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 placing them on ice. 
	NOTE: Prepare chemocompetent bacterial cells according to standard lab procedures. Alternatively, chemically competent bacterial cells can be purchased commercially.</li>
	<li>Add 50 ng of a plasmid encoding functionalized nanobodies. To allow sufficient site-specific biotinylation of the nanobody reporter during bacterial expression, also add a threefold excess (150 ng) of a bacterial expression plasmid encoding biotin ligase BirA (see also Table 1 for plasmid information). Gently pipet up and down. 
	NOTE: If nanobody biotinylation is not necessary or the nanobody reporters are devoid of a biotin acceptor peptide (BAP), co-transformation with a plasmid encoding BirA is not required.</li>
	<li>Incubate the bacterial cells for 30 min on ice.</li>
	<li>Heat shock the bacterial cells by placing them for 1 min at 42 &#176;C in a water bath or a heating block.</li>
	<li>Add 1 mL of room-temperature (RT) Luria broth (LB) medium and incubate the transformed bacteria in a thermoshaker for 1 h at 37 &#176;C to allow phenotypic expression of resistance genes. To prepare 1 L LB medium, balance 5 g of yeast extract, 10 g of tryptone and 10 g of NaCl, fill up with water and sterilize by autoclaving. If the functionalized nanobody has been subcloned in an expression vector containing resistance to ampicillin/carbenicillin, step 1.5 can be omitted.</li>
	<li>Pellet the bacteria at 11,000 x g for 1 min and resuspend in 100 &#181;L of fresh LB medium.</li>
	<li>Plate the suspended bacteria on LB plates containing the respective antibiotics (e.g., with 50 &#181;g/mL kanamycin and 50 &#181;g/mL carbenicillin when the bacteria have been co-transformed with nanobody reporter and BirA as stated above (step 1.2)).</li>
	<li>Place the LB plates in an incubator at 37 &#176;C and let grow overnight (O/N). 
	NOTE: The protocol can be paused here by storing the LB plate with grown colonies at 4 &#176;C. Use parafilm to seal LB plates.</li>
</ol>
2. Bacterial Liquid Culture and Induction of Functionalized Nanobody Expression
<ol>
	<li>Pick a bacterial colony containing the vector of interest from the plate and let it grow in a flask containing 20 mL of LB medium supplemented with antibiotics O/N in a shaking incubator at 37 &#176;C (see also step 1.7 regarding selection antibiotics).</li>
	<li>Next day, dilute the grown 20 mL bacterial culture into a flask containing 1 L of LB medium with selection antibiotics.</li>
	<li>Continue to incubate the bacterial culture at 37 &#176;C until it reaches an OD600 of 0.6-0.7. Allow the culture to cool down to RT before inducing protein expression.</li>
	<li>Induce protein expression of functionalized nanobodies and BirA by the addition of 1 mL of 1 M isopropyl-&#946;-d-thiogalactopyranosid (IPTG) to a final concentration of 1 mM of the inducer (1:1,000 dilution). Also add 10 mL of a 20 mM d-biotin stock solution resulting in 200 &#181;M d-biotin in the growth medium to allow biotinylation of the BAP epitope present in functionalized nanobodies (see also Table 1 for plasmid information) 
	NOTE: The d-biotin stock solution is prepared in ddH2O and brought into solution by drop-wise addition of 500 mM NaH2PO4. Alternatively, d-biotin can also be dissolved in DMSO. To produce nanobodies fused to APEX2 (VHH-APEX2), the LB medium must be complemented in addition with 1 mM 5-aminolevulinic acid (dALA) hydrochloride to promote heme incorporation. dALA is prepared as a 100 mM stock solution in ddH2O.</li>
	<li>Incubate the IPTG-induced 1 L bacterial culture at 30 &#176;C for 4 h for VHH-2xTS, at 20 &#176;C or RT O/N for VHH-APEX2, or at 16 &#176;C O/N for VHH-mCherry (see also Table 1 for plasmid information). 
	NOTE: Expression conditions for a new nanobody construct must be optimized by the experimenter.</li>
	<li>Transfer the bacterial culture from the flask into a 1 L centrifugation bottle and pellet the cells at 5,000 x g at 4 &#176;C for 45 min. Decant the supernatant and continue with the purification. 
	NOTE: The protocol can be paused here by storing the bacterial pellet at -80 &#176;C indefinitely. The pellet for VHH-APEX2 or VHH-mCherry is typically brownish (because of the incorporated heme) or pink, respectively.</li>
</ol>
3. Purification of Functionalized Nanobodies
<ol>
	<li>If necessary, thaw the frozen bacterial pellet on ice (see also step 2.6).</li>
	<li>Add 30 mL of ice-cold binding buffer (20 mM imidazole in 1x PBS) to the bacterial cell pellet and resuspend by pipetting up and down. Transfer resuspended bacteria into a labeled 50 mL tube.</li>
	<li>Supplement the 30 mL binding buffer with 200 &#181;g/mL lysozyme, 20 &#181;g/mL DNase I, 1 mM MgCl2 and 1 mM phenylmethylsulfonyl fluoride (PMSF) and incubate first for 10 min at RT, and then for 1 h at 4 &#176;C on an end-over-end shaker.</li>
	<li>Mechanically lyse bacterial cells in a 50 mL tube by placing a tip sonicator into the suspension. Apply constant 3x 1 min pulses with 1 min cooling periods in-between.</li>
	<li>Centrifuge the bacterial lysate at 15,000 x g at 4 &#176;C for 45 min to pellet the debris and intact cells. Transfer the sonicated bacterial cell lysate either into an appropriate centrifugation bottle for centrifugation or divide the lysate into several 5 mL tubes for tabletop centrifugation.</li>
	<li>Transfer the supernatant into a new 50 mL tube and discard the pelleted debris.</li>
	<li>Store the cleared lysate on ice while preparing purification by immobilized metal affinity chromatography (IMAC). To isolate histidine-tagged functionalized nanobodies, employ single-use His columns (see Table of Materials) allowing fast and simple gravity-flow purification. 
	NOTE: Alternatively, batch purification instead of commercial columns can be also used.</li>
	<li>Mounting His Columns at a Metal Stand.
	<ol>
		<li>Taper off the columns&#8217; storage solution, and equilibrate the His column with 10 mL of binding buffer (20 mM imidazole in 1x PBS).</li>
		<li>Empty by gravity flow (the flow-through can be discarded).</li>
	</ol>
	</li>
	<li>Gradually load the cleared bacterial lysate (~30 mL) onto the column. Empty by gravity flow (the flow-through can be discarded).</li>
	<li>Wash the His column 2x with 10 mL of binding buffer (20 mM imidazole in 1x PBS).</li>
	<li>Elute the nanobodies with 2 mL of elution buffer (500 mM imidazole in 1x PBS) into a 2 mL tube and apply a buffer exchange.</li>
	<li>Buffer Exchange.
	<ol>
		<li>Equilibrate a desalting column (see Table of Materials) placed in a 50 mL tube column adapter 5 times with 5 mL of 1x PBS.</li>
		<li>Allow the buffer to enter the packed bed. Discard the flow-through.</li>
		<li>After the fifth PBS equilibration, spin the column at 1,000 x g for 2 min.</li>
		<li>Discard the flow-through.</li>
		<li>Place the column with the adapter onto a new 50 mL tube. Load the 2 mL of eluted functionalized nanobody (from step 3.11) onto the PBS-equilibrated desalting column and spin at 1,000 x g for 2 min and collect the eluate. 
		NOTE: Instead of commercial desalting columns, dialysis can be applied to exchange buffer composition.</li>
	</ol>
	</li>
	<li>Determine the protein (nanobody) concentration of the eluate using the bicinchoninic (BCA) or Bradford assay according to the manufacturer&#8217;s instructions. 
	NOTE: For nanobody uptake assays by GFP reporter cell lines, a stock concentration of 2-10 mg/mL is optimal.</li>
</ol>
4. Validation of Functionalized Nanobody Expression (Coomassie Staining)
<ol>
	<li>Prepare 10-12.5% gels for sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) according to standard protocols. 
	NOTE: Alternatively, use commercially available precast gradient gels.</li>
	<li>Pipet 20 &#181;g of purified functionalized nanobody into a 1.5 mL tube and boil in sample buffer at 95 &#176;C for 5 min. 
	NOTE: As an example, add 10 &#181;L of a 2 mg/mL nanobody stock solution into a 1.5 mL tube with 10 &#181;L of 2x sample buffer. If the volume is too small, further dilute nanobody in PBS before adding sample buffer.</li>
	<li>Load the purified nanobody boiled in sample buffer onto an SDS-polyacrylamide gel, and run according to the standard PAGE protocol until the reference dye (e.g., bromophenol blue) has reached the end of the separating gel, followed by Coomassie staining and destaining.</li>
	<li>Coomassie Gel Staining and Destaining.
	<ol>
		<li>Uncast the gel and stain it with Coomassie staining solution (5% of a 10 g/L Coomassie stock solution in 10% acetic acid and 45% methanol in ddH2O) for 20-30 min at RT on a moving shaking platform. The gel should be completely covered in the staining solution.</li>
		<li>Destain the gel 2-3 times with destain solution (7.5% acetic acid and 15% methanol in ddH2O) for 1 h each at RT, before leaving the gel O/N for further destaining. 
		NOTE: Excess Coomassie can be efficiently soaked up by placing kitchen paper towels around the gel.</li>
	</ol>
	</li>
	<li>Image the gel with an imager or camera of choice. 
	NOTE: Nanobody expression can be further validated by immunoblot analysis using antibodies targeting its epitopes (see also Figure 1A).</li>
</ol>
5. Uptake of Functionalized Nanobodies by Cultured Cells for Fluorescence Staining
<ol>
	<li>In a cell culture hood, seed ~400,000 to 500,000 HeLa cells stably expressing a GFP-tagged reporter protein on 18-mm glass coverslips (No. 1.5H) in 35-mm dishes or 6-well clusters with complete medium containing antibiotics (Dulbecco&#8217;s Modified Eagle medium, DMEM, supplemented with 10% fetal calf serum (FCS), 100 units/mL streptomycin, 2 mM l-glutamine, and 1.5 &#181;g/mL puromycin). 
	NOTE: Here, we use HeLa cells stably expressing EGFP-Calnexin, EGFP-CDMPR and TfR-EGFP for illustration purposes. Apart from stable cell lines, also transiently transfected HeLa cells can be used. Stable cell lines can be co-cultivated at this step with parental non-transduced HeLa cells, for direct comparison.</li>
	<li>Incubate the cells O/N at 37 &#176;C in a 7.5% CO2 incubator to proliferate. 
	NOTE: Cells should have a confluency of ~80% the next day.</li>
	<li>Pipet 2 &#181;L of a 2 mg/mL nanobody stock solution into a 15 or 50 mL tube containing 2 mL of complete medium (this volume is sufficient to cover a 35 mm dish or one well of a 6-well cluster, adjust the volume of medium and nanobody proportionally to include more cell culture dishes or clusters). 
	NOTE: For the purpose of illustration, we here use VHH-mCherry, since no further antibody staining is required to see nanobody internalized by the EGFP reporters (see Figure 2C).</li>
	<li>Remove growth medium from the cells and add 2 mL of prewarmed complete medium containing 2 &#181;g/mL functionalized nanobody per 35 mm dish or 6-well unit.</li>
	<li>Incubate the cells for 1 h at 37 &#176;C in a 7.5% CO2 incubator to allow reporter-mediated nanobody uptake. 
	NOTE: Depending on the planned experiment, the time of nanobody uptake needs to be adjusted. For the experiment shown here, 1 h is sufficient to reach steady-state of nanobody uptake by EGFP-CDMPR and TfR-EGFP.</li>
	<li>Remove the medium and wash the cells 2-3 times with 1 mL of 1x PBS at RT.</li>
	<li>Fix the cells with 1 mL of 3% paraformaldehyde (PFA) for 10 min at RT.</li>
	<li>Remove PFA solution and quench remaining fixative with 1 mL of 50 mM NH4Cl in 1x PBS for 5 min at RT. 
	NOTE: 3% PFA should be disposed of separately as fixative waste.</li>
	<li>Wash the cells 3 times with 1 mL of 1x PBS at RT.</li>
	<li>Permeabilize the cells with 1 mL of 0.1% Triton X-100 in 1x PBS for 10 min at RT. 
	NOTE: Although no permeabilization is required, EGFP and mCherry fluorescence is better when afterwards the cells are embedded in mounting medium.</li>
	<li>Wash the cells 3 times with 1 mL of 1x PBS at RT.</li>
	<li>Place the coverslips with forceps on a drop (~100 &#181;L) of 1% BSA in 1x PBS containing 5 &#181;g/mL DAPI (4&#39;,6-diamidino-2-phenylindole) for 5 min at RT.</li>
	<li>Place the coverslips back into the dish/well and wash 3 times with 1 mL of 1x PBS at RT.</li>
	<li>Label the glass slides and mount the cells with Fluoromount-G. Let the mounting medium harden in the dark for 3-4 h and store the glass slides at 4 &#176;C under light protection.</li>
	<li>Image staining patterns using a microscope of choice (e.g., Point Scanning Confocal upright microscope).</li>
</ol>
6. Uptake of Functionalized Nanobodies by Cultured Cells (for Sulfation Analysis)
<ol>
	<li>In a cell culture hood, seed ~400,000 to 500,000 HeLa cells stably expressing a GFP-tagged reporter protein in 35 mm dishes or on 6-well clusters with complete medium containing antibiotics (Dulbecco&#8217;s Modified Eagle medium, DMEM, supplemented with 10% fetal calf serum (FCS), 100 units/mL streptomycin, 2 mM l-glutamine, and 1.5 &#181;g/mL puromycin). 
	NOTE: As mentioned above, we here use HeLa cells stably expressing EGFP-Calnexin, EGFP-CDMPR and TfR-EGFP for illustration purposes. Apart from stable cell lines, also transiently transfected HeLa can be used.</li>
	<li>Incubate the cells O/N at 37 &#176;C in a 7.5% CO2 incubator to proliferate. 
	NOTE: Cells should have a confluency of ~80% next day.</li>
	<li>Remove the complete medium, wash 2 times with 1x PBS at RT, and starve the cells with sulfate-free medium (SFM) for 1 h at 37 &#176;C in a 7.5% CO2 incubator. 
	NOTE: SFM is prepared by adding 10 mL of MEM amino acids (50x) solution, 5 mL of L-glutamine (200 mM), 5 mL of vitamin solution (100x), and 900 &#181;L of CaCl2&#183;2H2O to 500 mL of Eagle&#8217;s balanced salts. Pass through a 0.22 &#181;m filter and aliquot.</li>
	<li>In a ventilated hood or bench designated for radioactivity work, prepare sulfate labeling medium containing 0.5 mCi/mL [35S]sulfate as sodium salt in SFM. 
	CAUTION: Carefully handle all solutions containing radioactivity. Only work in designated hoods or benches. All material (tips, tubes, plates, etc.) or wash buffers in contact with radioactivity have to be collected and disposed of separately. Do this for all steps below (step 6.5-6.20) as well.</li>
	<li>Add VHH-2xTS in 1x PBS into sulfate labeling medium to a final concentration of 2 &#181;g/mL. 
	NOTE: As control, also include VHH-std or another nanobody devoid of TS sites to demonstrate specific labeling.</li>
	<li>Replace SFM by 0.7 mL of sulfate labeling medium containing 0.5 mCi/mL [35S]sulfate and 2 &#181;g/mL VHH-2xTS and incubate the cells for 1 h at 37 &#176;C in a 7.5% CO2 incubator designated for work with radioactivity. 
	NOTE: Depending on the planned experiment, the time of nanobody sulfation needs to be adjusted. In addition, to determine TGN arrival kinetics, labeling is performed for different times (e.g., for 10 min, 20 min, 30 min, etc.).</li>
	<li>Remove the labeling medium and wash the cells 2-3 times with ice-cold 1x PBS on a cooling plate or ice.</li>
	<li>Add 1 mL of lysis buffer (PBS containing 1% Triton X-100 and 0.5 % deoxycholate) supplemented with 2 mM PMSF and 1x protease inhibitor cocktail and incubate the cells for 10-15 min on a rocking platform at 4 &#176;C.</li>
	<li>Scrape and transfer the lysate into a 1.5 mL tube, vortex the lysate, and place for 10-15 min on an end-over-end rotator at 4 &#176;C.</li>
	<li>Prepare a postnuclear lysate by centrifugation at 10,000 x g for 10 min at 4 &#176;C.</li>
	<li>Using a new 1.5 mL tube, prepare 20-30 &#181;L of a slurry of Nickel beads in a 1.5 mL tube and wash once with 1x PBS by gently pelleting them at 1,000 x g for 1 min. 
	NOTE: Instead of Nickel beads, streptavidin beads (if nanobody is biotinylated) or protein A beads with an IgG against an epitope of the nanobody (T7- or HA-tag) can be used.</li>
	<li>Transfer the postnuclear lysate into a 1.5 mL tube containing Nickel beads and incubate for 1 h at 4 &#176;C on an end-over-end rotator to isolate the nanobodies. Remove an aliquot (50-100 &#181;L) of the postnuclear lysate and boil in SDS sample buffer for subsequent immunoblot analysis of total cell-associated nanobody, GFP reporter and loading control.</li>
	<li>Wash the beads 3 times with 1x PBS or lysis buffer by gently pelleting at 1,000 x g for 1 min. 
	NOTE: For more stringent washing of beads, 20 mM imidazole can be included in PBS or lysis buffer.</li>
	<li>Carefully remove all washing buffer from the beads, add 50 &#181;L of 2x sample buffer, and boil for 5 min at 95 &#176;C.</li>
	<li>Load both boiled beads on a midi 12.5% SDS-PAGE gel and run according to the standard PAGE protocol until the reference dye (e.g., bromophenol blue) has reached the end of the separating gel. 
	NOTE: Immunoblot analysis of total cell-associated nanobody, GFP reporter and loading control can also be performed using a mini 12.5% SDS-PAGE or precast gradient gel.</li>
	<li>Fix the separating gel in ~30 mL of fixation buffer (10% acetic acid and 45% methanol in ddH2O) for 1 h at RT, wash the gel three times with ~50 mL of deionized water and prepare for gel drying.</li>
	<li>Drying SDS-PAGE Gels. 
	<ol>
		<li>Carefully place the fixed gel on stretched cling film and cover it with precut filter paper.</li>
		<li>Place the gel with the filter paper down on top of the drying platform, place the vacuum cover, and switch on the vacuum pump for gel drying. Dry the gel for 3-5 h.</li>
		<li>Remove the cling film and place the dried gel into an autoradiography cassette equipped with a Phosphor Screen.</li>
	</ol>
	</li>
	<li>Image autoradiograph using a Phosphor Screen Imager. Follow the instructions of the manufacturer. 
	NOTE: Depending on the strength of the signal, dried gels need to be exposed longer with Phosphor screens.</li>
</ol>

The authors have nothing to disclose.

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&#039;,6-diamidino-2-phenylindole (DAPI)</td><td>Sigma-Aldrich</td><td>D9542</td><td>dissolved in 1x 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 NaH&lt;sub&gt;2&lt;/sub&gt;PO&lt;sub&gt;4 &lt;/sub&gt;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&amp;rsquo;s modified Eagle&amp;rsquo;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&#039;s balanced salts</td><td>Sigma-Aldrich</td><td>E6267</td><td></td></tr><tr><td>MEM amino acids (50x) solution</td><td>Sigma-Aldrich</td><td>M5550</td><td></td></tr><tr><td>MEM vitamin solution (100x)</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-&amp;beta;-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&amp;rsquo;s phosphate buffered saline (DPBS) w/o Ca&lt;sup&gt;2+&lt;/sup&gt;/Mg&lt;sup&gt;2+&lt;/sup&gt;</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 ...

analysis-endocytic-uptake-retrograde-transport-to-trans-golgi-network

Research

Education

JoVE Journal

JoVE Core

Cell Biology

Biochemistry

Unit 1

Intracellular Membrane Traffic

SCIENTISTS IN ACTION

9.0K Views.  University of Basel. Deze methode maakt de productie en toepassing van bacteriële uitgedrukte, gefunctionaliseerde nanolichamen mogelijk om retrograde eiwittransport van het celoppervlak naar intercellulaire compartimenten te analyseren. In het bijzonder het Trans-Golgi netwerk. Het belangrijkste voordeel van deze techniek is het gebruik van sulfatie. Een zeer specifieke, gecompartimenteerde en gevoelige post-vertaling wijziging om reporter nano-body complexe aankomst in het DTN-compartiment te beoordelen. Deze ...

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

Analyzing Cellular Internalization of Nanoparticles and Bacteria by Multi-spectral Imaging Flow Cytometry

Nanoparticulate systems have emerged as valuable tools in vaccine delivery through their ability to efficiently deliver cargo, including proteins, to antigen presenting cells1-5. Internalization of nanoparticles (NP) by antigen presenting cells is a critical step in generating an effective immune response to the encapsulated antigen. To determine how changes in nanoparticle formulation impact function, we sought to develop a high throughput, quantitative experimental protocol that was compatible with detecting internalized nanoparticles as well as bacteria. To date, two independent techniques, microscopy and flow cytometry, have been the methods used to study the phagocytosis of nanoparticles. The high throughput nature of flow cytometry generates robust statistical data. However, due to low resolution, it fails to accurately quantify internalized versus cell bound nanoparticles. Microscopy generates images with high spatial resolution; however, it is time consuming and involves small sample sizes6-8. Multi-spectral imaging flow cytometry (MIFC) is a new technology that incorporates aspects of both microscopy and flow cytometry that performs multi-color spectral fluorescence and bright field imaging simultaneously through a laminar core. This capability provides an accurate analysis of fluorescent signal intensities and spatial relationships between different structures and cellular features at high speed.
	Herein, we describe a method utilizing MIFC to characterize the cell populations that have internalized polyanhydride nanoparticles or Salmonella enterica serovar Typhimurium. We also describe the preparation of nanoparticle suspensions, cell labeling, acquisition on an ImageStreamX system and analysis of the data using the IDEAS application. We also demonstrate the application of a technique that can be used to differentiate the internalization pathways for nanoparticles and bacteria by using cytochalasin-D as an inhibitor of actin-mediated phagocytosis.

In this article, we describe a method utilizing multi-spectral imaging flow cytometry to quantify the internalization of polyanhydride nanoparticles or bacteria by RAW 264.7 cells.

Nanoparticulate  systems have emerged as valuable tools in vaccine delivery through their  ability to efficiently deliver cargo, including proteins, to ...

Bioengineering

Application of Fluorescent Nanoparticles to Study Remodeling of the Endo-lysosomal System by Intracellular Bacteria

Fluorescent nanoparticles (NPs) with desirable chemical, optical and mechanical properties are promising tools to label intracellular organelles. Here, we introduce a method using gold-BSA-rhodamine NPs to label the endo-lysosomal system of eukaryotic cells and monitor manipulations of host cellular pathways by the intracellular pathogen Salmonella enterica. The NPs were readily internalized by HeLa cells and localized in late endosomes/lysosomes. Salmonella infection induced rearrangement of the vesicles and accumulation of NPs in Salmonella-induced membrane structures. We deployed the Imaris software package for quantitative analyses of confocal microscopy images. The number of objects and their size distribution in non-infected cells were distinct from the ones in Salmonella-infected cells, indicating extremely remodeling of the endo-lysosomal system by WT Salmonella.

This article describes methods for the synthesis and fluorescent labeling of nanoparticles (NPs). The NPs were applied in pulse-chase experiments to label the endo-lysosomal system of eukaryotic cells. Manipulation of the endo-lysosomal system by activities of the intracellular pathogen Salmonella enterica were followed by live cell imaging and quantified.

Fluorescent nanoparticles (NPs) with desirable chemical, optical and mechanical properties are promising tools to label intracellular organelles. Here, we ...

Immunology and Infection

Differential Labeling of Cell-surface and Internalized Proteins after Antibody Feeding of Live Cultured Neurons

In order to demonstrate the cell-surface localization of a putative transmembrane receptor in cultured neurons, we labeled the protein on the surface of live neurons with a specific primary antibody raised against an extracellular portion of the protein. Given that receptors are trafficked to and from the surface, if cells are permeabilized after fixation then both cell-surface and internal protein will be detected by the same labeled secondary antibody. Here, we adapted a method used to study protein trafficking (&#8220;antibody feeding&#8221;) to differentially label protein that had been internalized by endocytosis during the antibody incubation step and protein that either remained on the cell surface or was trafficked to the surface during this period. The ability to distinguish these two pools of protein was made possible through the incorporation of an overnight blocking step with highly-concentrated unlabeled secondary antibody after an initial incubation of unpermeabilized neurons with a fluorescently-labeled secondary antibody. After the blocking step, permeabilization of the neurons allowed detection of the internalized pool with a fluorescent secondary antibody labeled with a different fluorophore. Using this technique we were able to obtain important information about the subcellular location of this putative receptor, revealing that it was, indeed, trafficked to the cell-surface in neurons. This technique is broadly applicable to a range of cell types and cell-surface proteins, providing a suitable antibody to an extracellular epitope is available.

We describe a method to label protein on the surface of living neurons using a specific polyclonal antibody to extracellular epitopes. Protein bound by the antibody on the cell surface and subsequently internalized via endocytosis can be distinguished from protein remaining on, or trafficked to, the surface during the incubation.

In order to demonstrate the cell-surface localization of a putative transmembrane receptor in cultured neurons, we labeled the protein on the surface of ...

Neuroscience

Quantification of Endosome and Lysosome Motilities in Cultured Neurons Using Fluorescent Probes

In the brain, membrane trafficking systems play important roles in regulating neuronal functions, such as neuronal morphology, synaptic plasticity, survival, and glial communications. To date, numerous studies have reported that defects in these systems cause various neuronal diseases. Thus, understanding the mechanisms underlying vesicle dynamics may provide influential clues that could aid in the treatment of several neuronal disorders. Here, we describe a method for quantifying vesicle motilities, such as motility distance and rate of movement, using a software plug-in for the ImageJ platform. To obtain images for quantification, we labeled neuronal endosome-lysosome structures with EGFP-tagged vesicle marker proteins and observed the movement of vesicles using a time-lapse microscopy. This method is highly useful and simplify measuring vesicle motility in neurites, such as axons and dendrites, as well as in the soma of both neurons and glial cells. Furthermore, this method can be applied to other cell lines, such as fibroblasts and endothelial cells. This approach could provide a valuable advancement of our understanding of membrane trafficking.

The investigation of membrane trafficking is crucial for understanding neuronal functions. Here, we introduce a method for quantifying vesicle motility in neurons. This is a convenient method that can be adapted to the quantification of membrane trafficking in the nervous system.

In the brain, membrane trafficking systems play important roles in regulating neuronal functions, such as neuronal morphology, synaptic plasticity, ...

Assay to Measure Nucleocytoplasmic Transport in Real Time within Motor Neuron-like NSC-34 Cells

Nucleocytoplasmic transport refers to the import and export of large molecules from the cell nucleus. Recently, a number of studies have shown a connection between amyotrophic lateral sclerosis (ALS) and impairments in the nucleocytoplasmic pathway. ALS is a neurodegenerative disease affecting the motor neurons and resulting in paralysis and ultimately in death, within 2-5 years on average. Most cases of ALS are sporadic, lacking any apparent genetic linkage, but 10% are inherited in a dominant manner. Recently, hexanucleotide repeat expansions (HREs) in the chromosome 9 open reading frame 72 (C9orf72) gene were identified as a genetic cause of ALS and frontotemporal dementia (FTD). Importantly, different groups have recently proposed that these mutants affect nucleocytoplasmic transport. These studies have mostly shown the final outcome and manifestations caused by HREs on nucleocytoplasmic transport, but they do not demonstrate nuclear transport dysfunction in real time. As a result, only severe nucleocytoplasmic transport deficiency can be determined, mostly due to high overexpression or exogenous protein insertion. 
This protocol describes a new and very sensitive assay to evaluate and quantify nucleocytoplasmic transport dysfunction in real time. The rate of import of a NLS-NES-GFP protein (shuttle-GFP) can be quantified in real time using fluorescent microscopy. This is performed by using an exportin inhibitor, thus allowing the shuttle GFP only to enter the nucleus. To validate the assay, the C9orf72 HRE translated dipeptide repeats, poly(GR) and poly(PR), which have been previously shown to disrupt nucleocytoplasmic transport, were used. Using the described assay, a 50% decrease in the nuclear import rate was observed compared to the control. Using this system, minute changes in nucleocytoplasmic transport can be examined and the ability of different factors to rescue (even partially) a nucleocytoplasmic transport defect can be determined.

This protocol describes a method to sensitively measure the nucleocytoplasmic transport rate within motor neuron-like NSC-34 cells by quantifying the real-time change in the nuclear import of a NLS-NES-GFP protein.

Nucleocytoplasmic transport refers to the import and export of large molecules from the cell nucleus. Recently, a number of studies have shown a ...

Determining Cell-surface Expression and Endocytic Rate of Proteins in Primary Astrocyte Cultures Using Biotinylation

Cell-surface proteins mediate a wide array of functions. In many cases, their activity is regulated by endocytic processes that modulate their levels at the plasma membrane. Here, we present detailed protocols for 2 methods that facilitate the study of such processes, both of which are based on the principle of the biotinylation of cell-surface proteins. The first is designed to allow for the semi-quantitative determination of the relative levels of a particular protein at the cell-surface. In it, the lysine residues of the plasma membrane proteins of cells are first labeled with a biotin moiety. Once the cells are lysed, these proteins may then be specifically precipitated via the use of agarose-immobilized streptavidin by exploiting the natural affinity of the latter for biotin. The proteins isolated in such a manner may then be analyzed via a standard western blotting approach. The second method provides a means of determining the endocytic rate of a particular cell-surface target over a period of time. Cell-surface proteins are first modified with a biotin derivative containing a cleavable disulfide bond. The cells are then shifted back to normal culture conditions, which causes the endocytic uptake of a proportion of biotinylated proteins. Next, the disulfide bonds of non-internalized biotin groups are reduced using the membrane-impermeable reducing agent glutathione. Via this approach, endocytosed proteins may thus be isolated and quantified with a high degree of specificity.

Two biotinylation-based methods, designed for determining the cell-surface expression and endocytic rate of proteins expressed at the plasma membrane, are presented in this report.

Cell-surface proteins mediate a wide array of functions. In many cases, their activity is regulated by endocytic processes that modulate their levels at ...

An Optical Assay for Synaptic Vesicle Recycling in Cultured Neurons Overexpressing Presynaptic Proteins

At active presynaptic nerve terminals, synaptic vesicles undergo cycles of exo- and endocytosis. During recycling, the luminal domains of SV transmembrane proteins become exposed at the cell surface. One of these proteins is Synaptotagmin-1 (Syt1). An antibody directed against the luminal domain of Syt1, once added to the culture medium, is taken up during the exo-endocytotic cycle. This uptake is proportional to the amount of SV recycling and can be quantified through immunofluorescence. Here, we combine Syt1 antibody uptake with double transfection of cultured hippocampal neurons. This allows us to (1) localize presynaptic sites based on expression of recombinant presynaptic marker Synaptophysin, (2) determine their functionality using Syt1 uptake, and (3) characterize the targeting and effects of a protein of interest, GFP-Rogdi.

We describe an optical assay for synaptic vesicle (SV) recycling in cultured neurons. Combining this protocol with double transfection to express a presynaptic marker and protein of interest allows us to locate presynaptic sites, their synaptic vesicle recycling capacity, and determine the role of the protein of interest.

At active presynaptic nerve terminals, synaptic vesicles undergo cycles of exo- and endocytosis. During recycling, the luminal domains of SV transmembrane ...

Tracking Drug-induced Changes in Receptor Post-internalization Trafficking by Colocalizational Analysis

The intracellular trafficking of receptors is a collection of complex and highly controlled processes. Receptor trafficking modulates signaling and overall cell responsiveness to ligands and is, itself, influenced by intra- and extracellular conditions, including ligand-induced signaling. Optimized for use with monolayer-plated cultured cells, but extendable to free-floating tissue slices, this protocol uses immunolabelling and colocalizational analysis to track changes in intracellular receptor trafficking following both chronic/prolonged and acute interventions, including exogenous drug treatment. After drug treatment, cells are double-immunolabelled for the receptor and for markers for the intracellular compartments of interest. Sequential confocal microscopy is then used to capture two-channel photomicrographs of individual cells, which are subjected to computerized colocalizational analysis to yield quantitative colocalization scores. These scores are normalized to permit pooling of independent replicates prior to statistical analysis. Representative photomicrographs may also be processed to generate illustrative figures. Here, we describe a powerful and flexible technique for quantitatively assessing induced receptor trafficking.

Receptor trafficking modulates signaling and cell responsiveness to ligands and is, itself, responsive to cell conditions, including ligand-induced signaling. Here, we describe a powerful and flexible technique for quantitatively assessing drug-induced receptor trafficking using immunolabeling and colocalizational analysis.

The intracellular trafficking of receptors is a collection of complex and highly controlled processes. Receptor trafficking modulates signaling and ...

Biology

The Microscopy-Based Assay to Study and Analyze the Recycling Endosomes using SNARE Trafficking

Recycling endosomes (REs) are tubular-vesicular organelles generated from early/sorting endosomes in all cell types. These organelles play a key role in the biogenesis of melanosomes, a lysosome-related organelle produced by melanocytes. REs deliver the melanocyte-specific cargo to premature melanosomes during their formation. Blockage in the generation of REs, observed in several mutants of Hermansky-Pudlak syndrome, results in hypopigmentation of skin, hair, and eye. Therefore, studying the dynamics (refer to number and length) of REs is useful to understand the function of these organelles in normal and disease conditions. In this study, we aim to measure the RE dynamics using a resident SNARE STX13.

Recycling endosomes are part of the endosomal tubular network. Here we present a method to quantify the dynamics of recycling endosomes using GFP-STX13 as an organelle marker.

Recycling endosomes (REs) are tubular-vesicular organelles generated from early/sorting endosomes in all cell types. These organelles play a key role in ...

An Introduction to Endocytosis and Exocytosis

Cells can take in substances from the extracellular environment by endocytosis and actively release molecules into it by exocytosis. Such processes involve lipid membrane-bound sacs called vesicles. Knowledge of the molecular architecture and mechanisms of both is key to understanding normal cell physiology, as well as the disease states that arise when they become defective. This video will first briefly review a few pivotal discoveries in the history of endo- and exocytosis research. Next, some key questions will be examined, followed by a discussion of the prominent methods used to investigate these problems, including cell labeling, fusion assays, and fluorescence imaging. Finally, it will explore current research being conducted by scientists in the field today.

Endocytosis and Exocytosis; Clathrin Coated Pits and SNAREs

Cells can take in substances from the extracellular environment by endocytosis and actively release molecules into it by exocytosis. Such processes ...

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

Samenvatting

Meer video's verkennen