe, der kan påvises ved EM og indtaste endocytiske vej på en tidsafhængig måde er afgørende for spec...

Det endosomal er et stort membran sortering apparat, der spiller flere vigtige cellulære roller, herunder handel med lysosomale enzym sortering receptorer og genbrug af plasmamembranen (PM) receptorer 1,2. Endosomes er opdelt i tre forskellige rum, dvs. de tidlige endosomes (EE), den afdøde endosomes (LE) og genbrug endosomes. Denne klassifikation er baseret på den tid det tager for endocytose materiale til at nå dem, om specifikke markørproteiner og på deres morfologi. Membraner, dvs protein-og lipid-dobbeltlag, internaliserede fra PM kan enten leveres til lysosomer via endosomes til nedbrydning eller genbruges tilbage. Membraner er også transporteres til endosomes fra Golgi og på samme måde, enten fortsætte til lysosomer eller blive hentet tilbage til Golgi. Desuden kan proteiner sorteres i luminale vesikler spirende indad fra den endosomale begrænsende membran, en proces, der fører til dannelsen afen underkategori af LE, de multivesikulære organer. Gæren endosomal er relativt mindre kompleks end den ene af høje eukaryote celler. Gær endosomer er opdelt i EE og LE. I modsætning til pattedyrsceller de ikke indeholder genanvendelse endosomer men også vævsspecifikke lysosom-relaterede organeller. Derfor har de et mindre komplekst netværk af ruter endosomale menneskehandel 3,4. Gær Derfor har repræsenteret, og repræsenterer stadig en fordelagtig eksperimentelt system til at studere nogle af de principper, membran trafik i endosomale system. Denne fordel understreges af det faktum, at mange gener involveret i den endosomale veje er indledningsvis isoleret med genetiske skærme i gær 5.. Mens gær endosomale system vildtype og mutant celler er blevet grundigt undersøgt ved hjælp af biokemiske og fluorescensmikroskopi tilgange har sin undersøgelse på det ultrastrukturelle niveau kun væretminimal. Morfologiske analyser er særligt relevante i gær, fordi de fleste af de endosomale organeller opdages så pointere strukturer ved fluorescens mikroskopi, som gør svært utvetydig identifikation 6. Desværre er det kun et begrænset antal antisera anerkendelse gær endosomale proteinmarkører arbejder i immuno-elektron-mikroskopi (IEM) præparater 7-10. For nogle proteiner, er dette problem blevet omgået ved den endogene tagging af genet af interesse, og anvendelsen af et antistof, der genkender tag for at opdage det 7,11,12. Men ofte proteiner påvises ved IEM på grund af deres lave ekspressionsniveauer. Deres overekspression er ikke en løsning, fordi denne tilgang kan fremkalde mis-lokaliseringer og / eller ændringer i organel morfologi / funktioner. Mærkningen af ​​endocytiske rum med en probe påvises ved elektronmikroskopi EM er således en effektiv løsning. Dette er en optimal løsning, især hvis PROBE går ind i endocytiske rute i en tidsafhængig måde, som gør det muligt at vide, hvornår det vil markere et bestemt organel 6. Optagelsen af positivt ladede nanogold ved gærsfæroplaster (dvs.. Gær, hvor cellevæggen er blevet enzymatisk fjernet) har med succes været anvendt til at identificere gær endosomale segmenter 10. Disse partikler stærkt binder til negativt ladede lipider, som udgør de biologiske membraner. Således positivt ladede nanogold associerer med PM, trænger cellen ved endocytose og passerer gennem EE og LE, før de når vacuole. Disse små guldpartikler dog ikke har en passende størrelse til at blive set af EM. At gøre dem synlige, kan deres størrelse udvides ved kemiske reaktioner, der fører til aflejring af sølv eller guld omkring guld sonden 13-15. Vi har udviklet og anvendt med succes en IEM tilgang baseret på Tokuyasu metode til at udføre subcellulærtlokalisering undersøgelser 8,16. Denne metode giver udfører immunogold mærkning på gærpræparater med en fremragende opløsning på morfologi 8,17-24. Vi har også etableret en procedure, der kombinerer denne IEM protokol med nanogold mærkning af gær endosomale systemet rummene 6. Ved hjælp af denne tilgang, vi har morfologisk karakteriseret forskellige underklasser af endosomes og ultrastrukturelt undersøgte mutanter med en endosomal handel defekt 6,25. Desuden har vi vist, at denne nanogold mærkning kan kombineres med immunogold labelings giver mulighed for at undersøge fordelingen af ​​et protein af interesse på de forskellige endosome subpopulationer. Her præsenterer vi, hvordan mærkningen af ​​gær endosomale-system med positivt ladede nanogold er praktisk udført.

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nanogold labeling of the yeast endosomal system for ultrastructural analyses

Endosomer er en af ​​de større membran sortering checkpoints i eukaryote celler, og de regulerer genanvendelse eller destruktion af proteiner meste fra plasmamembranen og Golgi. Som følge den endosomale system spiller en central rolle i at opretholde cellehomeostase, og mutationer i gener hører til dette netværk af organeller forbundet ved vesikulær transport forårsage alvorlige patologier, herunder cancer og neurobiologiske lidelser. Det er derfor af afgørende betydning for at forstå mekanismerne bag biogenese og organisering af endosomale system. Gæren Saccharomyces cerevisiae har været afgørende i denne opgave. Til specifikt mærke og analyserer på det ultrastrukturelle niveau endosomale system af denne model organisme, præsenterer vi her en detaljeret protokol for positivt ladede nanogold optagelse af spheroplaster efterfulgt af visualisering af disse partikler gennem en sølv ekstraudstyr reaktion. Denne metode er også et værdifuldt forl for den morfologiske undersøgelse af mutanter med defekter i endosomal menneskehandel. Desuden er det ikke kun gælder for ultrastrukturelle undersøgelser, men det kan også kombineres med immunguld labelings for protein localization undersøgelser.

Ifølge den præsenterede protokol kan morfologi gær endosome systemet være adgang ved transmission EM. Figur 1 viser forskellige typer af endosomale rum, der er blevet nået, og derfor er mærket med nanogold. Den sølvforstærket nanogold kan tydeligt ses som elektrondonorer tætte partikler. De optimale indstillinger bestemt for sølv-forstærkning reaktion tillader der guldpartikler i en homogen størrelse mellem 5 og 15 nm, som ikke interfererer med ultrastruktur gærcellen. Plasmamembranen samt ...

Watch this Scientific Journal Video about Nanogold Labeling of the Yeast Endosomal System for Ultrastructural Analyses at JoVE.com

Nanogold Labeling of the Yeast Endosomal System for Ultrastructural Analyses

Nanogold Mærkning af Gær endosomale System for Ultrastrukturelle Analyser

Gær, Saccharomyces cerevisiae, har været en vigtig model organisme for at identificere og studere gener, der regulerer den biogenese og funktioner endosomale system. Her præsenterer vi en detaljeret protokol for særlig mærkning af de endosomale rum til ultrastrukturelle studier.

Endosomes are one of the major membrane sorting checkpoints in eukaryotic cells and they regulate recycling or destruction of proteins mostly from the ...

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JoVE Journal

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