Several mGAT1 C-terminal fluorophore fusions display wild-type function and surface localization
We studied the subcellular localization of the mGAT1 C-terminal fluorophore addition constructs by laser-scanning confocal fluorescence microscopy () and compared their function to nonfluorescent wild-type mGAT1 in [3
H]GABA uptake assays (). With the optimized [3
H]GABA uptake assays described in Materials and methods, the major source of variability was apparently unavoidable variations in cell growth. Therefore, where appropriate, we used side-by-side assays (as in and ); in such assays, we believe that differences in substrate transport velocity of ≥25% reflect real differences in the membrane levels of functional transporters. The mGAT10
XFP constructs display fluorescence throughout the cytoplasm; at the periphery of the cells (that is, in the region near large amounts of plasma membrane), the fluorescence is brighter (). shows that this peripherally enhanced fluorescence does not represent complete plasma membrane insertion of mGAT10
XFP constructs: [3
H]GABA uptake of both mGAT10
YFP and mGAT10
CFP functioned less well (41 ± 1 and 60 ± 8%, respectively) compared with wild-type mGAT1. This poor function was not revealed by our previous expression system in HEK 293T cells (Chiu et al., 2002
), but a functional deficiency was observed when neurons of mGAT10
GFP knock-in mice exhibited reduced [3
H]GABA uptake compared with wild-type mice. We therefore believe that the present assay system has good relevance to several processes that control functional levels of mGAT1 expression in neurons.
Figure 3. Expression and subcellular localization of C-terminal XFP addition mGAT1 constructs in N2a cells. Each panel displays representative fluorescence microscopy images of live N2a cells expressing each C-terminal addition construct 48 h after transfection (more ...)
Figure 4. Functional characterization of C-terminal fusion mGAT1XFP constructs. (A) 20-min [3H]GABA uptake from N2a cells transfected with 100 ng/well of mGAT1 wild-type plasmid, an equimolar amount of the fluorescently tagged mGAT1 plasmids, or blank pcDNA3.1(+) (more ...)
Figure 5. Expression and functional characterization of C-terminal insertion mGAT1XFPCT constructs. (A) Each panel displays exemplar fluorescence microscopy images of live N2a cells expressing each of the C-terminal insertion constructs 48 h after transfection. (more ...)
We include data for mGAT10
GFP for primarily historical reasons. Its poor function in a knock-in mouse strain (Chiu et al., 2002
) provided a major motivation for the present study. Surprisingly, the mGAT10
GFP functioned even less well (22 ± 2% of wild type) than the corresponding YFP and CFP constructs. We have not systematically explored the source of the differences among the functional data for the three fluorophore fusions (YFP, CFP, and GFP), but as noted, the generally poor function of these mGAT10
XFP constructs was expected from the functional deficits of the knock-in mouse strain. Because the YFP-CFP FRET pair forms the basis of most measurements, we have not constructed additional GFP-containing mGAT1 mutants.
The constructs mGAT1XFP*, mGAT1XFP3, mGAT1XFP8, mGAT1XFP20, and mGAT1XFP28 exhibited strong localization in the cell periphery (). We determined that a large part of this fluorescence distribution arose from surface membrane expression. Three complementary assay types provide evidence of this point. (1) For each of these five constructs, [3H]GABA uptake from transfected N2a cells did not differ significantly from wild-type mGAT1 (). (2) Surface biotinylation experiments determined that 38–44% of the total expressed mGAT1XFP*, mGAT1XFP3, mGAT1XFP8, mGAT1XFP20, or mGAT1XFP28 protein inserted into the plasma membrane, which again did not differ significantly from wild-type mGAT1 (43%; ). (3) Concentration dependence of [3H]GABA uptake for mGAT1XFP*, mGAT1XFP8, mGAT1YFP3, and mGAT1YFP20 was, again, identical to wild-type mGAT1 (). Each panel of presents the concentration dependence data for two fluorescent constructs versus wild-type mGAT1 transfections performed in side-by-side assays. Within each set of experiments, the Km and Vmax for the fluorescently tagged transporters did not differ significantly from those of wild-type mGAT1.
On the other hand, mGAT1XFP45 constructs were expressed primarily inside the cell (). This point is reported by markedly impaired [3H]GABA uptake compared with wild-type mGAT1 (mGAT1CFP45, 21 ± 2%; mGAT1YFP45, 22 ± 2%; ) and by the very small fraction of the total mGAT1XFP45 protein that partitioned into the plasma membrane in biotinylation assays (2.8 ± 0.6% compared with 44 ± 5% for wild type; ). It should be noted that mGAT1XFP45 was particularly challenging to process during surface biotinylation experiments because the majority of the protein sample aggregated in the well of the acrylamide gels and did not separate under electrophoresis. Apparently, the mGAT1XFP45 constructs suffer from one or more major deficits.
Diminished mGAT1 function results if XFP is incorporated close to TM12
In the mGAT1565
CT and mGAT1570
CT constructs, the XFP insertion sites interrupted motifs that are important in the regulation of mGAT1 trafficking (Holton et al., 2005
; Farhan et al., 2007
; Boudanova et al., 2008
). However, the C terminus region interrupted by our mGAT1577
CT construct has little influence on mGAT1 oligomerization and trafficking, according to a previous report (Farhan et al., 2004
). displays the subcellular localization of the mGAT1565
CT, and mGAT1577
CT constructs. These three construct designs expressed varying degrees of concentrated fluorescence at the periphery of the cells close to the plasma membrane as well as in intracellular compartments. The intracellular localization was greatest in the mGAT1565
CT constructs, and in many instances, expression was confined entirely to intracellular compartments. This observation is exemplified by the mGAT1565
CT image presented in (21 of out of 79 cells imaged). Notably, all cells expressing mGAT1570
CT or mGAT1577
CT constructs exhibited some apparent plasma membrane localization, with mGAT1577
CT showing strong fluorescence concentrated in the cell periphery (), similar to that observed for mGAT1XFP3-mGAT1XFP28. We studied the [3
H]GABA uptake properties of the C-terminal YFP insertions (). mGAT1565
CT exhibited a marked deficit in uptake compared with wild-type mGAT1. Uptake from N2a cells expressing mGAT1570
CT was intermediate between mGAT1565
CT and wild-type mGAT1. Uptake from mGAT1577
CT-expressing cells did not differ significantly from wild-type mGAT1. In concentration–response experiments, the Km
CT also did not differ significantly from wild-type mGAT1, and its Vmax
was 78% that of wild type (). Thus, the presence of the XFP fluorophores between the C-terminal R565 and Q571 residues inhibited interactions required for normal trafficking of mGAT1 to the plasma membrane. Locating the XFP six residues more distally in the C terminus avoided such inhibitions and maintained mGAT1577
CT transporters at the plasma membrane in numbers almost equivalent to the wild-type nonfluorescent mGAT1.
N-terminal fluorophore additions impair mGAT1 function and trafficking
The N-terminally labeled XFP15mGAT1 fusions were primarily retained within intracellular regions () and displayed significantly reduced [3
H]GABA uptake compared with wild-type mGAT1 (CFP15mGAT1 35 ± 2% and YFP15mGAT1 28 ± 6%; ). This extended our previous experience with an N-terminally labeled GFPmGAT1 fusion that had only eight amino acids in its linker between the fluorophore and the mGAT1; the previous construct also failed to localize appropriately to the plasma membrane (Chiu et al., 2002
Acceptor photobleach FRET with fluorescent mGAT1 constructs shows that oligomerization is required for wild-type function
Dimerization/oligomerization of SLC6 transporter protomers is required for export of functional transporter from the ER to the plasma membrane (Farhan et al., 2006
; Bartholomäus et al., 2008
). Therefore, fluorescent mGAT1 constructs that behaved functionally like wild-type mGAT1 were expected to form oligomers of mGAT1 protomers. We studied the assembly of our fluorescent GAT1 constructs using the acceptor photobleach FRET technique (Nashmi et al., 2003
). We first investigated acceptor photobleach FRET between mGAT1XFP* pairs and between mGAT1XFP8 pairs, which are C-terminal GAT1–XFP fusion constructs that functioned like wild-type mGAT1 in both uptake and surface biotinylation experiments (). To quantify acceptor photobleach FRET between the mGAT1 oligomers, we constructed a scatter plot of the photobleach-induced changes in CFP and YFP fluorescence of each cell during the entire bleaching time, fitted a regression line to the data, and extrapolated to complete YFP photobleach (). The extrapolated values of CFP photorecovery for the mGAT1CFP*-mGAT1YFP* and the mGAT1CFP8-mGAT1YFP8 transfection pairs were 1.10 ± 0.01 and 1.26 ± 0.01, respectively (). Eq. 1 yields calculated FRET efficiencies of 9% for mGAT1CFP8/mGAT1YFP8 and 21% for mGAT1CFP*/mGAT1YFP*. For the C-terminal mGAT1XFP45 fusions, which have poor function, we detected no measurable FRET between mGAT1CFP45-mGAT1YFP45 coexpressed in N2a cells (). Likewise, no detectable acceptor photobleach FRET signal was recorded from cells coexpressing the CFP15mGAT1-YFP15mGAT1 constructs ().
Figure 6. FRET assessed by acceptor photobleach. (A) Plots of acceptor photobleach and donor photorecovery for mGAT1CFP*/mGAT1YFP* (n = 10) or (B) mGAT1CFP8/mGAT1YFP8 (n = 7) coexpressed in N2a cells. Data for each fluorophore were normalized to absolute fluorescence (more ...)
Pixel-based FRET reveals several components to the total FRET signal amplitude distribution
The acceptor photobleach FRET data gave a reasonable report of the average FRET signal from confocal images of whole N2a cells expressing fluorescent GAT1 protomers. However, acceptor photobleaching is not optimal for measurement of FRET at subcellular resolution in live cells. Attempting acceptor photobleach FRET in subcellularly differentiated ROIs would introduce error in the temporal resolution because imaging would be far slower than the dynamics of transporter trafficking. To determine FRET in specific ROIs, we used a spectrally resolved, sensitized emission FRET approach that determines FRET in a sample on a pixel-by-pixel basis and during the few microseconds required to measure each pixel (pixels are 69-nm squares). The spectrally unmixed images provide data similar to the three-filter method reported by Xia and Liu (2001)
, but using an optically more efficient, simultaneous detection of photons. Furthermore, we included the NFRET amplitude of each pixel as a datum in the analysis rather than averaging the signal amplitudes of all the pixels in an ROI. As shown below, this technique revealed that (a) FRET varies among subcellular compartments as defined by each ROI, and (b) within every ROI, the total NFRET distribution consists of two or three subcomponents, each with a distinct mean NFRET amplitude. For each fluorescent mGAT1 construct design studied, we analyzed the number of NFRET components, their amplitudes, and the proportion of the total NFRET distribution represented by each component. Comparing these data with the measured functional properties of each construct suggested that the subcomponents of NFRET distributions may represent mGAT1 dimers, higher-order mGAT1 oligomers, and mGAT1 oligomers interacting with PDZ domain–containing complexes, as explained in the Discussion and Appendix
Negative controls with noninteracting membrane proteins
For the negative control, we expressed two fluorescently tagged plasma membrane proteins, mGAT1 and the α4β2 nAChR, which do not interact in N2a cells (Drenan et al., 2008
). The mGAT1CFP8 construct was cotransfected with plasmids that assemble α4YFP/β2 nAChRs (Nashmi et al., 2003
; Khakh et al., 2005
; Drenan et al., 2008
; Son et al., 2009
). Control transfections of mGAT1CFP8/α4/β2 and mGAT1/α4YFP/β2 plasmids (250 ng of each plasmid) were also performed (a) to generate reference spectra and (b) to determine spectral bleedthrough as described previously. Both fluorescently tagged proteins maintained their normal expression pattern when coexpressed in N2a cells (): the fluorescence pattern for fluorescent α4YFPβ2 nAChRs was uniform with little enhancement at the plasma membrane (), as described previously in N2a cells (Drenan et al., 2008
; Son et al., 2009
), and the fluorescence pattern for mGAT1CFP8 resembled that of .
Figure 7. Pixel-by-pixel quantification of sensitized emission FRET between mGAT1CFP8 and α4YFPβ2 nAChRs. A negative control experiment. (A; from left to right) mGAT1CFP8 fluorescence and α4YFP nAChR subunit fluorescence unmixed from an (more ...)
We defined a whole cell ROI that encompassed all the fluorescent pixels in the cell minus the nonfluorescent cell nucleus. The mean NFRET for all pixels within this ROI for cells coexpressing mGAT1CFP8 and α4YFP/β2 nAChR was negative, indicating that no FRET occurred and that there was some overcorrection for donor and acceptor bleedthrough when there was no FRET ( and ).
Average NFRET for all pixels measured in each ROI
Because the mGAT1CFP8 construct localized strongly in the cell periphery, we used this fluorescence to define a second ROI that contributed “peripheral NFRET.” The peripheral ROI encompassed both the plasma membrane and a narrow annulus (~700 nm) of immediately adjacent cytoplasm. We use this description because constructs such as mGAT10
XFP exhibit concentrated fluorescence in the cell periphery due to pooling of transporter-containing vesicles within ~500 nm of the outer lipid bilayer of the cell, rather than because of efficient insertion into the plasma membrane (Chiu et al., 2002
). The calculated peripheral NFRET for coexpressed mGAT1CFP8 and α4YFP/β2 nAChR was also negative ( and ).
introduces frequency distributions of NFRET amplitudes from each pixel of several dozen cells. These data are binned to form all-pixel NFRET amplitude distributions: NFRET amplitude on the x axis and number of pixels on the y axis. Although the NFRET distributions for both ROIs for these negative control transfections were best fit with two Gaussians, both components had negative mean NFRET amplitudes (). These data confirm that the method detects no interaction between the α4β2 nAChR and GAT1 in intracellular regions or in the cell periphery.
Region-specific FRET quantification reveals high FRET efficiency in the periphery of cells expressing fluorescent GAT1 constructs that exhibit wild-type function
To investigate FRET between fluorescent GAT1 fusions, we defined four ROIs for each cell imaged. These were named the “whole cell ROI,” the “intracellular ROI,” the “perinuclear ROI,” and the “peripheral ROI.” The whole cell and peripheral ROIs were defined in the control experiments. Intracellular ROI is the space within the concentrated fluorescence at the cell periphery, but subtracting the dark space occupied by the cell nucleus, and is densely filled by ER (Fig. S1 A
). The perinuclear ROI describes a concentrated region of fluorescence in cells expressing the fluorescent mGAT1 constructs adjacent to the cell nucleus; according to organelle markers, this ROI comprises mainly ER and Golgi (Fig. S1 B). Fluorescence images of cells coexpressing the CFP and YFP variants of fluorescent GAT1 fusions were acquired and processed as described above.
Sensitized FRET from cells expressing the wild-type–like C-terminal fusion constructs mGAT1XFP* and mGAT1XFP8 confirmed earlier acceptor photobleach FRET findings that oligomerization was required to observe wild-type–like function from our fluorescent constructs (). In addition, for mGAT1XFP* and mGAT1XFP8, respectively, NFRET images showed that 30.1 and 43.3% of the whole cell ROI NFRET signal came from the peripheral ROI (, and ). The mean NFRET amplitudes for all pixels in an ROI for N2a cells expressing mGAT1XFP* or mGAT1XFP8 constructs were greatest in the peripheral ROI compared with the intracellular or the perinuclear ROIs (, and ). Specifically, the ratios of NFRET in the peripheral ROI were 1.6- and 1.7-fold greater than in the perinuclear ROIs for mGAT1XFP* and mGAT1XFP8, respectively. Also, the reported NFRET from pixels in the peripheral ROI of mGATXFP8-expressing cells had a much broader IQR than those from the perinuclear ROI ( and ). mGAT1 oligomerization was therefore detected in all ROIs, but an additional molecular event, specifically localized to the periphery of cells expressing mGAT1XFP* and mGAT1XFP8, resulted in elevated FRET in this region. Analysis of the NFRET distributions from each ROI determined that the NFRET from the intracellular and perinuclear ROIs of mGAT1XFP*- or mGAT1XFP8-expressing cells were best fit with two Gaussians, both reporting positive mean NFRET. However, the whole cell ROIs and the peripheral ROIs were best fit with three Gaussians (). The highest-amplitude NFRET component (1.2- to 1.8-fold greater mean NFRET than from the intermediate-amplitude component) represented 27–30% of the pixels in the peripheral ROIs (). Among the three subcellular ROIs, this highest-amplitude component appeared only in the peripheral ROI and was accompanied by an ~30% reduction in the peripheral ROI NFRET signal contributed by the lowest-amplitude component when compared with the intracellular or perinuclear ROIs (). Thus, for the wild-type–like functioning mGAT1XFP* and mGAT1XFP8, we infer that two different oligomerization events were described by the low- and intermediate-amplitude components that were common to the NFRET distributions of all examined ROIs (see Discussion and Appendix
). In addition, elevated mean NFRET in the peripheral ROI versus intracellular regions corresponded to the third high-amplitude component in the peripheral ROI NFRET distribution. We infer that a specific mGAT1 oligomerization or interaction event—the molecular correlate for the third high-amplitude NFRET component—is highly localized to the cell periphery.
Figure 8. Pixel-by-pixel quantification of sensitized emission FRET between mGAT1XFP* and mGAT1XFP8. (A; left two panels) mGAT1CFP* fluorescence and mGAT1YFP* fluorescence (calibration bars, ACUs). In the third panel, ROIs were used to determine NFRET. The red-shaded (more ...)
Sensitized NFRET from mGAT1XFP45, which reported little function and no FRET by acceptor photobleach analysis, was studied pixel by pixel (). This method did detect some mGAT1XFP45 oligomerization in small regions. These pixels were too few to influence the whole cell averaging algorithms that calculated FRET by acceptor photobleaching. The perinuclear ROI contained most of these small regions highlighted by the sensitized NFRET approach in mGAT1XFP45-expressing specimens. The mean NFRET amplitude for all pixels in the perinuclear ROI resembled that recorded for mGAT1XFP* or mGAT1XFP8 ( and ). For mGAT1XFP45, the FRETing pixels in the peripheral ROI contributed only 6.4% of the whole cell ROI NFRET signal, nonetheless indicating that some oligomerized mGAT1XFP45's were exported from the perinuclear region and could eventually contribute to the small but significant [3H]GABA uptake for this construct in the functional assays. The NFRET distributions for all ROIs of mGATXFP45-expressing cells were best fit with two Gaussians, the first of which reported a negative mean NFRET (). The positive population made up >75% of the NFRET signal in the peripheral and perinuclear ROIs. We observed that the mean NFRET of the positive Gaussian component was 1.4-fold larger in the peripheral ROI compared with the same component of the mGAT1XFP45 perinuclear ROI NFRET. Even though very few mGAT1XFP45 oligomers inserted into the plasma membrane, it appeared that those that did insert were subject to the same molecular event that caused increased FRET in the periphery of cells expressing wild-type functioning fluorescent mGAT1 constructs.
Figure 9. Pixel-by-pixel quantification of sensitized emission FRET between mGAT1XFP45. (A; from left to right) mGAT1CFP45 fluorescence and mGAT1YFP45 fluorescence (calibration bars, ACUs), ROIs used to determine NFRET (color coding as in ), and the NFRET (more ...)
Next, we examined the FRET of the mGAT15xxXFP5xxCT constructs, which show a graded improvement in function as the XFP moiety is moved downstream within the C terminus away from TM12 (). mGAT1565XFP566CT reported a robust mean NFRET amplitude for all pixels (≥0.17) in all ROIs examined (, and ), indicating that significant transporter oligomerization was occurring. However, the poor function of this construct, the completely internal distribution of fluorescence in 27% of cells expressing these constructs, and the minor component (16% of the whole cell ROI NFRET) contributed from the peripheral ROI lead us to conclude that the NFRET signal was mainly due to assembled intracellular mGAT1565XFP566CT rather than plasma membrane–inserted transporters, even in the peripheral ROI. The ratio of the mean NFRET for all pixels in the peripheral ROI compared with the perinuclear ROI was 0.99. mGAT1565XFP566CT NFRET distributions were best fit with two Gaussians in all ROIs examined (). In the peripheral and perinuclear ROIs, the two subpopulations contributed approximately equally to the total NFRET signal. In the intracellular ROI, the lower mean NFRET signal predominated.
Figure 10. Pixel-by-pixel quantification of sensitized emission FRET between mGAT1565XFP566CT and mGAT1570XFP571CT. (A; from left to right) mGAT1565CFP566CT fluorescence and mGAT1565YFP566CT fluorescence (calibration bars, ACUs), ROIs used to determine NFRET (color (more ...)
The mean NFRET amplitude from mGAT1570
CT-expressing cells (, and ) was less than that from those expressing mGAT1565
CT. This probably arose in part from the increased fluorophore separation in this construct. The XFP moiety is fused five residues more distal from the end of the TM12 helix in the mGAT1570
CT constructs. Exact intermolecular distances cannot be calculated from FRET efficiencies of proteins fused to GFP derivatives (Rizzo et al., 2006
). However, side-by-side FRET efficiency calculations (using Eq. 4) suggested that in the perinuclear ROIs, the apparent fluorophore separation in mGAT1570
CT oligomers (E = 7.9 ± 1.9%) is on average 1.25-fold greater than in mGAT1565
CT oligomers (E = 25 ± 3.5%). As for mGAT1565
CT, the mean NFRET amplitude from all pixels in cells expressing mGAT1570
CT was similar between the peripheral and perinuclear ROIs (ratio 1.0). Analysis of the NFRET distributions for each ROI determined that all were best fit with two Gaussians (). However, in the periphery, the two components did not appear to describe the same populations reported by the components of the other ROIs. The mean amplitude of the major peripheral NFRET component (98% of the signal) lay approximately halfway between the amplitudes of the two NFRET components of the perinuclear, intracellular, or whole cell ROIs. We suggest that two Gaussian components merged into one with an intermediate NFRET amplitude in the periphery because the populations mix at a level too fine to resolve in a 69-nm2
pixel (see Appendix
). The second component of the mGAT1570
CT peripheral NFRET signal had a mean amplitude (0.29 ± 0.09) twice that observed for the second Gaussian component of the perinuclear ROI NFRET (). Although contributing only a small fraction (2.3%) of the total signal for the peripheral ROI, the ratio of mean NFRET amplitude of this component versus the second component of the perinuclear or intracellular ROIs (2.1 and 1.7, respectively) resembled that of the highest-amplitude component of mGAT1XFP* or mGAT1XFP8 NFRET (). The small but measureable high-amplitude NFRET component in the mGAT1570
CT peripheral NFRET signal presumably reflects its impaired plasma membrane insertion, as determined by GABA uptake assays (), and is also consistent with the observation that mGAT1570
CT functions better than mGAT1565
mGAT1577XFP578CT, which exhibited almost wild-type function (), displayed strong mean NFRET for all pixels in the peripheral ROI, 1.7-fold greater than that observed in the perinuclear ROI (, and ). This NFRET amplitude was 1.5- to 1.8-fold stronger than that for the mean NFRET amplitude from all pixels in the peripheral ROI of mGAT1XFP*- or mGAT1XFP8-expressing cells (). As observed for those other wild-type functioning C-terminal fusions, the NFRET amplitude in the intracellular and whole cell ROIs was significantly less. The NFRET distributions from intracellular and perinuclear ROIs of mGAT1577XFP578CT-expressing N2a cells could be fitted with two Gaussians with low and intermediate mean NFRET amplitudes, the latter component providing approximately two thirds of the NFRET signal in the perinuclear ROI (). Importantly, in the peripheral ROI, a third Gaussian component appeared; this component was also detectable in the whole cell ROI (). This component had a mean NFRET amplitude (0.31 ± 0.04) twice that of the second intermediate-amplitude NFRET component and comprised ~30% of the peripheral NFRET signal. The relative distribution of transporter subpopulations for this construct, as determined by the components of NFRET in each ROI, resembled that for the wild-type functioning mGAT1XFP* and mGAT1XFP8 constructs ().
Figure 11. Pixel-by-pixel quantification of sensitized emission FRET between mGAT1577XFP578CT. (A; from left to right) mGAT1577CFP578CT fluorescence and mGAT1577YFP578CT fluorescence (calibration bars, ACUs), ROIs used to determine NFRET (color coding as in (more ...)
High NFRET in the peripheral ROI is independent of variance of YFP intensity
We examined whether high NFRET amplitudes measured in this study, particularly those at the periphery of cells expressing the wild-type functioning mGAT1XFP8 constructs, were large because of high local concentrations of YFP-tagged protomers in some domains. Line profile plots through cells expressing mGAT1XFP8 emphasized that the pixels with the greatest YFP signal intensity were usually located at the periphery of the cell (), as were the pixels with the highest amplitude net FRET (nF) or NFRET. We measured the colocalization of the YFP signal with both the nF signal and the NFRET signals. As a positive control, we determined that the mean Pearson product-moment correlation (r) for the colocalization of the CFP and YFP signals coexpressing mGAT1CFP8 and mGAT1YFP8 was 0.9 ± 0.01 (n = 31 cells) (not depicted). Colocalization analysis of the YFP fluorescence and the nF signal in the same group of cells reported that r = 0.5 ± 0.05 (). The square of r indicated that 25 ± 1.5% of the variance of nF in mGAT1XFP8-expressing cells is associated with changes in YFP intensity. When the relative contributions of the CFP and YFP intensities to the FRET signal in each pixel were normalized to generate the NRET images from the same cells, r = 0.01 ± 0.05 for IYFP versus NFRET colocalization (). This indicated that the normalization step in NFRET corrects for the influence of IYFP variance. One concludes that calculated NFRET was not artificially large due to high local IYFP in some ROIs.
Figure 12. Colocalization of IYFP with net FRET and NFRET signals. (A) Line profile analysis of YFP fluorescence. nF and NFRET measurements through the mGAT1XFP8-expressing cell in B illustrate the peripheral localization of the signals for all parameters. (B) Images (more ...)
Radial distance analysis of NFRET signal amplitude correlates to functional phenotype
The presence of the three NFRET components from only the fluorescent mGAT1 constructs with wild-type–like function suggested that one component arose from a mechanism that operates only in the cell periphery for mGAT1 oligomers, which are appropriately inserted into the plasma membrane. Apparently, this plasma membrane–associated component is also the component with the highest NFRET amplitude. We have already qualitatively observed that the largest amplitude NFRET signal occurred in the periphery of the cell (). As described in Materials and methods, we further analyzed NFRET images by grouping pixels into NFRET amplitude percentile groups (; look-up table in far right panel of ). The pixels contained within each bin or group are highlighted with the same color in the NFRET image (). We calculated each pixel's percent radial distance from the cell's center to the cell periphery. We analyzed the localization of NFRET amplitude for three representative constructs: mGAT1XFP8, which oligomerized efficiently as determined by acceptor photobleach and sensitized FRET analysis and functioned like wild-type nonlabeled mGAT1; mGAT1565XFP566CT, which showed strong FRET but compromised function; and mGAT1XFP45, which demonstrated weak function and weak FRET. The results of the analyses plotted in determined that the NFRET signals from pixels in cells expressing mGAT1XFP8 were the most peripherally located; the minimum percent distance from the center of mass for any NFRET amplitude percentile group was >85%. The pixels in the top fifth percentile of NFRET amplitude for mGAT1XFP8 were located a mean distance of 93% of the total radius from the center to mass to the edge of the cell, quantifying the statement that the highest-amplitude NFRET pixels were also the most peripherally located in the cell. Overall, the localization of NFRET signals of all amplitudes from pixels expressing mGAT1565XFP566CT trended toward the cell periphery rather than the center of mass, but significantly less so than from mGAT1XFP8. The trend followed a U-shaped pattern, with the bottom and top 20% NFRET pixel amplitudes being the most peripherally located (>70%), and the medium amplitude NFRET pixels residing in more intracellular locations. Localization of mGAT1XFP45 pixels that reported positive NFRET trended to more random distributions with increasing NFRET amplitude.
Correlation between function of fluorescent mGAT1 transporters and their NFRET properties
provides two ways to summarize the trends in NFRET and their relationship to function ([3H]GABA uptake) of mGAT1 constructs. Both panels of highlight the set of three correctly oligomerized, correctly trafficked, and correctly functioning fluorescent constructs: mGAT1XFP*, mGAT1XFP8, and mGAT577XFP578CT. In , we compare the function of each fluorescent construct (relative to nontagged wild-type mGAT1) versus the ratio of peripheral to perinuclear NFRET. The peripheral/perinuclear ratio was calculated from the mean NFRET for all pixels in each ROI. In this analysis, the fluorescent transporters could be classified into three groups:
Figure 14. The relationship between transporter function and NFRET. (A) Scatter plot of the ratio and mean (peripheral NFRET)/(perinuclear NFRET) for all pixels in the ROIs versus the percent function of each construct as compared with nonfluorescent wild-type mGAT1 (more ...)
(1) The three constructs with >75% wild-type function, as determined by [3H]GABA uptake, displayed a peripheral/perinuclear NFRET ratio ≥1.4.
(2) Constructs with compromised function (30–65% wild-type [3H]GABA uptake) demonstrated efficient oligomerization as determined by FRET quantification, but less efficient plasma membrane insertion, indicated by peripheral/perinuclear NFRET ratios close to 1.0.
(3) Represented by mGAT1XFP45, the constructs that display <30% of wild type function, limited oligomerization, and poor trafficking properties expressed a peripheral/perinuclear NFRET ratio of 0.1.
Wild-type functioning fluorescent transporters could therefore be clearly distinguished from constructs with poor oligomerization and/or trafficking: the former possess a peripheral/perinuclear NFRET ratio ≥1.4.
presents an additional hallmark of the three well-functioning constructs (as shown by [3H]GABA uptake levels). For these constructs, the peripheral ROI NFRET distributions could be fitted to three Gaussian components, and we have presented analyses that the highest-amplitude component arises from plasma membrane molecules. On the other hand, constructs with significantly reduced substrate transport rates (corresponding to reduced plasma membrane insertion) relative to wild-type GAT1 displayed peripheral ROI NFRET distributions comprising only two components (). The data suggest a threshold effect: if the peripheral NFRET distribution includes ≥~30% of the high-amplitude NFRET component, fluorescent mGAT1 molecules exhibit wild-type–like function ().
Nicotinic receptors also display a two-component NFRET distribution
Data presented to this point indicate that mGAT1, a membrane protein, displays only a two-component NFRET distribution if its oligomers are not inserted into the plasma membrane at normal wild-type levels. We assessed whether this conclusion holds for another membrane protein whose oligomerization has been studied in our laboratory in the N2a system: the heteropentameric mouse α4β2 nAChR (Nashmi et al., 2003
; Drenan et al., 2008
; Son et al., 2009
). Previous data indicate that α4β2 receptors are retained to a large extent in intracellular compartments (Nashmi et al., 2003
; Kuryatov et al., 2005
; Sallette et al., 2005
; Drenan et al., 2008
; Son et al., 2009
As with previous studies of this channel expressed in N2a cells (Drenan et al., 2008
; Son et al., 2009
), HEK 293 cells (Nashmi et al., 2003
), transfected neurons (Nashmi et al., 2003
; Khakh et al., 2005
), and α4YFP knock-in mice (Nashmi et al., 2007
), the fluorescence of both α4YFP and β2CFP nAChR subunits appeared evenly throughout the ER of N2a cells (), with a high degree of colocalization (Pearson product-moment correlation coefficient: r = 0.9 ± 0.02; n
= 11 cells) (not depicted). As described above (), the even fluorescence distribution throughout the cell provided only a single ROI per cell for this transfection: the ROI encompassed the entire cell without the nucleus. This was determined by the pixels exhibiting YFP fluorescence.
Robust FRET was observed throughout cells coexpressing α4YFPβ2CFP nAChR subunits; the mean NFRET for all pixels was 0.20 (, and ). The distribution of NFRET amplitudes for all analyzed pixels was best fit by two Gaussian components, with mean NFRET of 0.19 (representing 75% of all FRETing pixels) and 0.28 ().
Figure 15. Pixel-by-pixel quantification of sensitized emission FRET within α4YFPβ2CFP nAChRs. (A; from left to right) Panels display an N2a cell coexpressing β2CFP and α4YFP nAChR subunits with nonfluorescent wild-type mGAT1 (calibration (more ...)
C-terminal PDZ interactions are required to produce NFRET distributions comprising three Gaussian components
The PDZ type II–interacting domain at the distal C terminus of mGAT1 is necessary for at least two important transporter interactions in the cell periphery: with the exocyst complex (Farhan et al., 2004
) and with the actin cytoskeleton (Imoukhuede et al., 2009
). To determine whether an interaction of the C-terminal mGAT1 PDZ-interaction domain produced the observed third, high NFRET component in peripheral ROI NFRET distributions, we coexpressed mGAT10
CFP and mGAT10
YFP and examined the resulting subcellular NFRET profiles. As demonstrated earlier (), this construct design had compromised function. This occurs because the XFP moiety blocks access to the endogenous PDZ-interaction domain of mGAT1 (Imoukhuede et al., 2009
). displays representative images of the donor and acceptor fluorescence in an mGAT10
XFP-expressing cell together with the defined ROIs and calculated NFRET image. The ratio of mean NFRET amplitude for the peripheral versus the perinuclear regions for 63 cells expressing mGAT10
XFP was 0.9 ( and ), in contrast to 1.4 observed above for mGAT1XFP8 ( and ). Furthermore, the NFRET distributions for all ROIs from mGAT10
XFP-expressing cells were best fit by two Gaussian components. This indicated that mGAT1 transporters must possess a functional C-terminal PDZ-interacting domain to exhibit the third high-amplitude NFRET component in the cell periphery.
Figure 16. Pixel-by-pixel quantification of sensitized emission FRET between mGAT10XFP constructs. (A; from left to right) mGAT10CFP fluorescence and mGAT10YFP fluorescence (calibration bars, ACUs), ROIs used to determine NFRET (color coding as in ), and the (more ...)
PDZ-mediated tethering to the cytoskeleton locates third high-amplitude NFRET component in the cell periphery
A PDZ domain interaction occurs in the ezrin-mediated tethering of mGAT1 oligomers to the actin cytoskeleton (Imoukhuede et al., 2009
). We therefore investigated whether an intact cytoskeleton is required for the observed high NFRET component at the cell periphery.
We transfected N2a cells with the mGAT1XFP8 constructs and in half of the transfected dishes depolymerized the actin cytoskeleton with 5 µM latrunculin B (). As previously observed, mGAT1XFP8 showed detectable NFRET in all ROIs. The ratio of mean peripheral to perinuclear NFRET amplitude was 1.4 (, left; 67 cells, 4.7 and 1.8 × 105 pixels, respectively). 5 µM latrunculin B decreased this ratio to 0.9 (, right). The NFRET distributions for nontreated mGAT1XFP8-expressing N2a cells were fit with three Gaussian components in the whole cell and peripheral ROIs, with the highest mean amplitude NFRET component comprising ~30% of the total NFRET signal (). The intracellular and perinuclear ROIs were best fit with two Gaussian components (). Treatment of mGATXFP8-expressing cells with latrunculin B produced NFRET distributions from whole cell and peripheral ROIs that were best fit with two Gaussian components, similar to data for the fluorescent mGAT1 constructs with less than wild-type function (). Latrunculin B treatment therefore demonstrated that the polymerized actin cytoskeleton was required for the PDZ-interacting domain–mediated high NFRET component in the cell periphery.
Figure 17. Pixel-by-pixel quantification of sensitized emission FRET from mGAT1XFP8 when cells are treated with 5 µM latrunculin B. (A; top row, from left to right) mGAT1CFP8 fluorescence and mGAT1YFP8 fluorescence (calibration bars, ACUs), ROIs used to (more ...)
Interestingly, the perinuclear ROI of latrunculin B–treated cells was best fit by three Gaussian components (). A three-component perinuclear distribution was not found under other conditions in this study. The amplitude of the third component was equivalent to that seen in the periphery of nontreated cells and comprised a similar proportion of the total NFRET distribution in that ROI (~30%; ). This was not observed in the perinuclear ROI of mGAT10XFP-expressing cells, suggesting that the intact PDZ-interacting domain of mGAT1XFP8 plays a role.