Article |
Address correspondence to Mark L. Dell'Acqua, University of Colorado Health Sciences Center, Department of Pharmacology, C-236, 4200 E. Ninth Ave., SOM Rm. 2817C, Denver, CO 80262. Tel.: 303-315-3432. Fax: 303-315-7097. E-mail: mark.dellacqua{at}uchsc.edu
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Abstract |
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Key Words: calcineurin; cyclic AMPdependent protein kinases; glutamate; microscopy; fluorescence
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Introduction |
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One prototypic signaling scaffold that might have important functions in synaptic plasticity is A-kinase anchoring protein (AKAP) 79 (humanAKAP79/ratAKAP150) (Bregman et al., 1989; Carr et al., 1992). AKAP79 is a membrane/cortical cytoskeletontargeted anchoring protein that binds the cAMP-dependent protein kinase (PKA) regulatory (R) subunits, PKC, and protein phosphatase 2B/calcineurin catalytic A subunit (CaNA) (Coghlan et al., 1995; Klauck et al., 1996; Dell'Acqua et al., 1998; Gomez et al., 2002). In hippocampal neurons, AKAP79, PKA, and CaN are localized on postsynaptic dendritic spines with F-actin and PSD-95 family membrane-associated guanylate kinase (MAGUK) PDZ scaffolds (Gomez et al., 2002). AKAP79 is recruited to NMDA and AMPA glutamate receptors at synapses through binding the MAGUKs PSD-95 and synapse associated protein 97 (SAP97), respectively, and anchored PKA and CaN in these complexes participate in the regulation of AMPA receptor phosphorylation (Rosenmund et al., 1994; Colledge et al., 2000; Dell'Acqua et al., 2002; Tavalin et al., 2002). Importantly, PKA, CaN, and SAP97 have also been implicated in the regulation of AMPA receptor activity in LTP and LTD (Hayashi et al., 2000; Lee et al., 2000; Carroll et al., 2001). AKAP79 is targeted to the membrane cytoskeleton through an NH2-terminal basic domain that binds phosphatidylinositol-4,5-bisphosphate and F-actin (Dell'Acqua et al., 1998; Gomez et al., 2002). We have recently shown that AKAP79PKA synaptic targeting and association with MAGUKs depends on F-actin and is negatively regulated by NMDA receptor CaN signaling pathways that also control AMPA receptors in LTD (Beattie et al., 2000; Ehlers, 2000; Lin et al., 2000; Gomez et al., 2002). Thus, assembly and disassembly of AKAP79 complexes containing PKA, CaN, and MAGUKs may be central to synaptic plasticity.
Unfortunately, the biochemical precipitation techniques used to study the assembly of protein complexes are limited by steric hindrance from bulky fusion proteins or antibodies, use of detergents to solubilize complexes from cells, and poor yield of proteins precipitating through intermediate proteins. For instance, precipitation studies have shown limited amounts of CaN and PKA associated together with AKAP79 and have been unable to detect CaN and MAGUKs coprecipitating through the AKAP (Coghlan et al., 1995; Colledge et al., 2000). Even when biochemical methods work ideally, they still do not provide information about the existence or localization of the complex in intact living cells. Thus, precipitation methods cannot provide a complete, accurate depiction of the in vivo condition. As a first step in visualizing AKAP79 function in vivo, we have used microscopy techniques that allow observation of protein binding in living cells to provide direct evidence for membrane cytoskeletonlocalized molecular assembly of the PKAAKAP79CaN ternary complex in association with SAP97. In this work, we have employed both immunofluorescence and fluorescence resonance energy transfer (FRET) microscopy using proteins tagged with CFP and YFP variants of GFP to study signaling complex reconstitution in a model cell line.
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Results |
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The controls above demonstrating that FRET depends on CaN and PKA binding to AKAP79 use AKAP deletion mutants that also disrupt CFP/YFP colocalization. Thus, we performed controls confirming that image subtraction was measuring FRET and not colocalized CFP/YFP bleedthrough. We coexpressed AKAP79WTCFP with AKAP79WTYFP (Fig. 1 C, 1) or a construct fusing YFP onto the COOH terminus of the membrane-targeting domain of the unrelated AKAP18 (AKAP18[116]YFP; Fig. 1 F, 1) (Trotter et al., 1999). The CFP/YFP overlay images for cells expressing these protein pairs revealed significant colocalization of AKAP79CFP with AKAP79YFP (Fig. 2 C) or AKAP18YFP (Fig. 2 D) at the membrane. However, no FRETC was detected for these pairings despite membrane overlap (Fig. 2, C and D). Negative FRETNC values (<0; Table I) calculated from multiple images confirmed a lack of AKAP79 intermolecular FRET with itself or AKAP18. In contrast to AKAP79, AKAP18YFP also showed very strong localization to intracellular perinuclear/Golgi membranes (Fig. 2 D). Importantly, this strong Golgi YFP signal did not bleed into the FRETC image, further confirming the accuracy of image subtraction.
Confirmation of AKAP79 binding and FRET with CaN and PKA in fixed cells using YFP acceptor photobleaching
The advantage of the micro-FRET method is that it generates high-resolution, real-time images of sensitized FRET emission even if the proteinprotein interaction is unregulated. However, it is an indirect FRET measurement that relies on controls done in separate cells expressing donor or acceptor alone. FRET can also be directly measured within a single cell by determining the amount of CFP donor quenching that is relieved by YFP acceptor photobleaching (CFPpostbleach - CFPprebleach = CFP) and can be expressed as an apparent donor FRET efficiency percentage (%
CFP/CFPpostbleach). Unfortunately, this photobleaching method is not suitable for use in our live cell system because membrane ruffles and vesicles change shape and location during the 2 min required to bleach YFP. Nonetheless, in fixed cells, we were able to do single cell comparisons of FRETC determined by micro-FRET with apparent FRET efficiency (%
CFP/CFPpostbleach) determined by acceptor photobleaching.
As in live cells, coexpression of AKAP79CFP (Fig. 1 C, 1) with CaNAYFP (Fig. 1 D) or PKA-RIIYFP (Fig. 1 E) resulted in membrane anchoring of CaN (Fig. 3 A) or RII (Fig. 3 B) to AKAP79, detected by the micro-FRET method in fixed cells. AKAP79CaN and AKAP79RII FRETC signals (pseudo-color) at specific membrane locations are shown in images gated to the presence of YFP acceptor (blue underlay) to represent relative FRET intensities on a scale of blue (no FRET) to green (low FRET) to red (high FRET) (Fig. 3, A and B, top, second panel from right). Sites of high relative FRETC intensity (orange-red) included vesicles and plasma membrane ruffles where the donor and acceptors were highly colocalized. Micro-FRET measurements of FRETNC in membrane structures for these fixed samples were very reproducible across multiple experiments (n = 812) for AKAP79CFP paired with CaNYFP (14.0 ± 1.2) or RIIYFP (13.0 ± 1.6). This FRETNC value for AKAP79RII in fixed cells is very similar to that determined in live cells for RII (12.1 and 13.8; Table I), whereas the FRETNC value for AKAP79CaNA in fixed cells is larger than that determined in live cells for CaN (1.4 and 2.8; Table I). Thus, quantitative comparison of FRET for the same protein complex in live cells versus fixed cells is difficult because fixation may trap the complex in a form with somewhat altered chromophore separation and orientations compared with in living cells. Nonetheless, we can measure FRETC in fixed cells for AKAP79 binding to PKA and CaN.
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Direct binding of CaN to AKAP79 residues 321360 detected in living cells by CFP/YFP FRET
Our findings demonstrate the use of FRET to image binding between AKAP79 and CaN or PKA in both living and fixed cells, and from our controls with CaN and
PKA mutants, we can infer that these interactions involve the appropriate AKAP binding sites. However, we wished to compliment these "loss of function" controls with a "gain of function" approach using the AKAPCaN interaction as an example. Recently, we demonstrated that residues contained in 321360 of AKAP79 can bind CaN in vitro and inhibit signaling in cells, suggesting that this region is sufficient for AKAP79CaN anchoring (Dell'Acqua et al., 2002). To show that CYFRET observed between AKAP79 and CaN involves binding between AKAP79 residues 321360 and the CaNA subunit, we constructed three additional CFP donors (AKAP79[321360]CFP, Fig. 1 C, 4; AKAP18[116]CFP, Fig. 1 F, 1; and AKAP18[116]AKAP79[321360]CFP, Fig. 1 F, 2). In agreement with our previous studies, expression of the isolated CaN binding domain (321360) fused to CFP (Fig. 1 C, 4) produced an untargeted protein found in both the cytoplasm and the nucleus (Fig. 4, A and B). This (321360)CFP construct bound to CaNAYFP in the cytoplasm and prevented plasma membrane anchoring of CaNAYFP to AKAP79WT (Texas red antibody staining) (Fig. 4 A). In parallel live cells, where untagged AKAP79 cannot be seen, colocalization of (321360)CFP and CaNAYFP and FRETC were seen exclusively in the cytoplasm (Fig. 4 B). FRETNC measurements showed this FRET to be reproducible (13.3 ± 3.0; Table I).
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FRET imaging of the CaNAKAP79PKA ternary complex in living cells
The identified CaN (321360) and PKA (388409) binding sites on AKAP79 are in very close proximity to each other in primary sequence (Fig. 1 C, 1). Our FRET measurements above indicate that both proteins bound to AKAP79 are <50 Å from C/YFP at the AKAP COOH terminus. These findings raise the issue of whether PKA and CaN are bound to the same AKAP molecule simultaneously or if there is competition due to steric hindrance between the closely opposed binding sites. To address this issue, we sought to reconstitute CaNAKAP79PKA ternary complexes in COS7 cells. As a first step, we coexpressed AKAP79WTCFP (Fig. 1 C, 1) and PKA-RIIYFP (Fig. 1 E) with myc-tagged CaNA (Dell'Acqua et al., 2002). Cells were fixed and labeled with anti-myc antibodies to visualize CaNA along with AKAP79CFP and RIIYFP. These studies revealed that AKAP79WTCFP, PKA-RIIYFP, and mycCaNA were all colocalized in plasma membrane ruffles (Fig. 5 A, top). To show that this colocalization in plasma membrane structures was due to independent binding of CaN and PKA to their respective AKAP79 binding sites, we analyzed the PKA (Fig. 1 C, 2) and
CaN (Fig. 1 C, 3) mutants. Deletion of the PKA anchoring site on AKAP79 led to cytoplasmic localization of RIIYFP but retention of AKAP79CFP and mycCaNA at the membrane (Fig. 5 A, middle). Deletion of the CaN anchoring site on AKAP79 had the opposite effect, causing cytoplasmic localization of mycCaNA but maintaining membrane overlap for AKAP79CFP and RIIYFP (Fig. 5 A, bottom). These results do not support a model of competition between PKA and CaN binding to AKAP79 and suggest that these proteins can bind independently to closely spaced, but functionally separate, binding sites on the AKAP.
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Plasma membrane targeting and recruitment of SAP97 to the AKAP79 signaling scaffold
AKAP79 can bind MAGUK scaffold proteins including SAP97 and PSD-95 (Colledge et al., 2000). In particular, binding of AKAP79 to SAP97 is believed to recruit AKAP, PKA, and CaN to GluR1 subunits for the regulation of AMPA receptor phosphorylation (Colledge et al., 2000; Dell'Acqua et al., 2002; Tavalin et al., 2002). Using our transfection system, we set out to characterize the interaction between AKAP79 and SAP97 in living cells. In agreement with previous studies (Tiffany et al., 2000), SAP97YFP expressed alone was predominantly cytoplasmic (Fig. 6 A, top). However, expression of SAP97YFP with AKAP79CFP resulted in substantial SAP97 targeting to membrane ruffles in live cells (Fig. 6 A, bottom). In fixed cells, these sites of membrane colocalization of SAP97 with AKAP79 were found to be enriched in cortical F-actin stained with phalloidin (Fig. 6 B, top). We have previously shown that the highly-basic NH2 terminus of AKAP79 is necessary and sufficient for binding to phosphatidylinositol-4,5-bisphosphate and F-actin in vitro and targeting to dendritic spines in neurons and membrane ruffles in COS7 cells (Fig. 6 E; Dell'Acqua et al., 1998; Gomez et al., 2002). AKAP79 binds to the SAP97 MAGUK SH3 and GK domains (Fig. 6 F); however, the MAGUK binding site of AKAP79 is less defined but is thought to be located COOH terminal to the targeting domain (1153) and NH2 terminal to the CaN anchoring site (315360) (Fig. 6 E; Colledge et al., 2000). Thus, we expressed SAP97YFP with AKAP79(1153)CFP as an additional negative control to support the need for AKAP79MAGUK binding in SAP97 cortical targeting. As seen previously (Gomez et al., 2002), AKAP79(1153)CFP targeted to membrane ruffles enriched with F-actin (Fig. 6 B, middle); however, SAP97YFP remained cytoplasmic with little or no colocalization with cortical actin or the AKAP fragment (Fig. 6 B, bottom). Coexpression of SAP97YFP with a COOH-terminal fragment of AKAP79 ([150427]CFP) that is unable to localize to membranes or bind actin (Fig. 6 E; Gomez et al., 2002) resulted in no targeting of either protein to cortical actin (Fig. 6 B, bottom). This confirmed that binding of SAP97 to determinants found COOH terminal to the AKAP targeting domain must be required for SAP97 targeting to membrane ruffles with AKAP79 (Fig. 6 E).
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Discussion |
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Evidence for the existence of complexes containing AKAP79, PKA, CaN, and MAGUKs had previously been obtained using immunoprecipitation and GST precipitation with recombinant proteins and cell extracts (Coghlan et al., 1995; Colledge et al., 2000; Gomez et al., 2002). Due to the inherent limitations of precipitation reagents and the use of cell extracts in these biochemical approaches, results from such studies may not always be entirely representative of intact living cells. For instance, one previous in vitro precipitation study suggested competition between CaN and MAGUK binding to AKAP79 (Colledge et al., 2000), whereas a second electrophysiological study in live cells found no evidence for such competition (Tavalin et al., 2002). Earlier attempts to map the CaN binding site on AKAP79 using in vitro biochemical methods or the yeast two-hybrid system gave inconsistent results and were only able to narrow the binding site to the COOH-terminal two thirds of AKAP79 (Dell'Acqua et al., 1998; Kashishian et al., 1998). In contrast, our more recent studies combining cellular immunofluorescence with in vitro biochemical assays were successful in mapping the CaN anchoring site to a single 40-amino acid region in very close proximity to the PKA anchoring site (Dell'Acqua et al., 2002).
Thus, in order to get a more accurate view of AKAP79 scaffolding functions in vivo, we thought it was imperative to study the formation of AKAP79 complexes in intact cells using not only immunofluorescence, which shows protein colocalization on a scale of 200 nm, but also FRET microscopy, which detects true proteinprotein proximity within 5 nm (50 Å). Our current results show that by using micro-FRET, we can directly visualize, on this 5-nm scale, binding of AKAP79 to CaNA and PKA-RII in a ternary complex at the plasma membrane/cortical cytoskeleton of living cells. These binding interactions were confirmed by measurement of FRET in fixed cells using both micro-FRET and acceptor photobleaching methods. Importantly, the CaN and PKA binding interactions were shown to be through noncompeting sites near the AKAP COOH terminus. FRET signal strength is a function of not only the distance between chromophores but also the binding affinity of the proteinprotein interaction and chromophore orientations within the complex. Thus, differences in any of these parameters can explain the relative differences in FRET signals between acceptordonor pairs. For example, the weaker FRETNC that we measured for AKAP79CaN versus AKAP79PKA binding could reflect both the 2050-fold weaker reported affinity of AKAP79 for CaNA versus RII as well as the closer proximity of the PKA-RII binding site to the AKAP COOH terminus (Fig. 1 C, 1; Carr et al., 1992; Kashishian et al., 1998; Dell'Acqua et al., 2002). However, consistent with chromophore separation contributing to our observed differences in FRETNC, when the CaN binding site (321360) is fused directly to CFP with no intervening sequence (Fig. 1 C, 4; Fig. 1 F, 2), larger CaNAYFP FRETNC values (13.3 and 35.6) are measured than for full-length AKAP79CFP (2.8) (Table I). Extending this reasoning to the CaNAKAPPKA complex is problematic because the formation of a ternary complex is affected by the concentration of all three proteins and multiple binding affinities. Nonetheless, stronger FRETNC is seen for CaNACFP with RIIYFP in the AKAP79 ternary complex (9.1) versus with AKAP79YFP in a binary complex (1.4) (Table I). This result could be explained by the shorter distance between CaN and RII bound to the AKAP compared with the distance between CaN and the AKAP COOH terminus (Fig. 1 C, 1). It would also follow from similar FRETNC values (9.1 and 12.1) that the distances between RII and CaN bound to AKAP79 may be similar to the distance between bound RII and the AKAP79 COOH terminus. Interestingly, the spacings between the CaN and PKA binding sites and the PKA binding site and AKAP COOH terminus are in fact both 2030 amino acids in primary sequence (Fig. 1 C, 1).
By combining CFP/YFP imaging with immunofluorescence, our current work also shows that AKAP79 can regulate the membrane cytoskeletal targeting of SAP97 in concert with PKA and CaN and is not consistent with competition between CaN and MAGUK binding. Thus, in neurons, AKAP79 is likely to coordinate the assembly and targeting of PKACaNSAP97 complexes that are recruited to the COOH terminus of AMPA-GluR1 receptors through SAP97 PDZ domains (Fig. 6 F; Leonard et al., 1998). Recently a great deal of attention in the field of hippocampal synaptic plasticity has focused on regulation of the phosphorylation state and localization of AMPA receptors through PKA and CaN pathways that are likely to be organized by AKAP79MAGUK scaffolding. NMDA receptor Ca2+ influx in LTP, a synaptic strengthening process, leads to increased AMPA receptor activity and phosphorylation (Lee et al., 2000). In LTP, interactions of AMPA-GluR1 with SAP97 PDZ domains (Fig. 6 F) and phosphorylation by PKA may participate in both recruiting new receptors to the postsynaptic membrane and increasing channel activity (Banke et al., 2000; Hayashi et al., 2000; Passafaro et al., 2001; Shi et al., 2001). In contrast, NMDA receptor activation in LTD, a weakening of synaptic strength, leads to dephosphorylation of the GluR1 PKA site and removal of synaptic AMPA receptors by endocytosis through CaN activation (Beattie et al., 2000; Ehlers, 2000; Lee et al., 2000; Lin et al., 2000; Carroll et al., 2001). AKAP79 promotes similar PKA and CaN regulation of SAP97-linked AMPA-GluR1 receptors in heterologous systems and thus could play an important role in organizing PKA and CaN signals at synapses (Colledge et al., 2000; Dell'Acqua et al., 2002; Tavalin et al., 2002). In support of this model, we have recently shown that NMDACaN signaling pathways controlling AMPA receptors also negatively regulate the localization and association of the endogenous AKAP79/150PKA complex with MAGUKs in neurons (Gomez et al., 2002).
Localization of AKAP79/150 and AMPA receptors with MAGUKs at synapses is dependent on the actin cytoskeleton, and, interestingly, both SAP97 and AMPA-GluR1 can be linked to the cytoskeleton through 4.1 actin binding proteins (Lue et al., 1994; Shen et al., 2000; Zhou et al., 2001; Gomez et al., 2002). However, targeting of SAP97 to cortical actin in epithelial cells depends on an NH2-terminal domain (SAP97[165]) not present in other MAGUKs as well as secondarily on 4.1 binding (Fig. 6 E; Reuver and Garner, 1998; Wu et al., 1998). Thus, it has been proposed that common binding of SAP97 and GluR1 to 4.1 and actin may stabilize AMPA receptors at synapses. However, our findings show that AKAP79 can also regulate targeting of SAP97 to membrane cytoskeleton structures. Thus, the association of AKAP79 with SAP97 may be an additional targeting signal for both proteins that provides AKAPSAP97AMPA receptor complexes further attachment to the postsynaptic cytoskeleton.
As discussed above, the molecular compartmentalization of CaN and PKA in the AKAP79 complex and how they respond to cAMP and Ca2+ signals is likely to be very important for efficient bidirectional regulation of AMPA receptor phosphorylation and trafficking. AKAP79 binding to CaN inhibits phosphatase activity in vitro even with CaNB and Ca2+-calmodulin activators present (Coghlan et al., 1995; Dell'Acqua et al., 2002). In contrast, AKAP79 targets basally active PKA holoenzyme that can be further activated by elevated cAMP through increased C subunit release; however, it is unknown whether the inhibitory CaNA binding to AKAP79 is regulated by synaptic Ca2+ signals that activate CaN. Future applications of FRET imaging methods in transfected neurons will allow us to examine the regulation of CaNAKAP79PKA binding interactions and targeting by neuronal Ca2+ signaling pathways involved in synaptic plasticity.
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Materials and methods |
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COS7 cell culture and transfection
COS7 cells at 2050% confluency (2448 h after plating on glass coverslips in six-well plates) were transfected by calcium phosphate precipitation with cDNA expression constructs (0.52 µg each plasmid) for 416 h at 5% CO2, 37°C. Cells were washed twice with PBS, fed with DME, 10% FBS, and 1% penicillin/streptomycin (Invitrogen; GIBCO BRL), and grown for 2448 h before live cell imaging or fixation for immunocytochemistry.
Immunocytochemistry and digital fluorescence microscopy
COS7 cells were washed in PBS, fixed in 3.7% formaldehyde/PBS, and permeabilized in 0.2% Triton X-100/PBS. For staining of mycCaNA, mycSAP97, or AKAP79, cells were blocked in PBS + 3.0% BSA for 30 min, followed by incubation with primary antibodies (1:250 anti-myc [9E10] [Santa Cruz] or 1:1,000 anti-AKAP79 [918I] [Icos]) for 1 h, followed by washing in PBS. Next, coverslips were incubated for 1 h with secondary antibodies (goat anti-mouseTexas red, goat anti-rabbitTexas red, or goat anti-rabbitCy5 [Molecular Probes or Amersham Biosciences]) and, in some cases, 1:500 Texas redphalloidin (Molecular Probes) followed by washing in PBS. Coverslips were mounted on glass slides using the Pro-long anti-fade (Molecular Probes). Specific indirect immunofluorescence and/or intrinsic CFP and YFP fluorescence was detected with chroma filter sets using a Nikon TE-300 inverted microscope (100xplan-apo, oil, 1.4 NA) with Micromax (Princeton Instruments) or Sensicam (Cooke) digital CCD cameras and Slidebook 3.0 software (Intelligent Imaging Innovations). All images (including FRET images, see below) were exported from Slidebook in Tiff RGB format and figures assembled using Adobe Photoshop 5.5®.
CFP/YFP FRET microscopy and image analysis
For CYFRET imaging experiments in living cells, COS7 cells were transfected on 25-mm round glass coverslips and, 2448 h later, placed in an imaging chamber (Molecular Probes) at room temperature in media on the stage of the microscope described above. CYFRET image acquisition and analysis were done by the three-filter "micro-FRET" image subtraction method described by Sorkin et al. (2000). In brief, three images (100-ms or 250-ms exposure sets, 2 x 2 binning) were obtained: a YFP excitation/YFP emission image; a CFP excitation/CFP emission image; and a CFP excitation/YFP emission image (raw, uncorrected CYFRET). After imaging, background images were taken. Background-subtracted YFP and CFP images were then fractionally subtracted from raw CYFRET images based on measurements for CFP bleedthrough (0.500.56) and YFP cross-excitation (0.0150.02). This fractional subtraction generated corrected FRETC images, represented in monochrome or pseudo-color (gated to YFP acceptor levels), showing sensitized FRET within cells. The subtraction coefficients are rounded up from average cross-bleed values determined in cells expressing CFP- or YFP-tagged constructs alone. Thus, these coefficients result in the underestimation of FRETC signals for true FRET partners but prevent false positive detection of FRET.
Independent YFP acceptor photobleaching FRET measurement of CFP donor quenching was performed in fixed cells mounted without anti-fade reagent. YFP was selectively bleached by excitation at 535 nM for 2 min. The prebleach CFP image was then subtracted from the post-bleach CFP image to generate a pseudo-color image showing CFP quenching (CFP) due to FRET (gated to post-bleach CFP levels). Calculation of CFP quenching apparent FRET efficiency values from the photobleaching method and normalized FRETNC values from the micro-FRET method used Slidebook mask analysis. CFP quenching apparent FRET efficiency values were obtained by dividing the mean intensity for change in CFP fluorescence after YFP photobleaching by the post-bleach CFP mean intensity (CFPpostbleach - CFPprebleach =
CFP/CFPpostbleach) for masks covering entire cells. Values from multiple cells were averaged to give the mean apparent FRET efficiency expressed as a percentage ± SEM. FRETC intensity values were obtained by fractional subtraction (using coefficients described above) of mean intensities for CFP and YFP from raw CYFRET for masks covering membrane ruffles and vesicles. FRETC was converted to FRETNC by dividing with the product of CFP and YFP intensities (FRETNC = FRETC/CFP x YFP). FRETNC values were averaged for multiple cells to generate mean FRETNC values ± SEM (x10-5) (Table I). Negative FRETNC values are obtained when there is no FRET due to the overcorrection for CFP and YFP cross-bleeding. FRETNC is proportional to the equilibrium constant for the binding interaction (Gordon et al., 1998; Sorkin et al., 2000).
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Footnotes |
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Acknowledgments |
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This work was supported by grants from the American Heart Association (0130228N) and the National Institutes of Health/National Institute of Neurological Disorders and Stroke (NS40701) to M.L. Dell'Acqua.
Submitted: 27 September 2002
Revised: 25 November 2002
Accepted: 27 November 2002
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References |
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