JoVE Journal > Biochemistry
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Bioquímica

Analyse av Endocytic opptak og retrograd Transport til Trans-Golgi nettverket med Functionalized Nanobodies i kulturperler celler

Published: February 21, 2019
doi:
10.3791/59111
,
1Biozentrum,University of Basel

Summary

Retrograd transport av proteiner fra celleoverflaten til Golgi er avgjørende for å opprettholde membran homeostase. Her beskriver vi en metode for å analysere biokjemisk cellen overflaten til Golgi transport av rekombinante proteinene bruke functionalized nanobodies i HeLa celler.

Abstract

Transport av proteiner og membraner fra celleoverflaten av Golgi og utover er nødvendig for homeostase, organelle identitet og fysiologi. For å studere retrograd protein trafikk, har vi nylig utviklet en allsidig nanobody-baserte verktøykasse for å analysere fra celleoverflaten transport til Golgi komplekset, enten ved faste og levende celle imaging elektronmikroskop, eller biokjemisk. Vi utviklet functionalized anti-grønn fluorescerende protein (GFP) nanobodies-liten, monomerisk, høy affinitet protein bindemidler, som kan brukes på linjer uttrykke membran proteiner av interesse med en ekstracellulære GFP moiety. Derivatized nanobodies bundet til GFP journalister er spesielt internalisert og transportert piggyback langs journalister sortering ruter. Nanobodies var functionalized med fluorophores å følge retrograd transport av fluorescens mikroskopi og live bildebehandling, med ascorbate peroxidase 2 (APEX2) for å undersøke den ultrastructural lokaliseringen av reporter-nanobody komplekser av elektron mikroskopi, og med tyrosin sulfation (TS) motiver å vurdere kinetics trans-Golgi nettverk (TGN) ankomst. I denne metodologiske artikkelen skissere vi de generelle fremgangsmåten for å bacterially express og rense functionalized nanobodies. Vi illustrerer kraftig bruk av våre verktøy bruker mCherry – og TS-endret nanobodies å analysere endocytic opptak og TGN ankomsten av Last proteiner.

Introduction

Retrograd trafikk proteiner og lipider fra celleoverflaten til ulike intracellulær rom er avgjørende for vedlikehold av membran homeostase motvekt sekresjon og resirkulere komponenter i anterograd transport machineries1 , 2. etter internalisering via clathrin-avhengige eller -uavhengig endocytose, protein og lipid Last først fylle tidlig endosomes fra der de er ytterligere Omdirigert enten langs endo-lysosomale systemet, resirkulert til plasma membranen, eller målrettet trans-Golgi nettverket (TGN). Resirkulering fra endosomes og/eller celleoverflaten til TGN er en del av funksjonelle syklusen av en rekke anterograd transmembrane Last reseptorer, som kasjon-avhengig og uavhengig av cation mannose-6-fosfat receptors (CDMPR og CIMPR) levere nylig syntetisert lysosomale hydrolases fra TGN sent endosomes og lysosomer3,4,5, sortilin og SorLA6,7og Wntless (WLS) transport Wnt ligander til celleoverflaten 8 , 9 , 10 , 11. andre proteiner hentet tilbake til TGN er TGN46 og dens relaterte isoformene12,13,14, snarer (løselig N– ethylmaleimide-sensitive fusion faktor vedlegg reseptorer) 15 , 16 , 17, amyloid forløper protein (APP)18,19, progressiv ankylosis (ANK) protein20, metall transportører som ATP7A/B eller DMT121,22, og transmembrane behandler enzymer inkludert karboksypeptidase D, furin eller BACE123,24,25. Bortsett fra disse endogene proteiner kapre bakteriell og plante giftstoffer (f.eks Shiga og kolera gift, Risin og abrin) retrograd transport machineries å nå ER for retrotranslocation i stoffer26,27, 28,29.

For å direkte analysere retrograd trafikk, har vi utviklet et nanobody-baserte verktøysett for å merke og følg Last proteiner fra celleoverflaten til intracellulær rom30. Nanobodies representerer en ny familie av protein bindemidler avledet fra homodimeric tunge-kjede-bare antistoffer (hcAbs) som naturlig oppstår i camelids og cartilaginous fisker31,32. De utgjør variabel tunge-kjeden domenet (VHH) av hcAbs og har mange fordeler fremfor konvensjonelle antistoffer (f.eks IgGs): de er monomerisk, liten (~ 15 kDa) svært løselig, uten disulfide obligasjoner, kan bacterially uttrykt og valgt for høy affinitet bindende33,34,35,36. For å gjøre våre nanobody verktøyet allsidig og bredt aktuelt, ansatt vi functionalized anti-GFP nanobodies overflaten-etikett og spore proteiner merket med GFP på deres ekstracellulære/lumenal domene. Ved functionalization av nanobodies med mCherry, ascorbate peroxidase 2 (APEX2)37eller tyrosin sulfation (TS) sekvenser, retrograd transport av bonafide transmembrane Last proteiner kan analyseres av enten fast og lever celle bildebehandling, av elektronmikroskop, eller biokjemisk. Siden tyrosin sulfation formidlet av tyrosylprotein sulfotransferases (TPST1 og TPST2) er en posttranslational endring begrenset til trans-Golgi/TGN, kan vi direkte studere transport og kinetics proteiner av interesse fra celleoverflaten til dette intracellulær Golgi kupé38,39,40.

I denne metoder artikkelen beskriver vi enkel produksjon av functionalized nanobodies (VHH-2xTS-APEX2, – mCherry og derivater) egnet for en rekke programmer til å analysere retrograd transport i pattedyrceller30. We fokusere hovedsakelig på bruk av TS området endret nanobody for analyse av intracellulær trafikk fra celleoverflaten sulfation-rommet.

Protocol

1. bakteriell transformasjon med Functionalized Nanobodies Merk: Denne protokollen er optimalisert for uttrykket, rensing og analyse av functionalized anti-GFP nanobodies som beskrevet tidligere30. Derivatization med andre protein moieties kreve endring av denne standardprotokollen. Tine chemocompetent bakterier (~ 100 µL) egnet for protein uttrykk (f.eks Escherichia coli Rosetta BL21 (DE3) celler) ved å plassere dem på isen.<s…

Representative Results

Undersøke retrograd protein transport til ulike intracellulær destinasjoner, har vi nylig etablert en anti-GFP nanobody-basert verktøy for å merke og følg rekombinant fusion proteiner fra cellen overflaten30. Her viser vi bakteriell produksjonen av slike derivatized nanobodies og demonstrere programmet å studere endocytic opptak av fluorescens mikroskopi og immunoblotting, i tillegg til deres bruk å undersøke TGN ankomst av sulfation analyse. Sistnevnte pro…

Discussion

Nanobodies representerer en ny klasse av protein dokumentordner stillaser med mange fordeler fremfor konvensjonelle antistoffer: de er små, stabil, monomerisk, kan velges for høy affinitet og mangel disulfide obligasjoner33,35, 44 , 45. de brukes i en rekke programmer, slik som i celle kultur systemer og organismer utviklingsbiologi46,47</s…

Declarações

The authors have nothing to disclose.

Acknowledgements

Dette arbeidet ble støttet av Grant 31003A-162643 av Swiss National Science Foundation. Vi takker Nicole Beuret og den Biozentrum Imaging Core anlegg (IMCF) for støtte.

Materials

Anti-GFP antibody Sigma-Aldrich 118144600001 Product is distributed by Sigma-Aldrich, but manufactured by Roche
Anti-His6 antibody Bethyl Laboratories A190-114A
Anti-actin antibody EMD Millipore MAB1501
Goat anti-rabbit HRP Sigma-Aldrich A-0545
Goat anti-mouse HRP Sigma-Aldrich A-0168
4',6-diamidino-2-phenylindole (DAPI) Sigma-Aldrich D9542 dissolved in 1 x PBS/1%BSA
Dimethyl sulfoxide (DMSO) Applichem A3672
D-biotin Sigma-Aldrich B4501 dissolved in sterile 500 mM NaH2PO4 or DMSO
5-aminolevuilnic acid (dALA) hydrochloride Sigma-Aldrich A3785 dissolved in sterile water
DNase I Applichem A3778 dissolved in sterile water
Lysozyme Sigma-Aldrich 18037059001 Product is distributed by Sigma-Aldrich, but manufactured by Roche
Brefeldin A (BFA) Sigma-Aldrich B5936
Puromycin Invivogen ant-pr-1
Penicillin/Streptomycin Bioconcept 4-01F00-H
L-glutamine Applichem A3704
Dulbecco’s modified Eagle’s medium (DMEM) Sigma-Aldrich D5796
Fetal calf serum (FCS) Biowest S181B-500
Sulfur-35 as sodium sulfate Hartmann Analytics ARS0105 Product contains 5 mCi
Earle's balanced salts Sigma-Aldrich E6267
MEM amino acids (50 x) solution Sigma-Aldrich M5550
MEM vitamin solution (100 x) Sigma-Aldrich M6895
cOmplete, Mini Protease inhibitor cocktail Sigma-Aldrich 11836153001 Product is distributed by Sigma-Aldrich, but manufactured by Roche
Isopropyl-β-D-thiogalactopyranosid (IPTG) Applichem A1008 dissolved in sterile water, stock is 1 M
Carbenicillin disodium salt Applichem A1491 dissolved in sterile water, stock is 100 mg/mL
Kanamycin sulfate Applichem A1493 dissolved in sterile water, stock is 100 mg/mL
Coomassie-R (Brilliant Blue) Sigma-Aldrich B-0149
Paraformaldehyde (PFA) Applichem A3813
Bovine serum albumin (BSA) Sigma-Aldrich A2153
Fluoromount-G Southern Biotech 0100-01
Ni Sepharose High Performance GE Healthcare 17-5268-01
His GraviTrap columns GE Healthcare GE11-0033-99
His buffer kit GE Healthcare GE11-0034-00
Disposable PD10 desalting columns GE Healthcare GE17-0851-01
Mini-Protean TGX gels, 4-20%, 15-well Bio-Rad 456-1096
Dulbecco’s phosphate buffered saline (DPBS) w/o Ca2+/Mg2+ Sigma-Aldrich D8537
35-mm dishes Falcon 353001
6-well plates TPP 92406
Glass coverslips (No. 1.5H) VWR 631-0153
Phenylmethylsulfonyl fluoride (PMSF) Applichem A0999.0025 dissolved in 40% DMSO 60% isopropanol, stock in 500 mM
Tryptone Applichem A1553
Yeast extract Applichem A1552
Magnesium chloride hexahydrate Merck Millipore 105833 dissolved in sterile water, stock is 1 M
Calcium chloride dihydrate Merck Millipore 102382 dissolved in sterile water, stock is 1 M
Sodium chloride Merck Millipore 106404 dissolved in sterile water, stock is 5 M
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Tags

NanobodyEndocytic UptakeRetrograde TransportTrans-Golgi NetworkCell Surface ReceptorsFluorescence MicroscopyElectron MicroscopyTyrosine Sulfation

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Buser, D. P., Spiess, M. Analysis of Endocytic Uptake and Retrograde Transport to the Trans-Golgi Network Using Functionalized Nanobodies in Cultured Cells. J. Vis. Exp. (144), e59111, doi:10.3791/59111 (2019).
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