In this video, two methods will be presented, cell-surface biotinylation, and pulse-chase biotinylation. In the following demonstration, we will be using astrocytes. These are the most abundant glial cell types in the brain, and they happen to express our protein of interest which is Aquaporin-4.
The methods that we use in this paper could be applied to many different cell types, and those could include either cells in suspension or adherent cells. These techniques can be used to study the cell surface expression and internalization of virtually any transmembrane protein, as long as it possesses an extracellular domain accessible to biotin. The overall goal of this cell-surface biotinylation assay is to determine the effects of laminin on the expression of the water permeable channel Aquaporin-4 at the plasma membrane of astrocytes.
To begin this procedure, remove the astrocyte cultures from the incubator, and discard the medium by aspiration. Wash the cells thrice with four milliliters of chilled CM-PBS, and place the dishes on crushed ice. Remove the CM-PBS by aspiration, and then pipette two milliliters of biotin buffer into each well.
Gently tilt the dishes back and forth a few times to ensure complete coverage before placing the cultures on ice for 30 minutes. Afterward remove the biotin buffer, replace it with four milliliters of quenching buffer and keep the cultures on ice for 10 minutes. Next, aspirate and replace with the same amount of the quenching buffer before leaving the cultures on ice for 10 minutes.
Then, discard the quenching buffer and wash the cells thrice with four milliliters of chilled CM-PBS. Following this, scrape the cells into one milliliter of chilled CM-PBS and transfer the suspension to a microcentrifuge tube. Next, pellet the cells by centrifugation at 100 g for three minutes in a refrigerated microcentrifuge set to four degrees Celsius.
After that, discard the supernatant and resuspend the cells in 500 microliters of lysis buffer. Then, leave the samples on ice for 30 minutes, vortexing every five minutes. Following that, centrifuge the lysate at 14, 000 g for 10 minutes at four degrees Celsius to pellet any detergent and soluble materials.
Then, transfer the supernatant into a new microcentrifuge tube. Save 50 microliters of the lysate, and add 25 microliters of three X loading buffer to it. Subsequently, denature it by heating at 95 degrees Celsius in a dry bath.
This is the input fraction, containing both biotinylated cell-surface proteins as well as nonbiotinylated cytosolic proteins. Using a cut pipette tip, transfer 75 microliters of streptavidin agarose beads to the lysate, and incubate it at four degrees Celsius for three hours on a shaker. After that, pellet the streptavidin agarose beads by centrifugation at 1, 500 g for 30 seconds at four degrees Celsius.
Save 50 microliters of the supernatant and add 25 microliters of three X loading buffer. Then, denature it at 95 degrees Celsius in a water bath or a heating block. This represents the intracellular fraction, and is comprised primarily of nonbiotinylated cytosolic proteins.
Afterward, resuspend the pelleted beads in one milliliter of wash buffer and rock it for three minutes at four degrees Celsius. Pellet the beads and discard the supernatant. Repeat this process four more times to minimize the nonspecific binding of nonbiotinylated cytosolic proteins.
Add 50 microliters of loading buffer diluted to one X using lysis buffer. Release biotin and streptavidin from the beads by denaturing them at 95 degrees Celsius. This fraction should contain biotinylated cell-surface proteins only.
Subsequently, separate the input, cell surface, and intracellular fractions by SDS-PAGE and analyze by western blotting. The overall goal of the pulse-chase biotinylation experiment is to determine the approximate internalization rate of Aquaporin-4 in astrocytes. First, remove the astrocyte cultures from the incubator and aspirate the medium.
Then, wash the cells thrice with four milliliters of chilled CM-PBS and place the dishes on crushed ice. Aspirate the CM-PBS, and then pipette three milliliters of biotin buffer into each dish. Tilt the dishes back and forth a few times to ensure that buffer is well-distributed and leave them on ice for 30 minutes.
Then, aspirate the biotin buffer, and replace it with five milliliters of warm medium. Incubate one culture dish at 37 degrees Celsius for 15 minutes and a second dish at the same temperature for 30 minutes. Leave another dish at four degrees Celsius as the zero-minute sample.
At the end of the incubation period, discard the medium and wash the cells thrice with four milliliters of chilled CM-PBS. Afterwards, aspirate the CM-PBS, and then pipette six milliliters of reducing buffer over the cells, and leave the sample on ice for 15 minutes. Then, replace the medium with six milliliters of fresh reducing buffer, and place the sample on ice for an additional 15 minutes.
This step is critical as the glutathione contained within the reducing buffer will cleave the biotin moasis remaining at the cell surface. This ensures that only internalized proteins will be biotin labeled. Following that, remove the reducing solution, and replace it with six milliliters of quenching buffer.
Leave the sample on ice for another 15 minutes, and repeat the quenching step once more. Then, discard the quenching buffer, and wash the cells thrice, with four milliliters of chilled PBS. Aspirate the CM-PBS, and then scrape the cells into one milliliter of chilled PBS, using a cell lifter, and transfer the suspension into a microcentrifuge tube.
Pellet the cells by centrifugation at 100 g for three minutes. After three minutes, discard the supernatant, and resuspend the cells in 500 microliters of lysis buffer. Leave the sample on ice for 30 minutes, and vortex every five minutes.
Centrifuge the lysate at 14, 000 g, for 10 minutes at four degrees celsius, to pellet the detergent and soluble materials. Then, transfer the supernatant to a new microcentrifuge tube. Save 50 microliters of this lysate, and add loading buffer.
Subsequently, denature the cells at 95 degrees celsius, in a dry bath. This is the input fraction, containing both biotinylated endocytosed proteins, as well as nonbiotinylated proteins. Using a cut pipette tip, add 150 microliters of the streptavidin agarose slurry to the lysate, and incubate it at four degrees celsius for three hours, on a shaker.
After three hours, pellet the streptavidin agarose beads by centrifugation, at 1, 500 g for 30 seconds, at four degrees celsius. Resuspend the beads in one milliliter of wash buffer, and rock them for three minutes at four degrees celsius. Pellet the beads, and discard the supernatant.
Repeat this process four more times to minimize the non-specific binding of nonbiotinylated cytosolic proteins. Then, pellet the beads by centrifugation at 1, 500 g for 30 seconds, at four degrees celsius. Discard the overlying wash buffer, and add 50 microliters of one x loading buffer.
Release biotin and streptavidin from the beads, by denaturing at 95 degrees celsius. This fraction should contain internalized cell surface proteins only. Then, separate the input, cell surface, and unbound fractions, by SDS-PAGE, and analyze by Western blotting.
Shown here, are the cell surface, intracellular, and total fractions from the untreated astrocytes, and astrocytes treated with 24 nanomolar laminin, probed for Aquaporin-4, and beta-dystroglycan. This histogram illustrates the differences in the cell surface levels of Aquaporin-4, in the untreated and laminin treated astrocytes, normalized against beta-dytroglycan. In this figure, the internalized protein fractions collected at zero, 15, and 30 minutes, were probed for Aquaporin-4.
And this histogram, summarizes the internalization of Aquaporin-4, for four independent experiments, normalized against values for the zero minute time point. The asterisk indicates a statistically significant increase in the amount of Aquaporin-4, endocytosed between 15 and 30 minutes. When glutathione is used to break the disulfide bond in the cleavable biotin analog, the contribution of cell surface Aquaporin-4 to the biotinylated fraction, is sharply reduced.
Resulting in a clear difference between the zero minute, and 15 minute samples. When the step is omitted, the signal originating from the cell surface pool, renders the change between the two time points undetectable. Following this procedure, other methods like immunofluorescence and TIRF Microscopy could be applied to answer additional questions, including physical trafficking of the cargo.
