We recently characterized a labeling process for plasma membrane-localized KCa3.1 using BLAP-tagged channels in combination with recombinant BirA [28 ]. Briefly, cells were washed in PBS, followed by incubation in recombinant BirA for 30 min at room temperature, three PBS washes, incubation in streptavidin (10 μg/ml in PBS with BSA 1% ) at 4°C for 10 min and three washes in PBS with BSA 1% to remove unbound streptavidin. While this provides excellent labeling of channels, it takes nearly 50 min to complete and therefore needed to be streamlined for adaptation to a 96-well plate application. In light of this, we subcloned both BLAP-tagged KCa3.1 as well as BirA, in which a KDEL endoplasmic reticulum-retention motif (BirA-KDEL) had been added at the C-terminus (kindly provided in pDISPLAY), into the bicistronic plasmid, pBudCE4.1 (Invitrogen) behind the EF-1α and CMV promoters, respectively. BirA-KDEL was directly subcloned from pDISPLAY using Sal I and Hind III restriction sites. We then introduced a silent mutation into the Kpn I site that was transferred from the pDISPLAY vector during this cloning step. Finally, BLAP-KCa3.1 was PCR amplified from pcDNA3.1 and Kpn I, and Xho I restriction sites added in a single step and subcloned into pBudCE4.1 containing BirA-KDEL. Stable HEK293 cell lines were then selected using zeocin. With this approach, each subunit of the channel was biotinylated in the endoplasmic reticulum as it was synthesized via a covalent modification, resulting in a greater biotinylation efficiency compared with the addition of recombinant BirA. Cells expressing BirA-KDEL and KCa3.1-BLAP were seeded at a density of approximately 50% confluence (16 h prior to the experiment) into Nunc 96-well optical bottom black plates (Nalge Nunc International, Rochester, NY), which have been previously poly L-lysine coated. To ensure the highest biotinylation efficiency possible, 10-μM biotin was added to the growth media when the cells were seeded to eliminate the possibility that biotin supply is rate limiting.
To label plasma membrane channels, the growth media was removed, the cells were washed once with PBS with BSA 1% and the channels were labeled in a single step with streptavidin–Alexa555 (10 μg/ml in PBS with BSA 1%) at 4°C for 10 min followed by three washes in PBS with BSA 1% to remove unbound streptavidin. In this way, the total labeling time was reduced to approximately 12–15 min compared with the 50 min previously required. Cells treated in this way serve as controls (time = 0 min) in which all the channel is localized to the plasma membrane. To assess endocytosis of the channel, a subset of plates is returned to 37°C for 90 min in the absence or presence of the compound in order to be tested for its ability to inhibit endocytosis. We previously demonstrated that 90 min is sufficient to endocytose most of the labeled KCa3.1 in both HEK293 and endothelial cells [28 , Balut CM, Gao Y, Thibodeau PH, Murray SA, Devor DC. ESCRT-dependent targeting of plasma membrane localized KCa3.1 to the lysosomes. Manuscript Submitted]. It is important to note that the binding of streptavidin to biotin is essentially irreversible, with a Ka of 1015 M−1, such that it will stay attached to the channel throughout its endocytosis and subsequent degradation. To adapt our endocytosis assay to an automated analysis system we labeled the plasma membrane with WGA–Alexa488 (wheat germ agglutinin, 5 μg/ml, Invitrogen) in PBS with BSA 1% at 4°C, either at time 0 or after 90 min. Importantly, this WGA-labeling step is carried out prior to the cells being fixed and permeabilized, such that only the plasma membrane was labeled. For this, the cells are either immediately incubated in WGA–Alexa488 (time 0) or the growth medium is first removed and the cells washed once in PBS with BSA 1% (time 90 min) prior to WGA–Alexa488 labeling. Subsequently, the cells are washed three times in PBS with BSA 1% and once in PBS at 4°C to remove unbound WGA. This protocol allowed us to unequivocally define the membrane fluorescently and thus colocalize the channel with the plasma membrane as a means of assessing endocytosis. Subsequent to WGA-Alexa488 labeling, the cells were fixed and permeabilized in paraformaldehyde 2% with Triton X-100 0.1% for 15 min and the nuclei labeled with DAPI for 30 s. A schematic depicting this protocol and the labeling of plasma membrane channels is shown in Figure 1. Also shown is a high-resolution image at both time 0 and 90 min to illustrate the labeling of both KCa3.1 with streptavidin–Alexa555 and the plasma membrane with WGA–Alexa488 (Figure 2). As is apparent, at time 0, all of the channel is resident in the plasma membrane and colocalizes with WGA (the plasma membrane marker). After 90 min at 37°C, the majority of KCa3.1 has been endocytosed resulting in less colocalization with WGA–Alexa488.
Following labeling, individual cells within each well of the 96-well plate were imaged using a Nikon TiE inverted, widefield epifluoresence microscope with Plan–Neofluor objectives, a motorized six-position fluorescent filter cube turret with zero-pixel shift filters and a registered motorized stage with plate insert. The 12-bit grayscale images were captured using a Retiga 2000 camera (QImaging) and Volocity Acquisition software (v5.3.1, Perkin Elmer) that autofocused on the plasma membrane using the WGA–Alexa488 fluorescence, automatically capturing four to ten images per well. For these studies we utilized a 20× air objective with an numerical aperature of 0.5. Under the experimental conditions, both cellular autofluorescence and the autofluorescence associated with UBEI-41 were negligible and eliminated by thresholding prior to image collection. Fields that failed to autofocus or which were devoid of cells were ignored. Images were analyzed using the Volocity Quantification module using the colocalization analysis feature. Under the imaging conditions employed, there are 0.738 μm per pixel in both the x and y planes. Colocalization between the red and green channels is expressed as a Pearson’s correlation where a value of 1 indicates complete colocalization and 0 indicates no colocalization. A Pearson’s correlation coefficient was calculated for each experimental condition by averaging the coefficients for individual imaged fields. Initially, at time 0, KCa3.1 was exclusively localized to the membrane such that the Pearson’s correlation was high. However, given that KCa3.1 did not occupy all membrane space, colocalization will be less than 100% and was determined by expression level. Clearly, a high level of expression is necessary to obtain a large signal-to-noise level for these experiments, such that endocytosis can be clearly defined relative to time 0. This was obtained by having a stable cell line that expresses KCa3.1 in all cells driven by the EF-1α promoter coupled with high-efficiency labeling of the channel with biotin by utilizing the BirA-KDEL driven off a CMV promoter. The minimal starting colocalization, which will allow us to clearly define small-molecule modulators of endocytosis, has not been defined. Following endocytosis, colocalization of KCa3.1 with membrane fluorescence will be reduced such that modulators of this event can be defined.
Balut C.M., Gao Y., Luke C, & Devor D.C. (2010). Immunofluorescence-based assay to identify modulators of the number of plasma membrane KCa3.1 channels. Future medicinal chemistry, 2(5), 707-713.