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

1. Determining Relative Cell-surface Protein Expression in Astrocytes by Biotinylation

NOTE: Here, we illustrate the application of this biotinylation technique to the study of the effects of the extracellular matrix molecule laminin on the cell-surface localization of the water-permeable channel aquaporin-4 (AQP4). Specialized materials required for this assay include sulfo-NHS-LC-biotin and streptavidin-agarose resin (see Table of Materials).

1. Using the method outlined in Noel et al.¹², prepare cultures of cortical astrocytes approximately 2 weeks in advance of the assay, and grow them in 75 cm² vented culture flasks. When astrocytes are 80 – 90% confluent, detach them from the culture surface using 0.05% trypsin, and then passage them 1:3.
2. At 48 h prior to the assay, when cells are again 80 – 90% confluent, passage astrocytes 1:3 (accounting for the change in the culture format) onto 60 mm cell culture dishes so that they are >70% confluent on the day the experiment is to take place. Ensure that cells are evenly distributed between dishes.
3. At 16 h prior to assay, pipette laminin into culture medium to a final concentration of 24 nM, and incubate at 37 °C.
4. Immediately prior to assay, prepare the following, and then place on ice or refrigerate: CM-PBS (100 mg/L MgCl₂∙6H₂O and 100 mg/L CaCl₂ in 1X PBS, pH 7.4), biotin buffer (0.5 mg/mL sulfo-NHS-LC-biotin in CM-PBS), quenching buffer (50 mM NH₄Cl in CM-PBS), lysis buffer (25 mM Tris, pH 7.4, 25 mM glycine, 150 mM NaCl and 5 mM EDTA, 1% triton X-100, 1X protease inhibitor cocktail), 3X loading buffer (150 mM Tris, pH 6.8, 6% SDS, 30% glycerol, 300 mM DTT and 0.01% bromophenol blue), and wash buffer (10 mM Tris (pH 7.4), 1.5 mM EDTA, 150 mM NaCl, 1% Triton X-100, 1X protease inhibitor cocktail).
5. Remove dishes holding the astrocyte cultures from the incubator, and discard the medium.
6. Wash cells thrice with 4 mL chilled CM-PBS, and place the dishes on crushed ice.
7. Pipette 2 mL biotin buffer into each well, and gently tilt dishes back and forth a few times to ensure complete coverage. Leave on ice for 30 min.
8. Remove the biotin buffer using an aspirator, and replace with 4 mL quenching buffer. Leave on ice for 10 min.
9. Aspirate the quenching buffer, and replace with an equivalent volume of the same. Again, leave on ice for 10 min.
10. Discard the quenching buffer, and wash cells thrice with 4 mL chilled CM-PBS.
11. Scrape cells into 1 mL chilled CM-PBS using a cell lifter, and transfer the suspension to microcentrifuge tube.
12. Pellet cells by centrifugation at 100 x g for 3 min. Discard the supernatant, and re-suspend cells in 500 µL of lysis buffer.
13. Leave samples on ice for 30 min, vortexing every 5 min, or place them on an end-over-end rotator at 4 °C.
14. Centrifuge the lysate at 14,000 x g for 10 min at 4 °C to pellet any detergent-insoluble materials. Transfer the supernatant into a new microcentrifuge tube.
    1. Save 50 µL of this lysate and add loading buffer to it. Then denature it by heating at 95 °C in a dry bath; this is the "input" fraction, containing both biotinylated cell-surface proteins, as well as non-biotinylated cytosolic proteins.
15. Widen the opening of a pipette tip by cutting off approximately 0.5 cm of material from its end using a pair of sharp scissors. Using this pipette tip, transfer 75 µL of streptavidin-agarose beads (normally stored at 4 °C) to the lysate, and incubate at 4 °C for 3 h on shaker/rocker.
    1. As streptavidin-agarose is frequently sold as a slurry containing 50% beads by volume, suspended in an antimicrobial solution, triturate the slurry to ensure that the beads are evenly suspended, and then pipette 150 µL of the suspension into each sample.
16. Pellet streptavidin-agarose beads by centrifugation at 1500 x g for 30 s at 4 °C.
    1. Save 50 µL of the supernatant (add loading buffer and denature it at 95 °C in a water bath or heating block); this represents the "intracellular" fraction, and is comprised primarily of non-biotinylated cytosolic proteins.
17. Resuspend the pelleted beads in 1 mL wash buffer, and rock this for 3 min at 4 °C. Pellet beads (as in step 1.16), and discard the supernatant. Repeat this process 4x to minimize the nonspecific binding of nonbiotinylated cytosolic proteins.
18. Pellet the beads by centrifugation (1500 x g for 30 s at 4 °C), and discard the overlying wash buffer. Add 50 µL of 1X loading buffer (diluted using lysis buffer). Release biotin and streptavidin from beads by denaturing this at 95 °C; this fraction should contain biotinylated cell-surface proteins only ("cell-surface" fraction).
    1. Separate input, cell-surface, and intracellular fractions by SDS-PAGE ¹³, and analyze by western blotting ¹⁴.
       NOTE: While we used a 4 – 20% precast gradient gel in our experiments, a 12 – 14% separating gel with a 4% stacking layer (each containing 0.1% SDS) should suffice for the proteins of interest in this study. A molecular weight standard of the appropriate size range should also be used. Note that there can sometimes be an observable upshift in the apparent molecular masses of biotinylated proteins.

2. Determining Internalization Rate of Cell-surface Proteins in Astrocytes by Biotinylation

NOTE: In the following, we describe a typical pulse-chase biotinylation experiment used in this instance to track the endocytosis of AQP4 in astrocytes. This method is based on that used by Madrid et al.¹⁵. Specialized materials required include sulfo-NHS-SS-biotin, streptavidin-agarose resin, reduced glutathione, and iodoacetamide (see the Table of Materials).

1. Prepare cultures of mouse cortical astrocytes in 60 mm dishes using the methods outlined in the previous section. Ensure that cells are approximately 70% confluent on the day of the assay, and that each dish contains an equivalent number of cells.
2. Immediately prior to assay, prepare the following, and place on ice or refrigerate: CM-PBS (100 mg/L MgCl₂∙6H₂O and 100 mg/L CaCl₂ in 1X PBS, pH7.4), biotin buffer (0.5 mg/mL sulfo-NHS-SS-biotin in CM-PBS), reducing buffer (50 mM reduced glutathione, 75 mM NaCl and 75 mM NaOH), quenching buffer (50 mM iodoacetamide, 1% BSA, in CM-PBS), lysis buffer (25 mM Tris, pH 7.4, 25 mM glycine, 150 mM NaCl and 5 mM EDTA, 1% triton X-100, 1X protease inhibitor cocktail), 3X loading buffer (150 mM Tris, pH 6.8, 6% SDS, 30% glycerol, 300 mM DTT and 0.01% bromophenol blue), and wash buffer (10 mM Tris, pH 7.4, 1.5 mM EDTA, 150 mM NaCl, 1% triton X-100, 1X protease inhibitor cocktail).
3. Prepare fresh cell culture medium (DMEM supplemented with 10% Fetal Bbovine Serum (FBS), 1% penicillin/streptomycin, and 1% L-glutamine), and place it in a 37 °C water bath.
4. Remove astrocyte cultures from the incubator, and aspirate the medium using an aspirator.
5. Wash cells thrice with 4 mL chilled CM-PBS, and place the dishes on crushed ice.
6. Pipette 3 mL biotin buffer into each dish, tilt dishes back and forth a few times to ensure that buffer is well-distributed, and leave this on ice for 30 min.
7. Aspirate biotin buffer, and replace it with 5 mL warm medium. Incubate a culture dish at 37 °C for 15 min, and a second dish at the same temperature for 30 min. Leave another dish at 4 °C as the 0 min sample.
8. At the end of the incubation period, discard medium, and wash cells thrice with 4 mL chilled CM-PBS. Pipette 6 mL reducing buffer over cells, and leave on ice for 15 min.
9. Remove reducing buffer, and then replace with 6 mL fresh reducing buffer. Place on ice for an additional 15 min.
10. Remove reducing solution and replace with 6 mL quenching buffer. Leave on ice for 15 min.
11. Repeat quenching step once more.
12. Discard quenching buffer, and wash cells thrice with 4 mL chilled PBS.
13. Scrape cells into 1 mL chilled PBS using a cell lifter, and transfer the suspension into a microcentrifuge tube.
14. Pellet cells by centrifugation at 100 x g for 3 min. Discard the supernatant, and resuspend cells in 500 µL lysis buffer.
15. Leave this on ice for 30 min and vortex every 5 min. Alternatively, place the samples on an end-over-end rotator at 4 °C for this duration.
16. Centrifuge the lysate at 14,000 x g for 10 min at 4 °C to pellet the detergent-insoluble materials, then transfer supernatant to a new microcentrifuge tube. Save 50 µL of this lysate, add loading buffer to it and denature at 95 °C in a dry bath; this is the "input" fraction, containing both biotinylated endocytosed proteins, as well as nonbiotinylated proteins.
17. Using a cut pipette tip, add 150 µL of the streptavidin-agarose slurry to the lysate, and incubate at 4 °C for 3 h on shaker/rocker. See 1.15 for additional details on this step.
18. Pellet streptavidin-agarose beads by centrifugation at 1,500 x g for 30 s at 4 °C.
19. Resuspend beads in 1 mL wash buffer, and rock for 3 min at 4 °C. Pellet beads (as per step 2.18), and discard the supernatant. Repeat this process 4x to minimize the nonspecific binding of nonbiotinylated cytosolic proteins.
20. Pellet beads by centrifugation at 1,500 x g for 30 s, at 4 °C, and discard overlying wash buffer. Add 50 µL 1X loading buffer (diluted using lysis buffer). Release biotin and streptavidin from beads by denaturing at 95 °C; this fraction should contain internalized cell-surface proteins only ("endocytosed" fraction).
21. Separate input, cell-surface, and unbound fractions by SDS-PAGE¹³, and analyze by western blotting¹⁴.
    NOTE: While we used a 4 – 20% precast gradient gel in our experiments, a 12 – 14% separating gel with a 4% stacking layer (each containing 0.1% SDS) should suffice for the proteins of interest in this study. A molecular weight standard of the appropriate size range should also be used. Note that there can sometimes be an observable upshift in the apparent molecular masses of biotinylated proteins.