Analysis of Endocytic Uptake and Retrograde Transport to the Trans-Golgi Network Using Functionalized Nanobodies in Cultured Cells

Summary

Retrograde transport of proteins from the cell surface to the Golgi is essential to maintain membrane homeostasis. Here, we describe a method to biochemically analyze cell surface-to-Golgi transport of recombinant proteins using functionalized nanobodies in HeLa cells.

Abstract

Transport of proteins and membranes from the cell surface to the Golgi and beyond is essential for homeostasis, organelle identity and physiology. To study retrograde protein traffic, we have recently developed a versatile nanobody-based toolkit to analyze transport from the cell surface to the Golgi complex, either by fixed and live cell imaging, by electron microscopy, or biochemically. We engineered functionalized anti-green fluorescent protein (GFP) nanobodies — small, monomeric, high-affinity protein binders — that can be applied to cell lines expressing membrane proteins of interest with an extracellular GFP moiety. Derivatized nanobodies bound to the GFP reporters are specifically internalized and transported piggyback along the reporters' sorting routes. Nanobodies were functionalized with fluorophores to follow retrograde transport by fluorescence microscopy and live imaging, with ascorbate peroxidase 2 (APEX2) to investigate the ultrastructural localization of reporter-nanobody complexes by electron microscopy, and with tyrosine sulfation (TS) motifs to assess kinetics of trans-Golgi network (TGN) arrival. In this methodological article, we outline the general procedure to bacterially express and purify functionalized nanobodies. We illustrate the powerful use of our tool using the mCherry- and TS-modified nanobodies to analyze endocytic uptake and TGN arrival of cargo proteins.

Introduction

Retrograde traffic of proteins and lipids from the cell surface to various intracellular compartments is crucial for maintenance of membrane homeostasis to counterbalance secretion and to recycle components of anterograde transport machineries^1,2. Following internalization via clathrin-dependent or -independent endocytosis, protein and lipid cargo first populate early endosomes from where they are further redirected either along the endo-lysosomal system, recycled to the plasma membrane, or targeted to the trans-Golgi network (TGN). Recycling from endosomes and/or the cell surface to the TGN is part of the fun....

Protocol

1. Bacterial Transformation with Functionalized Nanobodies

NOTE: This protocol has been optimized for the expression, purification, and analysis of functionalized anti-GFP nanobodies as previously described³⁰. Derivatization with other protein moieties might require modification of this standard protocol.

1. Thaw chemocompetent bacteria (~100 µL) suited for protein expression (e.g., Escherichia coli Rosetta BL21 (DE3) cells) by pl.......

Discussion

Nanobodies represent an emerging class of protein binder scaffolds with many advantages over conventional antibodies: they are small, stable, monomeric, can be selected for high affinity and lack disulfide bonds^33,35,44,45. They are used in a number of applications, such as in cell culture systems and organisms in developmental biology^46,47

Materials

Name	Company	Catalog Number	Comments
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