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• W2036692012 abstract "The regulated process of protein import into the nucleus of a eukaryotic cell is mediated by specific nuclear localization signals (NLSs) that are recognized by protein-import receptors. In this study, we present fluorescence-based methods to quantitatively address the physicochemical details of NLS recognition by the receptor protein importin α (Impα) in living cells. First, by combining fluorescence recovery after photobleaching measurements and protein-concentration calibration, we quantitatively define nuclear import saturability and afford an affinity value for NLS-Impα binding. Second, by fluorescence lifetime imaging microscopy, we directly monitor the occurrence of NLS-Impα interaction and measure its effective dissociation constant (KD) in the actual cellular environment. Our kinetic and thermodynamic analyses independently indicate that the subsaturation of Impα with the expressed NLS cargo regulates nuclear import rates in living cells, in contrast to what can be predicted on the basis of available in vitro data. Finally, our experiments also provide evidence for the regulation of nuclear import mediated by the intrasteric importin β-binding domain of Impα and yield the first estimate of its autoinhibition energy in living cells. The regulated process of protein import into the nucleus of a eukaryotic cell is mediated by specific nuclear localization signals (NLSs) that are recognized by protein-import receptors. In this study, we present fluorescence-based methods to quantitatively address the physicochemical details of NLS recognition by the receptor protein importin α (Impα) in living cells. First, by combining fluorescence recovery after photobleaching measurements and protein-concentration calibration, we quantitatively define nuclear import saturability and afford an affinity value for NLS-Impα binding. Second, by fluorescence lifetime imaging microscopy, we directly monitor the occurrence of NLS-Impα interaction and measure its effective dissociation constant (KD) in the actual cellular environment. Our kinetic and thermodynamic analyses independently indicate that the subsaturation of Impα with the expressed NLS cargo regulates nuclear import rates in living cells, in contrast to what can be predicted on the basis of available in vitro data. Finally, our experiments also provide evidence for the regulation of nuclear import mediated by the intrasteric importin β-binding domain of Impα and yield the first estimate of its autoinhibition energy in living cells. Communication between nucleus and cytoplasm in eukaryotic cells is mediated by nuclear pore complexes (NPCs), 2The abbreviations used are: NPCnuclear pore complexNLSnuclear localization signalIBBimportin β-binding domainFRAPfluorescence recovery after photobleachingFLIMfluorescence lifetime imaging microscopyEGFPenhanced green fluorescent proteinA/Dacceptor/donor concentration ratioImpimportinFRETFörster resonance energy transferCHOChinese hamster ovaryEBFPenhanced blue fluorescent protein. large macromolecular assemblies that punctuate the nuclear envelope. Transport across the NPC not only localizes proteins destined to the nucleus or cytoplasm but also plays a key role in signal transduction pathways and in the regulation of major cellular processes. Small proteins (≤60–70 kDa) can move through the NPC by passive diffusion, whereas larger proteins require energy-consuming receptor-mediated mechanisms (1.Paine P.L. Moore L.C. Horowitz S.B. Nature. 1975; 254: 109-114Crossref PubMed Scopus (578) Google Scholar). nuclear pore complex nuclear localization signal importin β-binding domain fluorescence recovery after photobleaching fluorescence lifetime imaging microscopy enhanced green fluorescent protein acceptor/donor concentration ratio importin Förster resonance energy transfer Chinese hamster ovary enhanced blue fluorescent protein. A much studied mechanism for active translocation across the nuclear envelope is based on the presence of a “classical” nuclear localization sequence (NLS). A classical NLS can consist of one cluster of basic residues (monopartite) (2.Kalderon D. Roberts B.L. Richardson W.D. Smith A.E. Cell. 1984; 39: 499-509Abstract Full Text PDF PubMed Scopus (1868) Google Scholar) or of two clusters separated by 10–12 amino acids (bipartite) (3.Robbins J. Dilworth S.M. Laskey R.A. Dingwall C. Cell. 1991; 64: 615-623Abstract Full Text PDF PubMed Scopus (1247) Google Scholar). NLS-bearing cargoes are imported by a heterodimeric import receptor composed by importin α (Impα) and β (Impβ) (4.Görlich D. Kutay U. Annu. Rev. Cell Dev. Biol. 1999; 15: 607-660Crossref PubMed Scopus (1674) Google Scholar). The C-terminal region of Impα mediates direct recognition and binding to NLS-bearing proteins (5.Conti E. Uy M. Leighton L. Blobel G. Kuriyan J. Cell. 1998; 94: 193-204Abstract Full Text Full Text PDF PubMed Scopus (658) Google Scholar). Impα, however, also contains a small N-terminal autoinhibitory domain (named importin β-binding domain; IBB) that blocks this NLS-binding site (6.Kobe B. Nat. Struct. Biol. 1999; 6: 388-397Crossref PubMed Scopus (322) Google Scholar). Binding of Impβ to Impα suppresses this autoinhibitory blockade and allows Impα to bind cargo proteins with high affinity (7.Fanara P. Hodel M.R. Corbett A.H. Hodel A.E. J. Biol. Chem. 2000; 275: 21218-21223Abstract Full Text Full Text PDF PubMed Scopus (88) Google Scholar). Subsequent translocation of the Impα·Impβ cargo complex through the NPC is believed to be mediated by weak hydrophobic interactions between Impβ and nucleoporins (8.Bayliss R. Littlewood T. Stewart M. Cell. 2000; 102: 99-108Abstract Full Text Full Text PDF PubMed Scopus (392) Google Scholar). On the nuclear side of the NPC, RanGTP dislodges Impβ from the complex (9.Görlich D. Panté N. Kutay U. Aebi U. Bischoff F.R. EMBO J. 1996; 15: 5584-5594Crossref PubMed Scopus (533) Google Scholar). The Impα IBB domain then competes with the NLS for the binding site, facilitating the release of the NLS cargo into the nucleus (7.Fanara P. Hodel M.R. Corbett A.H. Hodel A.E. J. Biol. Chem. 2000; 275: 21218-21223Abstract Full Text Full Text PDF PubMed Scopus (88) Google Scholar, 10.Catimel B. Teh T. Fontes M.R. Jennings I.G. Jans D.A. Howlett G.J. Nice E.C. Kobe B. J. Biol. Chem. 2001; 276: 34189-34198Abstract Full Text Full Text PDF PubMed Scopus (136) Google Scholar). Impα cargo dissociation is further assisted by the high affinity binding of cellular apoptosis susceptibility (CAS) protein to NLS-free Impα. CAS is a carrier protein that drives Impα export in association with RanGTP (11.Kutay U. Bischoff F.R. Kostka S. Kraft R. Görlich D. Cell. 1997; 90: 1061-1071Abstract Full Text Full Text PDF PubMed Scopus (502) Google Scholar). Both the RanGTP·Impβ and RanGTP·CAS·Impα complexes translocate back to the cytoplasm (12.Bischoff F.R. Görlich D. FEBS Lett. 1997; 419: 249-254Crossref PubMed Scopus (205) Google Scholar, 13.Floer M. Blobel G. Rexach M. J. Biol. Chem. 1997; 272: 19538-19546Abstract Full Text Full Text PDF PubMed Scopus (118) Google Scholar), where they disassemble so that transport receptors can be recycled for another round of import. The asymmetric distribution of RanGTP between nucleus and cytoplasm provides the driving force leading to unidirectional cargo transport (14.Izaurralde E. Kutay U. von Kobbe C. Mattaj I.W. Görlich D. EMBO J. 1997; 16: 6535-6547Crossref PubMed Scopus (494) Google Scholar). This complex protein import mechanism is governed by the precise tuning of interactions between the various species that ensure its characteristic efficient unidirectional rate. A thorough description of nuclear import thus requires a quantitative analysis of the thermodynamic and kinetic aspects involved in these mechanisms. In particular, the NLS-Impα binding affinity is an important determinant of how efficiently cargo is transported into the nucleus. Indeed, recent models suggest that nuclear import rates are largely governed by the level of the NLS receptor Impα (15.Riddick G. Macara I.G. J. Cell Biol. 2005; 168: 1027-1038Crossref PubMed Scopus (97) Google Scholar). Concerning the thermodynamics of the process, a few reports based on in vitro-purified and -isolated proteins provided the binding affinity between different molecular components of the system (7.Fanara P. Hodel M.R. Corbett A.H. Hodel A.E. J. Biol. Chem. 2000; 275: 21218-21223Abstract Full Text Full Text PDF PubMed Scopus (88) Google Scholar, 10.Catimel B. Teh T. Fontes M.R. Jennings I.G. Jans D.A. Howlett G.J. Nice E.C. Kobe B. J. Biol. Chem. 2001; 276: 34189-34198Abstract Full Text Full Text PDF PubMed Scopus (136) Google Scholar, 16.Harreman M.T. Hodel M.R. Fanara P. Hodel A.E. Corbett A.H. J. Biol. Chem. 2003; 278: 5854-5863Abstract Full Text Full Text PDF PubMed Scopus (61) Google Scholar, 17.Timney B.L. Tetenbaum-Novatt J. Agate D.S. Williams R. Zhang W. Chait B.T. Rout M.P. J. Cell Biol. 2006; 175: 579-593Crossref PubMed Scopus (116) Google Scholar, 18.Hodel M.R. Corbett A.H. Hodel A.E. J. Biol. Chem. 2001; 276: 1317-1325Abstract Full Text Full Text PDF PubMed Scopus (271) Google Scholar, 19.Hu W. Jans D.A. J. Biol. Chem. 1999; 274: 15820-15827Abstract Full Text Full Text PDF PubMed Scopus (83) Google Scholar). High affinity interactions were shown between certain components of the nuclear import pathway (e.g. dissociation constant KD ≈ 40 nm for NLS-Impα binding (10.Catimel B. Teh T. Fontes M.R. Jennings I.G. Jans D.A. Howlett G.J. Nice E.C. Kobe B. J. Biol. Chem. 2001; 276: 34189-34198Abstract Full Text Full Text PDF PubMed Scopus (136) Google Scholar)). On the other hand, a recently published analysis of import kinetics in living yeast cells (17.Timney B.L. Tetenbaum-Novatt J. Agate D.S. Williams R. Zhang W. Chait B.T. Rout M.P. J. Cell Biol. 2006; 175: 579-593Crossref PubMed Scopus (116) Google Scholar) showed that the import machinery carries NLS cargo molecules into the nucleus at very low rates, a result that conflicts with the nanomolar dissociation constant values between the karyopherin (analogous of the mammalian importin) and the NLS; the same authors reported 20–100 nm KD values based on in vitro assays. Moreover, no saturation of import rates was observed, despite NLS cargoes being >20-fold more abundant than their carriers. In this article, we address the intriguing discrepancy that we believe is related to the peculiar conditions of the complex environment present in living cells. We analyze both thermodynamics and kinetics of the import process in living mammalian cell lines. A few high resolution imaging techniques are exploited to quantitatively study the nuclear import process, namely fluorescent recovery after photobleaching (FRAP) and fluorescence lifetime imaging (FLIM). FRAP is used to measure cargo import kinetics and yields an estimate of the NLS-Impα equilibrium constant in living cells. Indeed, FRAP allows one to monitor dynamic processes without perturbing their biochemical steady state because the latter does not depend on the emissive properties of the monitored probe. In the supplemental equations 1–19, we illustrate the mathematical model used to describe the nucleocytoplasmic exchange in the presence of both passive and active transport through the nuclear envelope. We shall refer to this model for the analysis of FRAP data presented in this study. FLIM allows the quantitative intracellular monitoring of the binding interactions encompassing Impα and NLS-bound cargo. Both FRAP and FLIM lead to NLS-Impα affinity values in the micromolar range, different from available in vitro estimates. We argue that the micromolar range actually characterizes complex formation in the cytoplasm and regulates nuclear import rates in living cells. Our experiments also support a model of nuclear import regulation mediated by the intrasteric IBB sequence in Impα and provide the first estimate of the autoinhibition energy in a living cell. Cloning of the EGFP, EBFP-EGFP (GFP2), NLS-EGFP, and NLS-EBFP-EGFP (NLS-GFP2) constructs used in this study was described in detail in a previous report (20.Cardarelli F. Serresi M. Bizzarri R. Giacca M. Beltram F. Mol. Ther. 2007; 15: 1313-1322Abstract Full Text Full Text PDF PubMed Scopus (69) Google Scholar). The mammalian expression vector encoding for NLS-mCherry fusion protein was generated in two steps. First, the mCherry template (generous gift of Roger Y. Tsien's laboratory) was amplified by PCR. The primers used (Sigma-Genosys) were 5′-CCC AAG CTT GGG ATG GTG AGC AAG GGC GAG GAG-3′ and 5′-CCG GAA TTC CGG TTA CTT GTA CAG CTC GTC CAT GCC-3′. Second, the mCherry template was inserted into HindIII-EcoRI sites of the NLS-EGFP pcDNA3 template (20.Cardarelli F. Serresi M. Bizzarri R. Giacca M. Beltram F. Mol. Ther. 2007; 15: 1313-1322Abstract Full Text Full Text PDF PubMed Scopus (69) Google Scholar). EGFP-Impα plasmid (mouse full-length mNPI2) was kindly provided by Yoshihiro Yoneda (Department of Frontier Biosciences, Osaka University). Detail about the plasmid can be found in Ref. 21.Miyamoto Y. Hieda M. Harreman M.T. Fukumoto M. Saiwaki T. Hodel A.E. Corbett A.H. Yoneda Y. EMBO J. 2002; 21: 5833-5842Crossref PubMed Scopus (88) Google Scholar. CHO-K1 were purchased from ATCC (CCL-61) and were grown in Ham's F12K medium supplemented with 10% fetal bovine serum at 37 °C and in 5% CO2. HeLa cells (CCL-2, ATCC) and U2OS cells (HTB-96, ATCC) were grown in Dulbecco's modified Eagle's medium supplemented with fetal bovine serum (10%) with glutamine (2 mm), penicillin (10 units/ml), and streptomycin (10 μg/ml), at 37 °C and 5% CO2 atmosphere. Transfections were carried out by using Lipofectamine reagent (Invitrogen) according to the manufacturer's instructions. For live imaging, 10 × 104 cells were plated 24 h before experiments onto 35-mm glass bottom dishes (WillCo-dish GWSt-3522). Cell fluorescence was measured using a Leica TCS SP2 inverted confocal microscope (Leica Microsystems AG, Wetzlar, Germany) interfaced with an Ar laser for excitation at 458, 476, 488, and 514 nm and with a helium-neon laser for excitation at 561 and 633 nm. Glass bottom Petri dishes containing transfected cells were mounted in a temperature-controlled chamber at 37 °C (Leica Microsystems) and viewed with a 40 × 1.25 numerical aperture oil immersion objective (Leica Microsystems). Images were collected at low excitation power and monitoring emission by means of the Acousto-Optical beam splitter (AOBS) detection system of the confocal microscope. The following collection ranges were adopted: 500–550 nm (EGFP) and 580–650 nm (mCherry). Background signal was subtracted in all images. The global concentrations of intracellular EGFP- and mCherry-linked proteins were determined by using the synthetic adduct fluorescein-glycine (“F-Gly”). F-Gly was obtained by reaction of the succinimidyl ester derivative of fluorescein (Invitrogen, Molecular Probes) and glycine (Sigma) in citrate buffer at pH 9. The fluorescence signal of known phosphate-buffered saline solutions of F-Gly was collected under the microscope using EGFP acquisition settings. Fluorescence counts were converted into absolute concentration values taking into account the difference in brightness (molar absorption times fluorescence quantum yield) between EGFP (or mCherry) and F-Gly. Each FRAP experiment started with a four-time line-averaged image (pre-bleach) of the cell followed by a single-point bleach (nonscanning) near the center of the nucleus with laser pulse at full power to photobleach most of the nuclear fluorescence. Fluorescence recovery was measured by starting a time lapse acquisition within a few milliseconds after bleaching, with the imaging settings described above. Our mathematical description of nucleocytoplasmic exchange in the presence of both passive diffusion and active transport shows that the concentration of fluorescent species in the cytoplasm and nucleoplasm follow a first-order kinetics (supplemental Equations 9–10). Hence, under the assumption of fluorescence proportionality to concentration, the collected FRAP curves in both compartments were fitted to a monoexponential equation, F(t)=F∞+(F0-F∞)exp(-t/τF)(Eq. 1) where superscripts 0 and ∞ label the fluorescence intensity collected at time 0 and asymptotically after bleaching, respectively. Fluorescence values were normalized by the signal of the entire cell at the same time to correct for bleaching caused by imaging and by pre-bleach fluorescence to verify the presence of an immobile fraction of fluorescent molecules within the nucleus. The nuclear volume (V) was calculated by assuming an ellipsoid shape for the nucleus with semiaxes dx, dy, and dz: V = (4/3)·π·dx·dy·dz. The three axes were estimated from confocal images of the nucleus. FLIM measurements were performed by illuminating the sample with a 468-nm pulsed laser diode at a 50 MHz repetition rate. Fluorescence emission was detected by means of fast photon-counting heads (H7422P-40, Hamamatsu) and time-correlated single photon counting electronics (SPC-830, Becker & Hickl, Berlin, Germany) at 500–540 nm (bandpass filter 510AF23, Omega Optical, Brattleboro, VT). Measurements were performed in living cells with the confocal system previously described with a 40× oil immersion objective (Leica Microsystems). Laser power was adjusted to yield photon-counting rates of ∼105 counts per second. Fluorescence decay was analyzed by the SPCImage (Becker & Hickl, Berlin, Germany) software package. Time-correlated single photon counting-detection was used to generate a lifetime map by fitting the fluorescence decay curve in each pixel of the image. Fluorescence decay curves of biological samples containing only unbound (EGFP-Impα) or only bound Impα (NLS-mCherry]·EGFP-Impα) were fitted within a monoexponential decay model; the result of the fitting procedure is thus a single fluorescence lifetime, characteristic of the Impα form (τF and τB, respectively). When a mix of unbound and bound Impα molecules were present, lifetime data were fitted to a biexponential decay law, F(t)=XB•e-tτB+XF•e-tτF(Eq. 2) where τF and τB were set to their previously determined values, and the amplitude coefficients XB and XF are the fitting parameters. The fluorescent fusion proteins used in this study were separately expressed in CHO-K1 cells, and their subcellular localization was analyzed by confocal microscopy (Fig. 1). As expected, fluorescent proteins (EGFP, GFP2, and mCherry) are evenly distributed in the cells (i.e. ratio between nucleoplasmic and cytoplasmic fluorescence Keq ∼ 1; Fig. 1A), whereas fluorescent proteins fused to the SV40 NLS are predominantly localized in the nucleus (i.e. Keq > 1; Fig. 1B). Nuclear accumulation is more evident with increasing cargo molecular weight (see Table 1 for the range of calculated Keq values), because nucleus-to-cytoplasm passive diffusion is impaired by a larger cargo size (20.Cardarelli F. Serresi M. Bizzarri R. Giacca M. Beltram F. Mol. Ther. 2007; 15: 1313-1322Abstract Full Text Full Text PDF PubMed Scopus (69) Google Scholar). FRAP analysis of mCherry and NLS-mCherry nucleus/cytoplasm shuttling kinetics and cytoplasmic diffusivity (data not shown) yielded transport parameters in keeping with previously reported results on analogous EGFP-based constructs (22.Cardarelli F. Serresi M. Bizzarri R. Beltram F. Traffic. 2008; 9: 528-539Crossref PubMed Scopus (39) Google Scholar). EGFP-Impα is localized in both compartments (with a moderate nuclear enrichment; Fig. 1C). Data show Impα accumulation on the nuclear envelope that can be linked to its binding to NPC components, such as nucleoporins and importin β (23.Ciciarello M. Mangiacasale R. Thibier C. Guarguaglini G. Marchetti E. Di Fiore B. Lavia P. J. Cell Sci. 2004; 117: 6511-6522Crossref PubMed Scopus (69) Google Scholar). This Impα localization is consistent with existing studies in living cells (21.Miyamoto Y. Hieda M. Harreman M.T. Fukumoto M. Saiwaki T. Hodel A.E. Corbett A.H. Yoneda Y. EMBO J. 2002; 21: 5833-5842Crossref PubMed Scopus (88) Google Scholar).TABLE 1Thermodynamic and kinetic parameters derived for NLS-tagged EGFP and GFP2 cargoesKeqKD*vC→Nμmμm3/sNLS-EGFP2.2–4.616 ± 7294 ± 24NLS-GFP27.5–20.420 ± 6304 ± 26 Open table in a new tab Our FRAP analysis requires the quantitative determination of the concentration of GFP-tagged proteins in a living cell (in this case, EGFP-Impα or NLS-EGFP and NLS-mCherry). One possible way to calibrate GFP-tagged proteins makes use of GFP solutions of known concentration in the cell medium. Confocal imaging of GFPs, however, usually exhibits a gradient in the fluorescence intensity along the optical axis (z) that is usually attributed to aspecific adsorption of the protein onto the bottom of the Petri dish (Fig. 2A, right panel). A time-consuming pretreatment of the Petri dish with an adsorption blocking agent such as bovine serum albumin or chlorosilane is required whenever GFPs need be used for calibration (24.Brown C.M. Dalal R.B. Hebert B. Digman M.A. Horwitz A.R. Gratton E. J. Microsc. 2008; 229: 78-91Crossref PubMed Scopus (145) Google Scholar), but this treatment is not usually compatible with cell viability. Here, we overcame this problem by means of a novel method based on the stable and biocompatible F-Gly. F-Gly displays a homogeneous distribution of fluorescence intensity along the z axis when confocally imaged (Fig. 2A, left panel) and retains most spectral characteristics of EGFP. Importantly, F-Gly does not permeate into cells and can therefore be added to cell cultures and imaged at the same focal height of cells (Fig. 2B) without affecting the intracellular determination of EGFP. This prevents the effect of optical aberration on fluorescence determination at different focal heights from the coverslip. Following this procedure, we carried out the calibration of intracellular EGFP concentration by using increasing amounts of F-Gly in the cell medium. The brightness ratio between EGFP and F-Gly was easily determined by steady state fluorescent measurements in a cuvette adopting the same excitation and collection ranges of experiments conducted in living cells. The concentration of mCherry was in turn calculated by using the measured brightness ratio between EGFP and mCherry in two excitation/emission setups. We found EGFP-Impα concentration values ranging from 1–2 μm up to 20–30 μm in highly transfected cells. Transfected NLS-mCherry generally yielded a wider range of expression levels, ranging from low micromolar concentration up to 200–300 μm. FRAP analysis of nucleocytoplasmic shuttling was performed on cells expressing the NLS-EGFP construct (or NLS-GFP2, see “Experimental Procedures”). The nuclear fluorescence was photobleached, and the subsequent recovery was monitored by time-lapse imaging (Fig. 3A). The fluorescence recovery in the nucleoplasm and/or the decay in the cytoplasm were well fitted by monoexponential equations (Fig. 3B), consistently with the kinetic behavior of our model of nucleocytoplasmic exchange (supplemental equations 9–10). The ratio between the asymptotic nuclear and cytoplasmic fluorescence, the recovery constant, the nuclear volume, and the global concentration of NLS molecules in the cytoplasm (denoted here by CNLS) were combined to calculate ΦC→N and PN, which represent the excess flux of cargo toward the nucleus solely due to active transport and nuclear envelope permeability, respectively (for definitions and more details, refer to supplemental data). Our model of nucleocytoplasmic diffusion links the latter two parameters to the concentration of cargo molecules in the cytoplasm bound to the importin complex (denoted here by [NLS·Impα]) according to ΦC→N=[NLS•Impα](VC→N-PX)(Eq. 3) where vC→N (μm3/s) is the maximum rate for active transport toward the nucleus (i.e. the rate achievable when all NLS-EGFP is bound to Impα). For each cell, ΦC→N (mol/s) was determined experimentally and plotted against CNLS. (The latter was obtained from the fluorescence emission making use of our calibration procedure; Fig. 3D.) Notably, CNLS spans a large interval of concentrations on account of the large expression variability of the NLS-EGFP cargo and so does the excess active transport flux ΦC→N. Fig. 3D shows that ΦC→N increases linearly up to CNLS ≈ 15–20 μm and indicates that, at this concentration, the endogenous Impα/β transport system is operating below maximum capacity; at higher NLS-EGFP cytoplasmic density values, import rates deviate from linearity and finally reach a saturation value, as expected for a binding isotherm. If we assume a single binding equilibrium between the NLS cargo and Impα (Scheme 1), [NLS·Impα] can be expressed as a function of CNLS, the global cytoplasmic concentration of Impα (CImpα), and the binding dissociation constant KD*. [NLS•Impα]=12(CImpα+CNLS+KD*) -12[(CImpα+CNLS+KD*)2-4CIMPαCNLS]0.5(Eq. 4) To recover the biochemically relevant parameter KD*, we fitted the ΦC→N versus CNLS curve with Equations 3 and 4, setting CImpα = 1 μm, the reported value of global Impα concentration in a living cell (25.Percipalle P. Butler P.J. Finch J.T. Jans D.A. Rhodes D. J. Mol. Biol. 1999; 292: 263-273Crossref PubMed Scopus (20) Google Scholar). Our fitting analysis yields KD* = 16 ± 7 μm and vC→N = 300 μm3/s, as reported in Table 1 (for a mean nuclear volume of 800 femtoliters, this vC→N value corresponds to ∼1.5 × 108 molecules/s). As shown in Fig. 4A, we found an analogous trend in ΦC→N versus CNLS for NLS-GFP2, yielding a similar value for the maximum rate of active transport toward the nucleus, vC→N (Table 1). We previously reported that this 2-fold increase in cargo size has a severe impact on passive diffusion kinetics in living cells (20.Cardarelli F. Serresi M. Bizzarri R. Giacca M. Beltram F. Mol. Ther. 2007; 15: 1313-1322Abstract Full Text Full Text PDF PubMed Scopus (69) Google Scholar). Conversely, we show here that the kinetics of NLS cargo nuclear import by the endogenous Impα/β machinery is not significantly affected by the same increase in cargo size. Notably, the trend of ΦC→N versus CNLS does not depend on the specific cell line chosen (Fig. 4B).FIGURE 4The FRAP assay: application to NLS-GFP2 and other cell lines. A, despite its influence on the shuttling kinetics (reported in Ref. 20.Cardarelli F. Serresi M. Bizzarri R. Giacca M. Beltram F. Mol. Ther. 2007; 15: 1313-1322Abstract Full Text Full Text PDF PubMed Scopus (69) Google Scholar), GFP2 cargo has no effect on the saturation behavior of nuclear import rates. Fitting of cumulative NLS-GFP2 data to Equation 4 (solid black line) yielded KD* = 20 ± 6 μm. B, the same FRAP analysis of nuclear import was conducted in other two cell lines: HeLa (black dots) and U2OS (red dots). Nuclear import saturability in these cell lines is consistent with that observed in CHO-K1 cells.View Large Image Figure ViewerDownload Hi-res image Download (PPT) Available in vitro assays testing the binding equilibrium of NLS and Impα (the latter in the Impβ-activated form) indicate that the thermodynamic dissociation constant (KD) is in the nanomolar range (17.Timney B.L. Tetenbaum-Novatt J. Agate D.S. Williams R. Zhang W. Chait B.T. Rout M.P. J. Cell Biol. 2006; 175: 579-593Crossref PubMed Scopus (116) Google Scholar), in contrast with our observation of a micromolar binding range. We wish to stress, however, that KD* can indeed be very different from KD, because of the complex and heterogeneous nature of the intracellular environment. On account of this, KD* will be henceforth referred to as the “effective dissociation constant” of the NLS-Impα binding equilibrium. The origin of this difference and the relevance of the KD* value will be discussed in the following sections. We developed a Förster resonance energy transfer (FRET)-by-FLIM method to quantitatively monitor protein-protein interactions in living cells and obtain a separate independent measurement of the affinity of NLS-Impα binding in physiological conditions. We used EGFP as the donor fluorophore (high brightness and photostability and monoexponential lifetime) fused to Impα and mCherry as the acceptor (fast maturation, large absorption, and high photostability (26.Shaner N.C. Campbell R.E. Steinbach P.A. Giepmans B.N. Palmer A.E. Tsien R.Y. Nat. Biotechnol. 2004; 22: 1567-1572Crossref PubMed Scopus (3507) Google Scholar, 27.Albertazzi L. Arosio D. Marchetti L. Ricci F. Beltram F. Photochem. Photobiol. 2009; 85: 287-297Crossref PubMed Scopus (99) Google Scholar)) fused to the NLS. A quantitative description of the NLS-Impα interaction was obtained by extracting from the FLIM data the molar fractions of free and bound Impα, i.e. XF and XB, respectively (Equation 2). Following the procedure described under “Experimental Procedures,” we first determined τF and τB, the donor lifetime values corresponding to free EGFP-Impα and to its bound form, respectively. To measure the lifetime of donor, we recorded intensity and lifetime images of cells expressing only EGFP-Impα (Fig. 5A). As the expected fluorescence decay was well fitted by a monoexponential function yielding τF = 2.57 ± 0.01 ns (mean ± S.D. for n = 12 analyzed cells, Table 2). The EGFP lifetime is quite homogeneous cell wide (Fig. 5A, color-coded image) consistently with the known insensitivity of EGFP emission to the local details of the cellular environment (see Fig. 5B for the experimental lifetime dispersion).TABLE 2Average (mean ± S.D.) lifetime values (ns) from the whole population of observed cellsmCherryNLS-mCherry (−ATP)EGFP-Impα2.57 ± 0.012.55 ± 0.032.19 ± 0.04" @default.
• W2036692012 created "2016-06-24" @default.
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• W2036692012 date "2009-12-01" @default.
• W2036692012 modified "2023-09-27" @default.
• W2036692012 title "Probing Nuclear Localization Signal-Importin α Binding Equilibria in Living Cells" @default.
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