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

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 placing them on ice.
   NOTE: Prepare chemocompetent bacterial cells according to standard lab procedures. Alternatively, chemically competent bacterial cells can be purchased commercially.
2. 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.
3. Incubate the bacterial cells for 30 min on ice.
4. Heat shock the bacterial cells by placing them for 1 min at 42 °C in a water bath or a heating block.
5. Add 1 mL of room-temperature (RT) Luria broth (LB) medium and incubate the transformed bacteria in a thermoshaker for 1 h at 37 °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.
6. Pellet the bacteria at 11,000 x g for 1 min and resuspend in 100 µL of fresh LB medium.
7. Plate the suspended bacteria on LB plates containing the respective antibiotics (e.g., with 50 µg/mL kanamycin and 50 µg/mL carbenicillin when the bacteria have been co-transformed with nanobody reporter and BirA as stated above (step 1.2)).
8. Place the LB plates in an incubator at 37 °C and let grow overnight (O/N).
   NOTE: The protocol can be paused here by storing the LB plate with grown colonies at 4 °C. Use parafilm to seal LB plates.

2. Bacterial Liquid Culture and Induction of Functionalized Nanobody Expression

1. 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 °C (see also step 1.7 regarding selection antibiotics).
2. Next day, dilute the grown 20 mL bacterial culture into a flask containing 1 L of LB medium with selection antibiotics.
3. Continue to incubate the bacterial culture at 37 °C until it reaches an OD₆₀₀ of 0.6-0.7. Allow the culture to cool down to RT before inducing protein expression.
4. Induce protein expression of functionalized nanobodies and BirA by the addition of 1 mL of 1 M isopropyl-β-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 µM 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.
5. Incubate the IPTG-induced 1 L bacterial culture at 30 °C for 4 h for VHH-2xTS, at 20 °C or RT O/N for VHH-APEX2, or at 16 °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.
6. Transfer the bacterial culture from the flask into a 1 L centrifugation bottle and pellet the cells at 5,000 x g at 4 °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 °C indefinitely. The pellet for VHH-APEX2 or VHH-mCherry is typically brownish (because of the incorporated heme) or pink, respectively.

3. Purification of Functionalized Nanobodies

1. If necessary, thaw the frozen bacterial pellet on ice (see also step 2.6).
2. 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.
3. Supplement the 30 mL binding buffer with 200 µg/mL lysozyme, 20 µg/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 °C on an end-over-end shaker.
4. 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.
5. Centrifuge the bacterial lysate at 15,000 x g at 4 °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.
6. Transfer the supernatant into a new 50 mL tube and discard the pelleted debris.
7. 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.
8. Mounting His Columns at a Metal Stand.
   1. Taper off the columns’ storage solution, and equilibrate the His column with 10 mL of binding buffer (20 mM imidazole in 1x PBS).
   2. Empty by gravity flow (the flow-through can be discarded).
9. Gradually load the cleared bacterial lysate (~30 mL) onto the column. Empty by gravity flow (the flow-through can be discarded).
10. Wash the His column 2x with 10 mL of binding buffer (20 mM imidazole in 1x PBS).
11. 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.
12. Buffer Exchange.
    1. Equilibrate a desalting column (see Table of Materials) placed in a 50 mL tube column adapter 5 times with 5 mL of 1x PBS.
    2. Allow the buffer to enter the packed bed. Discard the flow-through.
    3. After the fifth PBS equilibration, spin the column at 1,000 x g for 2 min.
    4. Discard the flow-through.
    5. 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.
13. Determine the protein (nanobody) concentration of the eluate using the bicinchoninic (BCA) or Bradford assay according to the manufacturer’s instructions.
    NOTE: For nanobody uptake assays by GFP reporter cell lines, a stock concentration of 2-10 mg/mL is optimal.

4. Validation of Functionalized Nanobody Expression (Coomassie Staining)

1. 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.
2. Pipet 20 µg of purified functionalized nanobody into a 1.5 mL tube and boil in sample buffer at 95 °C for 5 min.
   NOTE: As an example, add 10 µL of a 2 mg/mL nanobody stock solution into a 1.5 mL tube with 10 µL of 2x sample buffer. If the volume is too small, further dilute nanobody in PBS before adding sample buffer.
3. 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.
4. Coomassie Gel Staining and Destaining.
   1. 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.
   2. 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.
5. 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).

5. Uptake of Functionalized Nanobodies by Cultured Cells for Fluorescence Staining

1. 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’s Modified Eagle medium, DMEM, supplemented with 10% fetal calf serum (FCS), 100 units/mL streptomycin, 2 mM l-glutamine, and 1.5 µg/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.
2. Incubate the cells O/N at 37 °C in a 7.5% CO₂ incubator to proliferate.
   NOTE: Cells should have a confluency of ~80% the next day.
3. Pipet 2 µL 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).
4. Remove growth medium from the cells and add 2 mL of prewarmed complete medium containing 2 µg/mL functionalized nanobody per 35 mm dish or 6-well unit.
5. Incubate the cells for 1 h at 37 °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.
6. Remove the medium and wash the cells 2-3 times with 1 mL of 1x PBS at RT.
7. Fix the cells with 1 mL of 3% paraformaldehyde (PFA) for 10 min at RT.
8. 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.
9. Wash the cells 3 times with 1 mL of 1x PBS at RT.
10. 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.
11. Wash the cells 3 times with 1 mL of 1x PBS at RT.
12. Place the coverslips with forceps on a drop (~100 µL) of 1% BSA in 1x PBS containing 5 µg/mL DAPI (4',6-diamidino-2-phenylindole) for 5 min at RT.
13. Place the coverslips back into the dish/well and wash 3 times with 1 mL of 1x PBS at RT.
14. 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 °C under light protection.
15. Image staining patterns using a microscope of choice (e.g., Point Scanning Confocal upright microscope).

6. Uptake of Functionalized Nanobodies by Cultured Cells (for Sulfation Analysis)

1. 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’s Modified Eagle medium, DMEM, supplemented with 10% fetal calf serum (FCS), 100 units/mL streptomycin, 2 mM l-glutamine, and 1.5 µg/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.
2. Incubate the cells O/N at 37 °C in a 7.5% CO₂ incubator to proliferate.
   NOTE: Cells should have a confluency of ~80% next day.
3. 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 °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 µL of CaCl₂·2H₂O to 500 mL of Eagle’s balanced salts. Pass through a 0.22 µm filter and aliquot.
4. 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.
5. Add VHH-2xTS in 1x PBS into sulfate labeling medium to a final concentration of 2 µg/mL.
   NOTE: As control, also include VHH-std or another nanobody devoid of TS sites to demonstrate specific labeling.
6. Replace SFM by 0.7 mL of sulfate labeling medium containing 0.5 mCi/mL [³⁵S]sulfate and 2 µg/mL VHH-2xTS and incubate the cells for 1 h at 37 °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.).
7. Remove the labeling medium and wash the cells 2-3 times with ice-cold 1x PBS on a cooling plate or ice.
8. 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 °C.
9. 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 °C.
10. Prepare a postnuclear lysate by centrifugation at 10,000 x g for 10 min at 4 °C.
11. Using a new 1.5 mL tube, prepare 20-30 µL 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.
12. Transfer the postnuclear lysate into a 1.5 mL tube containing Nickel beads and incubate for 1 h at 4 °C on an end-over-end rotator to isolate the nanobodies. Remove an aliquot (50-100 µL) of the postnuclear lysate and boil in SDS sample buffer for subsequent immunoblot analysis of total cell-associated nanobody, GFP reporter and loading control.
13. 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.
14. Carefully remove all washing buffer from the beads, add 50 µL of 2x sample buffer, and boil for 5 min at 95 °C.
15. 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.
16. 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.
17. Drying SDS-PAGE Gels.
    1. Carefully place the fixed gel on stretched cling film and cover it with precut filter paper.
    2. 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.
    3. Remove the cling film and place the dried gel into an autoradiography cassette equipped with a Phosphor Screen.
18. 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.