Cellular responses to external stimuli heavily rely on the set of proteins that are expressed on the cell surface in a given moment. Accordingly, the number, the sub-cellular localization, and subunit composition of these proteins are dynamically controlled. Here, we will use an antibody feeding approach to quantify the level of surface expressed receptors in cell cultures, as long as the internalization and recycling resume.
This is a very versatile protocol, that provides mechanistic information of the regulation of surface expressed proteins. In our example, we will study glutamine receptors in primary hippocampal neurons. This protocol can be adapted to any protein possessing an anagenic extracellular epitope, if antibodies aren't available, transvection of tagged constructs can be useful for both labeling and studying specific mutations.
In our examples, we use antibodies against endogenous GluA1, and tagged Glu12 B.To begin this procedure, transfer the cover slips cell side up to a paraffin film covered tray, to save reagents, and facilitate manipulation. Save and maintain conditioned media at 37 degrees Celsius for incubation and washing steps. Incubate the cells with primary antibodies diluted in conditioned media, at room temperature, for 15 minutes.
After this, use a vacuum pipette to carefully aspirate off the antibody containing media, and wash the cells three times with conditioned media. If studying surface versus intracellular receptor expression fix the cells with paraformaldehyde after this step, as outlined in the text protocol. Maintain the cells in conditioned media without antibodies, and return them to the incubator at 37 degrees Celsius to allow for internalization.
If studying internalization process, fix the cells with paraformaldehyde after this step, as outlined in the text protocol. To block the epitopes on the primary antibody attached to the surface expressed receptors that have not been internalized, incubate cells with unconjugated Fab anti-IgG antibody fragments diluted in conditioned media, for 20 minutes at room temperature. After this, wash the cells three times with conditioned media.
Incubate the washed wells with conditioned media containing 80 micromolar Dynasore, at 37 degrees Celsius, to allow for recycling of internalized receptors. After finishing the modifications on live cells, wash the cells once with PBS plus. Add a solution of 4%paraformaldehyde and 4%sucrose in PBS to the cells, and incubate in a fume hood at room temperature for seven to eight minutes, to fix the cells.
Then wash the cells three times with regular PBS. Add a solution of 10%Normal Goat Serum in PBS to the cells, and incubate at room temperature for 30 minutes, to block non-specific binding sites. Next, incubate the cells with fluorescently tagged secondary antibody diluted in 3%NGS in PBS at room temperature for one hour, to label primary antibody label receptors.
After this, wash the cells three times with PBS. To begin labeling the intracellular receptors, add a solution of 0.25%TritonX in PBS, and incubate at room temperature for five to 10 minutes, to permeabilize the cells. Then block with 10%NGS at room temperature for 30 minutes.
If studying surface versus total, incubate the cells with the same primary antibody used previously, diluted with 3%NGS in PBS, at room temperature for one hour, to label the intracellular receptors. Next, wash the cells three times with PBS. Label with second fluorescently tagged secondary antibody, diluted in 3%NGS in PBS, at room temperature for one hour.
After this wash the cells three times with PBS. First gently place the cover slips, cell side down, on 12 to 15 microliters of the appropriate mounting media to mount the cells. Then image the cells on the appropriate confocal microscope, and analyze the cells as outlined in the text protocol.
This protocol to study glutamate receptor trafficking is based on differential labeling of receptors, expressed at the cell surface, and those expressed in internal membranes. After acquiring confocal images, the fluorescent signal can be easily quantified to show an increase in surface expression, relative to intracellular population, following chemical LTP. No signal for PSD-95 can be obtained in non-permeabilized cells, demonstrating the integrity of the plasma membrane.
This indicates that the signal obtained for surface GluA1 indeed corresponds to surface expressed receptors. Importantly, a minimal signal for internalized GluA1 can be observed under non-permeabilization conditions, showing that all surface epitopes are occupied by the initial round of antibody labeling. Receptors that have been internalized are then identified.
This exampled highlights that GluN2B phosphorylation at S1480 promotes receptor internalization. As the phosphomimetic mutant S1480E displayed a much higher internalization ratio compared to wild-type receptors. As expected, no signal is obtained for internalized receptors in the control.
Next, recycled receptors and internalized receptors are identified. The generated recycling ratio shows that GluN2B S1480E has no effect on NMDAR recycling. In the control, a strong signal can be observed for surface expressed GluN2B in the absence of Fab blocking.
This signal disappears in Fab-treated cultures, demonstrating that the blocking protocol is sufficient to completely block the surface expressed epitopes, and that the surface signals observed after recycling indeed correspond to receptors trafficked back to the plasma membrane. The current protocol is just one of the methods available for studying the regulation of surface expressed proteins. Other approaches can be taken to expand or verify the data obtained here.
Those include biochemical methods, as biotinylation, life imaging techniques, or functional approaches, like electrophysiology or calcium imaging. We use PFA to freeze the localization of receptors, however PFA is a known carcinogen, so it's crucial to perform steps using PFA under a fume hood, while wearing appropriate PPE.
