Name: analysis of endocytic uptake and retrograde transport to the trans-golgi network using functionalized nanobodies in cultured cells
Uploaded: 2019-02-21T15:00:00
Description: Watch this Scientific Journal Video about Analysis of Endocytic Uptake and Retrograde Transport to the Trans-Golgi Network Using Functionalized Nanobodies in Cultured Cells at JoVE.com

This work was supported by Grant 31003A-162643 by the Swiss National Science Foundation. We thank Nicole Beuret and the Biozentrum Imaging Core Facility (IMCF) for support.

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.
<ol>
	<li>Thaw chemocompetent bacteria (~100 &#181;L) suited for protein expression (e.g., Escherichia coli Rosetta BL21 (DE3) cells) by pl...

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 bonds33,35,44,45. They are used in a number of applications, such as in cell culture systems and organisms in developmental biology46,47</...

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 machineries1,2. 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 functional cycle of a number of anterograde transmembrane cargo receptors, such as the cation-dependent and cation-independent mannose-6-phosphate receptors (CDMPR and CIMPR) delivering newly synthesized lysosomal hydrolases from the TGN to late endosomes and lysosomes3,4,5, sortilin and SorLA6,7, and Wntless (WLS) transporting Wnt ligands to the cell surface8,9,10,11. Other proteins retrieved back to the TGN are TGN46 and its related isoforms12,13,14, SNAREs (soluble N-ethylmaleimide-sensitive fusion factor attachment receptors)15,16,17, amyloid precursor protein (APP)18,19, progressive ankylosis (ANK) protein20, metal transporters such as ATP7A/B or DMT121,22, and transmembrane processing enzymes including carboxypeptidase D, furin or BACE123,24,25. Apart from these endogenous proteins, bacterial and plant toxins (e.g., Shiga and cholera toxin, ricin and abrin) hijack retrograde transport machineries to reach the ER for retrotranslocation into the cytosol26,27,28,29.
In order to directly analyze retrograde traffic, we have previously developed a nanobody-based toolkit to label and follow cargo proteins from the cell surface to intracellular compartments30. Nanobodies represent a new family of protein binders derived from homodimeric heavy-chain-only antibodies (hcAbs) that naturally occur in camelids and cartilaginous fishes31,32. They constitute the variable heavy-chain domain (VHH) of hcAbs and have many advantages over conventional antibodies (e.g., IgGs): They are monomeric, small (~15 kDa), highly soluble, devoid of disulfide bonds, can be bacterially expressed, and selected for high-affinity binding33,34,35,36. To make our nanobody tool versatile and broadly applicable, we employed functionalized anti-GFP nanobodies to surface-label and track proteins tagged with GFP at their extracellular/lumenal domain. By functionalization of nanobodies with mCherry, ascorbate peroxidase 2 (APEX2)37, or tyrosine sulfation (TS) sequences, retrograde transport of bonafide transmembrane cargo proteins can be analyzed by either fixed and live cell imaging, by electron microscopy, or biochemically. Since tyrosine sulfation mediated by tyrosylprotein sulfotransferases (TPST1 and TPST2) is a posttranslational modification restricted to the trans-Golgi/TGN, we can directly study transport and kinetics of proteins of interest from the cell surface to this intracellular Golgi compartment38,39,40.
In this methods article, we describe the ease of production of functionalized nanobodies (VHH-2xTS, -APEX2, -mCherry and derivatives) suited for a number of applications to analyze retrograde transport in mammalian cells30. We mainly focus on the use of TS site-modified nanobody for analysis of intracellular traffic from the cell surface to the compartment of sulfation.

<table >
	
		
		
		
		
	
	<tbody>
		<tr >
			<td >Anti-GFP antibody</td>
			<td >Sigma-Aldrich</td>
			<td >118144600001</td>
			<td >Product is distributed by Sigma-Aldrich, but manufactured by Roche</td>
		</tr>
		<tr >
			<td >Anti-His6 antibody</td>
			<td >Bethyl Laboratories</td>
			<td >A190-114A</td>
			<td ></td>
		</tr>
		<tr >
			<td >Anti-actin antibody</td>
			<td >EMD Millipore</td>
			<td >MAB1501</td>
			<td ></td>
		</tr>
		<tr >
			<td >Goat anti-rabbit HRP</td>
			<td >Sigma-Aldrich</td>
			<td >A-0545</td>
			<td ></td>
		</tr>
		<tr >
			<td >Goat anti-mouse HRP</td>
			<td >Sigma-Aldrich</td>
			<td >A-0168</td>
			<td ></td>
		</tr>
		<tr >
			<td >4&#39;,6-diamidino-2-phenylindole (DAPI)</td>
			<td >Sigma-Aldrich</td>
			<td >D9542</td>
			<td >dissolved in 1 x PBS/1%BSA</td>
		</tr>
		<tr >
			<td >Dimethyl sulfoxide (DMSO)</td>
			<td >Applichem</td>
			<td >A3672</td>
			<td ></td>
		</tr>
		<tr >
			<td >D-biotin</td>
			<td >Sigma-Aldrich</td>
			<td >B4501</td>
			<td >dissolved in sterile 500 mM NaH2PO4 or DMSO</td>
		</tr>
		<tr >
			<td >5-aminolevuilnic acid (dALA) hydrochloride</td>
			<td >Sigma-Aldrich</td>
			<td >A3785</td>
			<td >dissolved in sterile water</td>
		</tr>
		<tr >
			<td >DNase I</td>
			<td >Applichem</td>
			<td >A3778</td>
			<td >dissolved in sterile water</td>
		</tr>
		<tr >
			<td >Lysozyme</td>
			<td >Sigma-Aldrich</td>
			<td >18037059001</td>
			<td >Product is distributed by Sigma-Aldrich, but manufactured by Roche</td>
		</tr>
		<tr >
			<td >Brefeldin A (BFA)</td>
			<td >Sigma-Aldrich</td>
			<td >B5936</td>
			<td ></td>
		</tr>
		<tr >
			<td >Puromycin</td>
			<td >Invivogen</td>
			<td >ant-pr-1</td>
			<td ></td>
		</tr>
		<tr >
			<td >Penicillin/Streptomycin</td>
			<td >Bioconcept</td>
			<td >4-01F00-H</td>
			<td ></td>
		</tr>
		<tr >
			<td >L-glutamine</td>
			<td >Applichem</td>
			<td >A3704</td>
			<td ></td>
		</tr>
		<tr >
			<td >Dulbecco&#8217;s modified Eagle&#8217;s medium (DMEM)</td>
			<td >Sigma-Aldrich</td>
			<td >D5796</td>
			<td ></td>
		</tr>
		<tr >
			<td >Fetal calf serum (FCS)</td>
			<td >Biowest</td>
			<td >S181B-500</td>
			<td ></td>
		</tr>
		<tr >
			<td >Sulfur-35 as sodium sulfate</td>
			<td >Hartmann Analytics</td>
			<td >ARS0105</td>
			<td >Product contains 5 mCi</td>
		</tr>
		<tr >
			<td >Earle&#39;s balanced salts</td>
			<td >Sigma-Aldrich</td>
			<td >E6267</td>
			<td ></td>
		</tr>
		<tr >
			<td >MEM amino acids (50 x) solution</td>
			<td >Sigma-Aldrich</td>
			<td >M5550</td>
			<td ></td>
		</tr>
		<tr >
			<td >MEM vitamin solution (100 x)</td>
			<td >Sigma-Aldrich</td>
			<td >M6895</td>
			<td ></td>
		</tr>
		<tr >
			<td >cOmplete, Mini Protease inhibitor cocktail</td>
			<td >Sigma-Aldrich</td>
			<td >11836153001</td>
			<td >Product is distributed by Sigma-Aldrich, but manufactured by Roche</td>
		</tr>
		<tr >
			<td >Isopropyl-&#946;-D-thiogalactopyranosid (IPTG)</td>
			<td >Applichem</td>
			<td >A1008</td>
			<td >dissolved in sterile water, stock is 1 M</td>
		</tr>
		<tr >
			<td >Carbenicillin disodium salt</td>
			<td >Applichem</td>
			<td >A1491</td>
			<td >dissolved in sterile water, stock is 100 mg/mL</td>
		</tr>
		<tr >
			<td >Kanamycin sulfate</td>
			<td >Applichem</td>
			<td >A1493</td>
			<td >dissolved in sterile water, stock is 100 mg/mL</td>
		</tr>
		<tr >
			<td >Coomassie-R (Brilliant Blue)</td>
			<td >Sigma-Aldrich</td>
			<td >B-0149</td>
			<td ></td>
		</tr>
		<tr >
			<td >Paraformaldehyde (PFA)</td>
			<td >Applichem</td>
			<td >A3813</td>
			<td ></td>
		</tr>
		<tr >
			<td >Bovine serum albumin (BSA)</td>
			<td >Sigma-Aldrich</td>
			<td >A2153</td>
			<td ></td>
		</tr>
		<tr >
			<td >Fluoromount-G</td>
			<td >Southern Biotech</td>
			<td >0100-01</td>
			<td ></td>
		</tr>
		<tr >
			<td >Ni Sepharose High Performance</td>
			<td >GE Healthcare</td>
			<td >17-5268-01</td>
			<td ></td>
		</tr>
		<tr >
			<td >His GraviTrap columns</td>
			<td >GE Healthcare</td>
			<td >GE11-0033-99</td>
			<td ></td>
		</tr>
		<tr >
			<td >His buffer kit</td>
			<td >GE Healthcare</td>
			<td >GE11-0034-00</td>
			<td ></td>
		</tr>
		<tr >
			<td >Disposable PD10 desalting columns</td>
			<td >GE Healthcare</td>
			<td >GE17-0851-01</td>
			<td ></td>
		</tr>
		<tr >
			<td >Mini-Protean TGX gels, 4-20%, 15-well</td>
			<td >Bio-Rad</td>
			<td >456-1096</td>
			<td ></td>
		</tr>
		<tr >
			<td >Dulbecco&#8217;s phosphate buffered saline (DPBS) w/o Ca2+/Mg2+</td>
			<td >Sigma-Aldrich</td>
			<td >D8537</td>
			<td ></td>
		</tr>
		<tr >
			<td >35-mm dishes</td>
			<td >Falcon</td>
			<td >353001</td>
			<td ></td>
		</tr>
		<tr >
			<td >6-well plates</td>
			<td >TPP</td>
			<td >92406</td>
			<td ></td>
		</tr>
		<tr >
			<td >Glass coverslips (No. 1.5H)</td>
			<td >VWR</td>
			<td >631-0153</td>
			<td ></td>
		</tr>
		<tr >
			<td >Phenylmethylsulfonyl fluoride (PMSF)</td>
			<td >Applichem</td>
			<td >A0999.0025</td>
			<td >dissolved in 40% DMSO 60% isopropanol, stock in 500 mM</td>
		</tr>
		<tr >
			<td >Tryptone</td>
			<td >Applichem</td>
			<td >A1553</td>
			<td ></td>
		</tr>
		<tr >
			<td >Yeast extract</td>
			<td >Applichem</td>
			<td >A1552</td>
			<td></td>
		</tr>
		<tr >
			<td >Magnesium chloride hexahydrate</td>
			<td >Merck Millipore</td>
			<td >105833</td>
			<td>dissolved in sterile water, stock is 1 M</td>
		</tr>
		<tr >
			<td >Calcium chloride dihydrate</td>
			<td >Merck Millipore</td>
			<td >102382</td>
			<td>dissolved in sterile water, stock is 1 M</td>
		</tr>
		<tr >
			<td >Sodium chloride</td>
			<td >Merck Millipore</td>
			<td >106404</td>
			<td>dissolved in sterile water, stock is 5 M</td>
		</tr>
	</tbody>
</table>

analysis of endocytic uptake and retrograde transport to the trans-golgi network using functionalized nanobodies in cultured cells

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 &#8212; small, monomeric, high-affinity protein binders &#8212; 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&#39; 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.

To investigate retrograde protein transport to various intracellular destinations, we have recently established an anti-GFP nanobody-based tool to label and follow recombinant fusion proteins from the cell surface30. Here, we demonstrate the bacterial production of such derivatized nanobodies and demonstrate their application to study endocytic uptake by fluorescence microscopy and immunoblotting, as well as their use to investigate TGN arrival by sulfation analysi...

Watch this Scientific Journal Video about Analysis of Endocytic Uptake and Retrograde Transport to the Trans-Golgi Network Using Functionalized Nanobodies in Cultured Cells at JoVE.com

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

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.

Transport of proteins and membranes from the cell surface to the Golgi and beyond is essential for homeostasis, organelle identity and physiology. To ...

This method enables the production and application of bacterial expressed, functionalised nano-bodies to analyze retrograde protein transport from the cell surface to intercellular compartments.In particular the Trans-Golgi network.The main advantage of this technique is the use of sulfation.A highly specific, compartmentalized, and sensitive post-translation modification to assess reporter nano-body complex arrival in the DTN compartment.This method in combination with other derivatised nano-bodies for life cell imaging and electron microscopy can also be used to study endocitic processes of disease-related cell surface receptors.Our repertoire of functionalised nano-bodies has been engineered to study retrograde transport in cultured cells.However, these tools may also be applied in multicellular organisms.To begin this procedure, pick a bacterial colony containing the vector of interest from the plate and inoculate a flask containing 20 milliliters of LB medium supplemented with antibiotics.Incubate overnight at 37

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