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Biochemistry

Analisi dell'endocitosi assorbimento e trasporto retrogrado alla rete Trans-Golgi utilizzando Nanobodies funzionalizzati in cellule coltivate

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

Summary

Trasporto retrogrado delle proteine di superficie delle cellule di Golgi è essenziale per mantenere l’omeostasi della membrana. Qui, descriviamo un metodo per analizzare biochimicamente trasporti di superficie–Golgi cellulare di proteine ricombinanti utilizzando nanobodies funzionalizzati in cellule HeLa.

Abstract

Trasporto di proteine e membrane dalla superficie delle cellule di Golgi e oltre è essenziale per l’omeostasi, organello identità e fisiologia. Per studiare il traffico della proteina retrograda, abbiamo recentemente sviluppato un versatile basato su nanobody toolkit per analizzare il trasporto dalla superficie delle cellule al complesso di Golgi, mediante imaging cellulare fisso e dal vivo, da microscopia elettronica, o biochimicamente. Abbiamo progettato funzionalizzati anti-verde proteina fluorescente (GFP) nanobodies — raccoglitori di proteine ad alta affinità, monomerico e piccoli — che possono essere applicati alle linee cellulari che esprimono le proteine di membrana di interesse con una parte extracellulare di GFP. Derivatizzato nanobodies associato ai reporter GFP sono specificamente interiorizzato e trasportato sulle spalle lungo rotte ordinamento il reporter. Nanobodies sono stati funzionalizzati con fluorofori seguire trasporto retrogrado mediante microscopia a fluorescenza e immagini, con ascorbato perossidasi 2 (APEX2) per studiare la localizzazione ultrastrutturale dei reporter-nanobody complessi da elettrone dal vivo microscopia e con motivi di solfatazione (TS) di tirosina per valutare la cinetica dell’arrivo di trans-Golgi network (TGN). In questo articolo metodologico, descriviamo la procedura generale per batterico esprimere e purificare nanobodies funzionalizzati. Vi illustriamo l’uso potente del nostro strumento utilizzando il nanobodies mCherry – e TS-modificato per analizzare l’assorbimento endocitosi e TGN arrivo di proteine cargo.

Introduction

Retrogrado traffico di proteine e di lipidi dalla superficie delle cellule ai vari compartimenti intracellulari è cruciale per il mantenimento dell’omeostasi di membrana per controbilanciare la secrezione e riciclare componenti di anterograda trasporto macchinari1 , 2. a seguito di internalizzazione tramite endocitosi clatrina-dipendente o – indipendente, carico della proteina e del lipido innanzitutto popolare presto endosomes da dove si trovano ulteriori reindirizzamento sia lungo il sistema endolisosomiale, riciclato alla membrana plasmatica, o mirati alla rete trans-Golgi (TGN). Riciclaggio da endosomi e/o di superficie delle cellule a livello di TGN è parte del ciclo funzionale di un numero di recettori transmembrana carico anterograda, quali i recettori di mannosio-6-fosfato catione-dipendenti e indipendenti del catione (CDMPR e CIMPR) consegna recentemente sintetizzato idrolasi lisosomiale da TGN tardi endosomi e lisosomi3,4,5, sortilina e SorLA6,7e Wntless (WLS) trasporto di ligandi di Wnt alla superficie delle cellule 8 , 9 , 10 , 11. altre proteine Estratto torna a TGN sono TGN46 e sue isoforme correlate12,13,14, Rullanti ( N– ethylmaleimide-sensibili fusione fattore allegato recettori solubili) 15 , 16 , 17, precursore dell’amiloide (APP) di proteina18,19, anchilosi progressiva (ANK) proteina20, trasportatori metallici quali ATP7A/B o DMT121,22e del transmembrane elaborazione enzimi tra cui carbossipeptidasi D, furin o BACE123,24,25. Oltre a queste proteine endogene, tossine batteriche e pianta (ad es., tossina Shiga e colera, ricina e abrina) dirottare macchinari di trasporto retrogrado per raggiungere il pronto soccorso per dislocazione nel cytosol26,27, 28,29.

Al fine di analizzare direttamente il traffico retrograda, precedentemente abbiamo sviluppato un kit di strumenti basati su nanobody per etichettare e seguire le proteine cargo dalla superficie delle cellule per compartimenti intracellulari30. Nanobodies rappresentano una nuova famiglia di proteine leganti derivati da omodimeriche pesante-catena-solo gli anticorpi (hcAbs) che si presentano naturalmente in camelidi e pesci cartilaginei31,32. Essi costituiscono il dominio variabile di catene pesanti (VHH) di hcAbs e presentano molti vantaggi sopra gli anticorpi convenzionali (ad es., IgG): sono monomerici, piccolo (~ 15 kDa), altamente solubile, privo di legami bisolfurico, può essere espresso batterico e selezionato per legame ad alta affinità33,34,35,36. Per rendere il nostro strumento di nanobody versatile e ampiamente applicabili, abbiamo impiegato nanobodies funzionalizzati anti-GFP per le proteine di superficie-etichetta e pista con GFP al loro dominio extracellulare/lumenal etichettate. Dalla funzionalizzazione di nanobodies con mCherry, ascorbato perossidasi 2 (APEX2) trasporto retrogrado37, o sequenze di solfatazione (TS) tirosina di proteine transmembrana carico bonafide può essere analizzato da uno fisso e live imaging cellulare, di la microscopia elettronica, o biochimicamente. Poiché la solfatazione di tirosina mediata da tyrosylprotein sulfotransferasi (TPST1 e TPST2) è una modificazione post traduzionale limitato a trans-Golgi/TGN, possiamo studiare direttamente trasporti e cinetica di proteine di interesse dalla superficie delle cellule a questo intracellulare Golgi vano38,39,40.

In questo articolo di metodi, descriviamo la facilità di produzione di nanobodies funzionalizzati (VHH-2xTS, – APEX2, – mCherry e derivati), adatto per un numero di applicazioni per analizzare il trasporto retrogrado in cellule di mammifero30. Ci concentriamo principalmente sull’uso di TS per volta il sito nanobody per l’analisi del traffico intracellulare dalla superficie delle cellule al comparto della solfatazione.

Protocol

1. batterica trasformazione con Nanobodies funzionalizzati Nota: Questo protocollo è stato ottimizzato per l’espressione, la purificazione e l’analisi di nanobodies funzionalizzati anti-GFP come descritto in precedenza30. Derivatizzazione con altre parti di proteine potrebbero essere necessarie modifiche del presente protocollo standard. Scongelare i batteri chemocompetent (~ 100 µ l) adatti per l’espressione della proteina (ad es., Esch…

Representative Results

Per studiare il trasporto retrogrado proteina per varie destinazioni intracellulare, recentemente abbiamo stabilito uno strumento anti-GFP nanobody per etichettare e seguire le proteine di fusione ricombinante dalla superficie cellulare30. Qui, dimostriamo la produzione batterica di tali derivatizzati nanobodies e dimostrare la loro applicazione per studiare l’assorbimento endocitosi di microscopia di fluorescenza e immunoblotting, nonché il loro uso di investigar…

Discussion

Nanobodies rappresentano una classe emergente di proteina legante ponteggi con molti vantaggi rispetto ai convenzionali anticorpi: sono piccole, stabile, monomerico, possono essere selezionati per legami disolfuro ad alta affinità e mancanza33,35, 44 , 45. sono utilizzati in numerose applicazioni, come nei sistemi di coltura delle cellule e negli organismi in biologia dello sviluppo<sup class=…

Disclosures

The authors have nothing to disclose.

Acknowledgements

Questo lavoro è stato supportato da Grant 31003A-162643 di Swiss National Science Foundation. Ringraziamo Nicole Beuret e Biozentrum Imaging Core Facility (IMCF) per il supporto.

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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