Article |
Address correspondence to Bernd Nürnberg, Institut für Physiologische Chemie II, Klinikum der Heinrich-Heine-Universität, Universitätsstr. 1, Gebäude 22.03, 40 225 Düsseldorf. Tel.: 49-211-811-2724. Fax: 49-211-811-2726. E-mail: bernd.nuernberg{at}uni-duesseldorf.de
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Abstract |
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Key Words: G protein; fluorescence imaging; phosphoinositide 3-kinase; FRET; PH domain
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Introduction |
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PtdIns-3,4,5-P3 represents the major product of class I PI3Ks in vivo. These lipid kinases are under tight control of cell surface receptors, including receptor tyrosine kinases (RTK) or G proteincoupled receptors (GPCR; Katso et al., 2001). All class I members are heterodimers consisting of a p110 catalytic and a p85- or p101-type noncatalytic subunit. The class IA p110 isoforms , ß, and
form a complex with p85 adaptor subunits, whereas the only class IB member (p110
) is associated with a p101 noncatalytic subunit. The p85 subunit binds to tyrosine-phosphorylated RTKs. This interaction has two consequences resulting in activation of the PI3K. First, the cytosolic enzyme translocates to the inner leaflet of the plasma membrane, giving p110 access to its lipid substrate (Gillham et al., 1999). Second, the interaction with the tyrosine-phosphorylated receptor induces a conformational change of p85 that results in disinhibition of p110 enzymatic activity (Yu et al., 1998). Because the p85 regulatory subunit inhibits the p110 catalytic subunit, it is feasible that constitutively membrane-associated class IA p110 mutants trigger downstream responses characteristic of growth factor action (Klippel et al., 1996).
Similar to class IA PI3Ks, unstimulated PI3K is predominantly localized in the cytosol, whereas GPCRs induced an increase of PI3K
in the membrane fraction (Al-Aoukaty et al., 1999; Naccache et al., 2000). GPCRs activate PI3K
through direct interaction with Gß
, whereas a stimulation by G
is quantitatively less important (Stoyanov et al., 1995; Stephens et al., 1997; Leopoldt et al., 1998). However, little is known about the activating mechanism, e.g., it remains elusive whether Gß
functions as a membrane anchor or an allosteric regulator of PI3K
. Although the p101 subunit has been proposed to act as an indispensable adaptor linking Gß
with p110
(Stephens et al., 1997), in vitro studies have suggested that p101 is not mandatory for Gß
-induced stimulation of PI3K
(Stoyanov et al., 1995). Moreover, a direct interaction of Gß
with NH2- and COOH-terminal regions of p110
has been demonstrated (Leopoldt et al., 1998). Further support for a direct interaction of Gß
with the p110 catalytic subunit came from the observation that the p110ß isoform can also be stimulated by Gß
, regardless of whether p85 is present or not (Kurosu et al., 1997; Maier et al., 1999, 2000). Nevertheless, the noncatalytic p101 subunit of PI3K
binds tightly to Gß
, suggesting specific roles in G proteininduced regulation of this lipid kinase. For instance, in vitro studies showed that Gß
-stimulated p110
potently produced PtdIns-3-P, whereas the Gß
-activated heterodimeric p110
/p101 counterpart potently catalyzed the formation of PtdIns-3,4,5-P3 (Maier et al., 1999). This suggests that p101 may affect the interaction of Gß
-stimulated p110
with the lipid interface.
Therefore, the exact roles of Gß and PI3K
subunits in G proteininduced activation of PI3K
in living cells remain unclear. Hence, we asked whether Gß
functions as a membrane anchor and/or allosteric activator, and whether p101 is required for proper function of p110
in vivo. To tackle these questions, we examined cellular events leading to activation of PI3K
using fluorescent fusion proteins in living cells.
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Results |
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In vivo dimerization of PI3K subunits
To directly demonstrate heterodimerization of PI3K subunits in living cells, we used fluorescence resonance energy transfer (FRET). FRET between two fluorophores, e.g., CFP and YFP, is restricted to distances of <100 Å, and therefore, provides direct evidence for a proteinprotein interaction (Teruel and Meyer, 2000). We coexpressed CFP- and YFP-tagged PI3K
subunits and determined FRET by following the donor (CFP) recovery during acceptor (YFP) bleach (Fig. 3 A). All combinations showed significant FRET, although quantitative differences were evident.
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Gß dimers recruit PI3K
to membranes via the p101 subunit
Current concepts of PI3K activation are based on the recruitment of the cytosolic lipid kinase to the plasma membrane (Klippel et al., 1996). In the case of PI3K, the major stimulus is assumed to be Gß
, which is membrane-bound and has been shown to directly bind to both kinase subunits under in vitro conditions (Stephens et al., 1997; Maier et al., 1999). Therefore, we asked whether overexpression of free Gß
directs YFP-fused PI3K
subunits to the cell membrane in vivo. Surprisingly, p110
was not membrane-localized in the presence of coexpressed Gß
in HEK cells (Fig. 4 A, top). In contrast, Gß
recruited p101 to the membrane, resulting in accumulation of NH2- or COOH-terminally YFP-tagged p101 at both plasma- and endomembranes (Fig. 4 A, bottom, and Fig. 4 B). This corresponds to the subcellular distribution of overexpressed Gß
dimers (see below), suggesting that p101 interacted with membrane-bound Gß
.
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Interestingly, coexpression of heterodimeric PI3K together with Gß
produced a rounded morphology and detachment of the cells (Fig. 4 C). This morphological change was reversed by treatment with 100 nM of the PI3K inhibitor wortmannin in approximately half of the cells within 30 min, and not seen when a kinase-deficient YFP-p110
-K833R mutant was used (Fig. 4 C). Hence, the morphological change was related to the enzymatic activity of PI3K
stimulated by the coexpressed Gß
.
p101 is required for G proteinmediated activation of p110 in vivo
Because changes in morphology are not a very reliable measure of PI3K activity, we examined phosphorylation of endogenous protein kinase B (PKB or Akt) as an established PI3K-specific read-out system. To avoid detachment of the cells as a consequence of constitutive PI3K stimulation by overexpressed Gß
, we transiently stimulated the heterologously expressed Gi-coupled formyl-methionyl-leucyl-phenylalanine (fMLP) receptor, which is known to activate PI3K
(Stephens et al., 1993). Stimulation with fMLP induced Akt phosphorylation in HEK cells in the same range as with FCS (Fig. 5 A). However, Akt phosphorylation was only seen when the receptor was coexpressed with both PI3K
subunits, and not with p110
alone. These data imply that p101 is required for GPCR-induced activation of PI3K
in vivo.
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Translocation of an isolated PH domain in single cells may be a more sensitive read-out for PtdIns-3,4,5-P3 production than the Akt phosphorylation assay. Hence, we used the GFP-GRP1PH translocation approach to reexamine the requirement of p101 for receptor-induced PI3K activation (Fig. 5 A). Nevertheless, an fMLP-induced membrane recruitment of GFP-GRP1PH in cells coexpressing the receptor and p110
(but not p101) was not visible (Fig. 5 B, fourth panel). This finding confirms that the noncatalytic p101 subunit is required for GPCR-mediated stimulation of PI3K
in living cells.
To further strengthen the findings, we replaced GFP-GRP1PH by the GFP-fused PH domain of Bruton's tyrosine kinase (BtkPH), which has also been described to bind PtdIns-3,4,5-P3 with high specificity and affinity (Várnai et al., 1999). As expected, fluorescent BtkPH was distributed equally between cytosol and nucleus in HEK cells (Fig. 5 C). However, cells coexpressing the receptor and both PI3K subunits exhibited a slight basal membrane localization of BtkPH-CFP followed by a prominent translocation from the cytosol to the membrane on exposure to fMLP. Similar to GFP-GRP1PH, no fMLP-induced redistribution of BtkPH-CFP was seen in the absence of p101.
Activation of a membrane-targeted p110-CAAX in vivo
The presented data suggest that in living cells, p101 is an indispensable adaptor for GPCR-induced translocation and activation of class IB PI3K, which is equivalent to the role of p85 in RTK-induced class IA PI3K activation. Vice versa, Gß
behaves as a membrane anchor recruiting PI3K
through association with p101. So far, these experiments did not clarify whether membrane recruitment itself is sufficient for activation of the enzyme or whether additional allosteric stimulation is required. To tackle this question, we generated p110
mutants containing a COOH-terminal isoprenylation signal, i.e., a CAAX-box motif, which constitutively localizes p110
to the plasma membrane. p110
-CAAX accumulated at the plasma membrane of HEK cells, and was able to complex with p101 (Fig. 6, top). Coexpression of Gß
together with YFP-p110
-CAAX increased not only fluorescent staining of endomembranes, but also produced a wortmannin-sensitive rounding of the cells (Fig. 6, bottom) even in the absence of p101 (not depicted), which was similar to the effect after coexpression of Gß
with p110
/p101 dimers (see Fig. 4 C). These observations imply that membrane-bound PI3K
is not fully active, but can be further activated by Gß
.
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Next, we sought to confirm our principal findings using nontransformed cells in primary culture with a higher degree of differentiation than HEK cells. Vascular smooth muscle (VSM) cells were injected with plasmids encoding the fMLP receptor, p101, p110 or p110
-CAAX, and GFP-GRP1PH in different combinations, and redistribution of the fluorescent PtdIns-3,4,5-P3 sensor was monitored (Fig. 8). Again, this series of experiments confirmed that p101 is required for GPCR-mediated activation of PI3K
in living cells, whereas membrane-targeted p110
can be stimulated even in the absence of p101.
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Discussion |
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A p110-GFP construct has been previously reported (Metjian et al., 1999), but data describing a functional interaction of the fusion protein with G proteins are not available. Therefore, we systematically tagged each PI3K
subunit and found heterodimerization, enzymatic activity, and Gß
-sensitivity of the fusion proteins unaffected by the tags, whereas a GFP-tagged Gß complexed to G
did not interact with PI3K
(unpublished data). In resting cells, fluorescent PI3K
was predominantly detected in the cytosol. This resembles the native situation in which the vast majority of the endogenous PI3K
pool has been assigned to the cytosol (Stephens et al., 1994, 1997; Tang and Downes, 1997). Thus, the GFP-tagged proteins are appropriate tools to study the cellular events leading to activation of PI3K
.
Recent analysis of p110 crystals gave insights into the three-dimensional structure of the catalytic PI3K
subunit (Walker et al., 1999; Pacold et al., 2000). However, the structure of p101 and the molecular determinants for the interaction of both PI3K
subunits are currently unknown. In vitro data derived from p101 and p110
deletion mutants suggest that large areas of p101 may interact with the NH2-terminal side of p110
(Krugmann et al., 1999). FRET data presented here indicate that NH2 and both COOH termini of p110
and p101 are in close proximity. The latter finding is of interest because the p110
COOH terminus harbors the catalytic domain. Previous data have implied that not only Ras (Pacold et al., 2000), but also Gß
(Leopoldt et al., 1998) directly interact with the COOH terminus of p110
. Therefore, interaction of p101 with the catalytic domain of p110
would be in line with the functional data suggesting that p101 affects interaction of Gß
-stimulated p110
with the lipid interface (Maier et al., 1999).
Gß is considered to be the principal, direct stimulus of GPCR-induced PI3K
enzymatic activity. Convincing evidence for this assumption has come from reconstitution of purified proteins, demonstrating that in vitro, the enzymatic activity of heterodimeric and monomeric PI3K
is significantly stimulated by Gß
(Stoyanov et al., 1995; Stephens et al., 1997; Tang and Downes, 1997; Leopoldt et al., 1998). However, when we challenged the concept in living cells, marked differences in the sensitivities between heterodimeric and monomeric PI3K
emerged. The G proteincoupled fMLP receptor activated p110
/p101, but not p110
, as evident from the translocation of fluorescent PtdIns-3,4,5-P3 sensors or the stimulation of Akt phosphorylation. Likewise, coexpressed Gß
recruited p110
/p101, but not p110
, to membranes of HEK cells. Interestingly, PI3Kß, another Gß
-regulated PI3K, did not translocate to the membrane on GPCRs or Gß
stimulation, whereas RTKs induced membrane recruitment in vivo (unpublished data).
Does membrane recruitment of PI3K by itself result in constitutive activation of p110
? To answer this intriguing question, we used an isoprenylated mutant of p110
, i.e., p110
-CAAX, which is permanently attached to the membrane, and thereby, is in close proximity to its lipid substrates. Expression of p110
-CAAX only slightly enhanced membrane association of fluorescent PtdIns-3,4,5-P3 sensors in HEK or VSM cells. Notably, it did not affect the morphology of HEK cells. In contrast, coexpression of Gß
together with p110
-CAAX induced morphological changes comparable to cells transfected with plasmids encoding Gß
and p110
/p101. Furthermore, stimulation of the cells with fMLP stimulated Akt phosphorylation and membrane recruitment of PH domains regardless of whether p110
/p101 or p110
-CAAX was the effector. This establishes a second role for Gß
, i.e., the activation of the membrane-attached catalytic p110
subunit even in the absence of p101 (Fig. 9). Although Gß
interacts with either monomeric PI3K
subunit through individual binding sites, it does not exclude the possibility that heterodimeric PI3K
forms either a common or different binding site(s) for Gß
. Attempts to determine the stoichiometry of the interaction between heterodimeric PI3K
and one or more Gß
were inconclusive. One possible reason may be a weak affinity between p110
and Gß
, whereas p101 binds at least with moderate affinity to Gß
as determined by copurification studies (Stephens et al., 1997; Krugmann et al., 1999; Maier et al., 1999) and by a Biacore plasmon resonance approach (unpublished data).
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Increasing evidences suggest that monomeric p110 may also function as a downstream regulator of GPCR-dependent signal transduction pathways in cells (Lopez-Ilasaca et al., 1997, 1998; Bondeva et al., 1998; Murga et al., 1998; Baier et al., 1999). However, under in vivo conditions the p101 subunit seems mandatory for G proteinmediated activation of PI3K
by mediating membrane recruitment. Therefore, the question arises of how monomeric p110
translocates to the membrane. In this context, recent in vitro evidence points to the possibility that membrane attachment of p110
may involve binding to anionic phospholipids (Kirsch et al., 2001). In line with this finding, the majority of PI3K
is already associated with lipid vesicles in the absence of Gß
under in vitro conditions, and vesicle-bound PI3K
can still be activated by Gß
(Krugmann et al., 2002). Alternatively, p110
may be recruited by binding to membrane-anchored proteins other than Gß
. In this respect, p110
, like all other class I PI3Ks, is directly activated by GTP-bound Ras (Pacold et al., 2000).
In conclusion, we present in vivo evidence that Gß may stimulate PI3K
in a dual and complementary way, i.e., by recruitment to the membrane, and by activation of the membrane-bound enzyme (Fig. 9). In this scenario, the p101 subunit functions as an adaptor molecule necessary to recruit the catalytic subunit to the plasma membrane through high affinity interaction with Gß
. In turn, direct interaction between Gß
and the membrane-attached catalytic p110
subunit contributes to a final activation of the enzyme by a mechanism other than translocation.
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Materials and methods |
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The human fMLP receptor cDNA (Boulay et al., 1990) was amplified with the primers 5'-GCC ACC ATG GAG ACA AAT TCC TCT CTC-3' and 5'-TCA CTT TGC CTG TAA CTC CAC-3' and subcloned in pcDNA3 via HindIII and XhoI. The cDNAs of Gi2 (Conklin et al., 1993), human Gß1 (Codina et al., 1986), and bovine G
2 (Gautam et al., 1989) were subcloned into pcDNA3. Plasmids for GFP-GRP1PH and Ras N17 are described elsewhere (Ridley et al., 1992; Gray et al., 1999).
For generation of expression plasmids encoding CFP-tagged Gß1 and BtkPH, restriction sites were introduced by PCR using the indicated primers, the AdvantageTM II PCR enzyme system (CLONTECH Laboratories, Inc.) and the pGEM®-T Easy Vector (Promega) for first subcloning. The Gß1 cDNA was amplified using the primers 5'-TAC AAG TCC GGA CAA GCT TCC ATG AGT GAG CTT GAC CAG TTA CGG C-3' and 5'-CGG GAT CCG TCG ACC CAT GGT GGC GTT AGT TCC AGA TCT TGA GGA AGC-3', allowing for in-frame subcloning into the HindIII and BamHI sites of pECFP-C1. The cDNA encoding the PH domain of human Btk (Várnai et al., 1999) and adjacent 5' untranslated bases was amplified from cDNA of dibuturyl-cAMPdifferentiated HL-60 cells using the primers 5'-CCA AGT CCT GGC ATC TCA ATG CAT CTG-3' and 5'-TGG AGA CTG GTG CTG CTG CTG GCT C-3'. A nested PCR was performed using the primers 5'-GGA AGA TCT CGA GCC ACC ATG GCC GCA GTG ATT CTG G-3' and 5'-GGG GAT CCC GGG CCC GAG GTT TTA AGC TTC CAT TCC TGT TCT CC-3', allowing for in-frame subcloning into the XhoI and BamHI sites of pECFP-N1 (CLONTECH Laboratories, Inc.). The cDNA inserts and flanking regions of the resulting CFP-Gß1 and BtkPH-CFP constructs were confirmed by sequencing.
Cell culture, transfection, and intranuclear microinjection
HEK 293 cells (American Type Culture Collection) were grown at 37°C with 5% CO2 in DME or in MEM with Earle's salts supplemented with 10% FCS, 100 µg/ml streptomycin, and 100 U/ml penicillin. All transfections were done with a FuGENETM 6 transfection reagent (Roche) following the manufacturer's recommendations. For fluorescence microscopy experiments, cells were seeded on glass coverslips. For confocal imaging of the subcellular localization of the PI3K subunits, cells were transfected with 0.1 µg of plasmid encoding YFP-tagged PI3K
subunits, 0.2 µg of plasmid for the untagged complementary subunit, and 1 µg each of plasmid for Gß1 and G
2 (except for the experiment shown in Fig. 4 B; 0.1 µg YFP-p101 + 2 µg Gß1 or G
2 alone, or 0.5 µg Gß1 + 0.5 µg G
2 + 1 µg G
i2). For monitoring PH domain translocation, or Akt- or ERK phosphorylation, HEK cells were transfected with 0.2 µg plasmid encoding the fMLP receptor, and 0.4 µg each of the plasmids encoding the PI3K
subunits and the fluorescent PH domain. The total amount of transfected cDNA was always kept constant (2.5 µg/well) by the addition of empty expression vector. For FRET analysis, cells were transfected with 1.5 µg of plasmid encoding the YFP-tagged p110
and 0.5 µg of plasmid for CFP-tagged p101. All experiments were performed one or two days after transfection.
VSM cells from neonatal rat aortas were cultured as described previously (Reusch et al., 2001). Cells were seeded on glass coverslips and microinjected with the plasmids for GFP-GRP1PH (0.4 µg/ml), fMLP receptor (0.05 µg/ml), p110 (0.05 µg/ml), and p101 (0.05 µg/ml) using an Eppendorf micromanipulator and transjector. Confocal images were taken one day after microinjection.
Immunoblot analysis of whole-cell lysates, gel filtration, and membrane fractions
p110/p101 titration; transfected HEK cells were lysed in Laemmli buffer and whole-cell lysates including nuclei were subjected to a 10% SDS-PAGE. Proteins were blotted on nitrocellulose membranes, probed with anti-GFP (CLONTECH Laboratories, Inc.) and peroxidase-coupled antirabbit IgG antibodies (Sigma-Aldrich) and visualized by ECL (Amersham Biosciences). Akt and ERK phosphorylation; HEK 293 cells were transfected as indicated with the plasmids encoding the fMLP receptor (0.2 µg), PI3K
subunits (each 0.4 µg), and H-Ras N17 (1.5 µg). Cells were serum-starved overnight, then stimulated as indicated, and lysed in Laemmli buffer. Whole-cell lysates were subjected to SDS-PAGE and immunoblotting, and probed with antiPhospho-Akt or antiPhospho-ERK1/2 and anti-ERK1/2 as described previously (Reusch et al., 2001). Gel filtration; HEK cells were transfected with equal amounts of the plasmids for YFP-p110
(or p110
-YFP) and CFP-p101 (or pcDNA3). Cells were washed in PBS and lysed in 20 mM Hepes, pH 7.5, 150 mM NaCl, 10 mM ß-mercaptoethanol, 4 mM EDTA, 0.2% polyoxyethylene-10-laurylether (C12E10), 200 µM Pefabloc® SC, 30 µg/ml TPCK, 30 µg/ml trypsin inhibitor, and 50 µg/ml benzamidine by repetitive aspiration through a 26-gauge needle. Cytosols were prepared (100,000 g for 30 min at 4°C), and subjected to gel filtration on a 24-ml Superdex 200 column and eluted with the same buffer on an ÄKTATM purifier system (Amersham Biosciences). Fractions were collected, and aliquots were subjected to SDS-PAGE and immunoblotting with anti-GFP. Preparation of membrane fractions; HEK cells were transfected as indicated with the plasmids for YFP-p110
-K833R (0.1 µg), p101 (0.2 µg), Gß1 (1.0 µg), and G
2 (1.0 µg). Cells were washed with PBS and lysed in PBS with protease inhibitors (see above) by repetitive aspiration through a 26-gauge needle. Membranes were prepared (16,000 g for 20 min at 4°C), redissolved in Laemmli buffer, subjected to SDS-PAGE and immunoblotting, and probed with anti-p110
(see next section) and anti-Gßcommon (AS398; Leopoldt et al., 1997).
In vitro PI3K assay
HEK cells were transfected with equal amounts of the plasmids encoding p110 and p101 (or empty pcDNA3 to keep the total amount of transfected cDNA constant). Cell lysates were subjected to immunoprecipitation using a monoclonal anti-p110
antibody (Bondev et al., 2001). In brief, protein A Sepharose was preincubated with or without antibody, washed, incubated overnight with cleared lysates, and washed again. The in vitro PI3K assay was performed as described previously (Maier et al., 1999). Radioactive PtdIns-3,4,5-P3 was visualized with a PhosphorImager (Fuji Bas-Reader 1500; Ray Test).
Fluorescence imaging and detection of FRET
Glass coverslips were mounted on a custom-made chamber and covered with a Hepes-buffered solution containing 138 mM NaCl, 6 mM KCl, 1 mM MgCl2, 1 mM CaCl2, 5.5 mM glucose, 10 mM Hepes, pH 7.5, and 2 mg/ml BSA. For confocal imaging, an inverted confocal laser scanning microscope with a Plan-Apochromat 63x/1.4 objective (model LSM 510; Carl Zeiss MicroImaging, Inc.) was used. YFP or GFP were excited at 488 nm, and fluorescence emission was detected through a 505-nm long pass filter. CFP was excited 458 nm, and detected through a 475-nm long pass filter. Pinholes were adjusted to yield optical sections of 0.51.5 µm.
FRET analysis was performed using an inverted microscope with a Plan-Apochromat 63x/1.4 objective (Axiovert 100; Carl Zeiss MicroImaging, Inc.). CFP and YFP were alternately excited at 440 and 500 nm with a monochromator (Polychrome II; TILL Photonics) in combination with a dual reflectivity dichroic mirror (<460 nm and 500520 nm; Chroma Technology Corp.). Emitted light was filtered through 475505-nm (CFP) and 535565-nm (YFP) band pass filters, changed by a motorized filter wheel (Lambda 10/2; Sutter Instrument Co.), and detected by a cooled CCD camera (Imago; TILL Photonics). FRET was assessed as recovery of CFP (donor) fluorescence during YFP (acceptor) bleach. First, CFP and YFP fluorescences without acceptor bleach were recorded during 30 cycles with a 1020-ms exposure. Then, 60 cycles were recorded with an additional 2-s illumination per cycle at 510 nm to bleach YFP.
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Footnotes |
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Acknowledgments |
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This work was supported by the Deutsche Forschungsgemeinschaft and Fonds der Chemischen Industrie.
Submitted: 21 October 2002
Revised: 2 December 2002
Accepted: 2 December 2002
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