In Vivo Study of HIV-1 Tat Arginine-rich Motif Unveils Its Transport Properties
Authors: Francesco Cardarelli, Michela Serresi, Ranieri Bizzarri, Mauro Giacca, Fabio Beltram
Journal: Molecular Therapy (2007)

Tat-derived peptides have attracted much interest as molecular carriers for intracellular delivery as they incorporate specific attributes required for efficient cargo delivery to sub-cellular domains. Little is known, however, about intracellular trafficking and interactions of Tat peptide–tagged cargoes, although some in vitro studies have suggested the relevance of active processes in Tat peptide–driven nuclear translocation. These issues are addressed by comparing Tat peptide–induced transport properties with well-established passive diffusion and active import benchmarks in living cells. Specifically, we examine several constructs of increasing molecular weight (MW) both below and above the threshold for passive diffusion through the nuclear pore. The resulting sub-cellular localization is analyzed by confocal imaging, and construct intracellular dynamics is investigated by fluorescence recovery after photobleaching (FRAP) real-time imaging. Our experiments yield the characteristic transport parameters of Tat peptide intra-cytoplasm dynamics and nucleus/cytoplasm shuttling. These results allow us to elucidate the mechanism of Tat peptide–driven nuclear permeation, demonstrating that it crosses the nuclear envelope (NE) by passive diffusion. Finally, we discuss the limitations of this route in terms of acceptable cargo size. Tat-derived peptides have attracted much interest as molecular carriers for intracellular delivery as they incorporate specific attributes required for efficient cargo delivery to sub-cellular domains. Little is known, however, about intracellular trafficking and interactions of Tat peptide–tagged cargoes, although some in vitro studies have suggested the relevance of active processes in Tat peptide–driven nuclear translocation. These issues are addressed by comparing Tat peptide–induced transport properties with well-established passive diffusion and active import benchmarks in living cells. Specifically, we examine several constructs of increasing molecular weight (MW) both below and above the threshold for passive diffusion through the nuclear pore. The resulting sub-cellular localization is analyzed by confocal imaging, and construct intracellular dynamics is investigated by fluorescence recovery after photobleaching (FRAP) real-time imaging. Our experiments yield the characteristic transport parameters of Tat peptide intra-cytoplasm dynamics and nucleus/cytoplasm shuttling. These results allow us to elucidate the mechanism of Tat peptide–driven nuclear permeation, demonstrating that it crosses the nuclear envelope (NE) by passive diffusion. Finally, we discuss the limitations of this route in terms of acceptable cargo size. IntroductionThe Tat protein of human immunodeficiency virus type 1 is a powerful transcriptional activator of viral gene expression and enhances the rate of human immunodeficiency virus type 1 long terminal repeat–specific transcription.1Raha T Cheng SW Green MR HIV-1 Tat stimulates transcription complex assembly through recruitment of TBP in the absence of TAFs.PLoS Biol. 2005; 3: e44Crossref PubMed Scopus (94) Google Scholar,2Brady J Kashanchi F Tat gets the "green" light on transcription initiation.Retrovirology. 2005; 2: 69Crossref PubMed Scopus (130) Google Scholar In addition to this critical role as a transcription factor, a growing body of evidence suggests that Tat plays a role as an extracellular protein. In fact, Tat is readily taken up by cells through interactions with heparin sulfate proteoglycans displayed on the cell membrane and then develops a number of peculiar interactions with specific cell proteins.3Tyagi M Rusnati M Presta M Giacca M Internalization of HIV-1 tat requires cell surface heparan sulfate proteoglycans.J Biol Chem. 2001; 276: 3254-3261Crossref PubMed Scopus (618) Google Scholar,4Marcello A Cinelli RA Ferrari A Signorelli A Tyagi M Pellegrini V et al.Visualization of in vivo direct interaction between HIV-1 TAT and human cyclin T1 in specific subcellular compartments by fluorescence resonance energy transfer.J Biol Chem. 2001; 276: 39220-39225Crossref PubMed Scopus (54) Google ScholarCell penetration depends upon the integrity of the basic region of the protein (YGRKKRRQRRR, basic residues in bold). This arginine-rich sequence also corresponds to the presumed nuclear localization signal (NLS) and the trans-activation responsive region binding domain of the protein.5Jeang KT Xiao H Rich EA Multifaceted activities of the HIV-1 transactivator of transcription, Tat.J Biol Chem. 1999; 274: 28837-28840Crossref PubMed Scopus (374) Google Scholar The growing interest in the cell-penetrating properties of this peptide is due to its ability to drive the internalization of a large variety of cargoes chemically coupled or fused to it in mammalian and human cell types.6Brooks H Lebleu B Vives E Tat peptide-mediated cellular delivery: back to basics.Adv Drug Deliv Rev. 2005; 57: 559-577Crossref PubMed Scopus (566) Google Scholar Data indicate that the Tat peptide can determine cellular uptake of non-permeant molecules and lead to the expected biological response, consistent with the internalization of intact peptide-cargo fusions.7Caron NJ Torrente Y Camirand G Bujold M Chapdelaine P Leriche K et al.Intracellular delivery of a Tat-eGFP fusion protein into muscle cells.Mol Ther. 2001; 3: 310-318Abstract Full Text Full Text PDF PubMed Scopus (130) Google Scholar,8Del Gaizo V Payne RM A novel TAT-mitochondrial signal sequence fusion protein is processed, stays in mitochondria, and crosses the placenta.Mol Ther. 2003; 6: 720-730Abstract Full Text Full Text PDF Scopus (98) Google Scholar Despite the variety of biotechnological applications of the Tat peptide as a delivery vector, little is known about the intracellular behavior and fate of Tat peptide–based chimeras. As mentioned, the Tat peptide is a putative NLS. So far, two distinct classes of NLSs have been identified: a single stretch of basic residues, exemplified by the NLS of the simian virus 40 (SV40) large tumor/antigen, and two clusters of basic residues separated by a spacer region of 10–12 amino acids, as typified by Xenopus laevis nucleoplasmin.9Kalderon D Roberts BL Richardson WD Smith AE A short amino acid sequence able to specify nuclear location.Cell. 1984; 39: 499-509Abstract Full Text PDF PubMed Scopus (1850) Google Scholar,10Robbins J Dilworth SM Laskey RA Dingwall C Two interdependent basic domains in nucleoplasmin nuclear targeting sequence: identification of a class of bipartite nuclear targeting sequence.Cell. 1991; 64: 615-623Abstract Full Text PDF PubMed Scopus (1240) Google Scholar These signals are usually recognized by the cytoplasmic receptor proteins Imp-α and Imp-β and actively translocated through the pore channel.11Gorlich D Kostka S Kraft R Dingwall C Laskey RA Hartmann E et al.Two different subunits of importin cooperate to recognize nuclear localization signals and bind them to the nuclear envelope.Curr Biol. 1995; 5: 383-392Abstract Full Text Full Text PDF PubMed Scopus (413) Google Scholar,12Gorlich D Vogel F Mills AD Hartmann E Laskey RA Distinct functions for the two importin subunits in nuclear protein import.Nature. 1995; 377: 246-248Crossref PubMed Scopus (409) Google Scholar The directionality of nuclear import is thought to be conferred by the asymmetric distribution of the guanosine triphosphate– and guanosine diphosphate–bound forms of protein Ran between cytoplasm and nucleus.13Smith A Brownawell A Macara IG Nuclear import of Ran is mediated by the transport factor NTF2.Curr Biol. 1998; 8: 1403-1406Abstract Full Text Full Text PDF PubMed Google Scholar,14Izaurralde E Kutay U von Kobbe C Mattaj IW Gorlich D The asymmetric distribution of the constituents of the Ran system is essential for transport into and out of the nucleus.EMBO J. 1997; 16: 6535-6547Crossref PubMed Scopus (491) Google Scholar The nuclear pore complex (NPC) also mediates passive bi-directional diffusion of proteins of molecular masses of up to 60–70 kd.15Paine PL Moore LC Horowitz SB Nuclear envelope permeability.Nature. 1975; 254: 109-114Crossref PubMed Scopus (573) Google ScholarSo far, the mechanism directing the nuclear import of the Tat peptide has been studied only by in vitro assays, with contradictory results. Truant and Cullen proposed an active nuclear import mechanism involving cytosolic factors (Imp-β, but not Imp-α) and suggested the existence of a novel class of basic NLS sequences that function independently of Imp-α.16Truant R Cullen BR The arginine-rich domains present in human immunodeficiency virus type 1 Tat and Rev function as direct importin beta-dependent nuclear localization signals.Mol Cell Biol. 1999; 19: 1210-1217Crossref PubMed Google Scholar In contrast, Efthymiadis et al. reported that the Tat peptide can target a large heterologous protein (β-galactosidase from Escherichia coli, 120 kd) to the nucleus in the absence of both Imp-β and Imp-α. An entirely novel pathway was invoked that involves energy consumption and binding to nuclear components.17Efthymiadis A Briggs LJ Jans DA The HIV-1 Tat nuclear localization sequence confers novel nuclear import properties.J Biol Chem. 1998; 273: 1623-1628Crossref PubMed Scopus (156) Google ScholarIn this work we chose a different approach that allowed us to investigate Tat peptide intracellular transport properties in living cells without the need to disrupt the integrity of the cytoplasmic or nuclear membranes. We designed green fluorescent protein (GFP)–based fusion proteins of different molecular sizes, both below and above the passive diffusion size limit. These constructs represent a benchmark for intracellular passive diffusion behavior when expressed in cells and were also used as cargoes for prototypical active import by fusing them to the NLS of SV40. The behavior of these two classes of constructs was compared with that of the same GFP cargoes fused to the Tat peptide. We analyzed the sub-cellular localization of all constructs by confocal imaging and addressed their intracellular dynamics using the fluorescence recovery after photobleaching (FRAP) method. Photobleaching is an irreversible photochemical change in the fluorophore structure so that it is no longer fluorescent. In a FRAP experiment, fluorescent molecules in a selected region of the cell are irreversibly photobleached using a high-power laser beam. The subsequent movement of surrounding non-bleached fluorescent molecules into the photobleached area is recorded at low laser power. Results from these experiments allow one to probe the trafficking properties of a given protein within living cells.18Lippincott-Schwartz J Snapp E Kenworthy A Studying protein dynamics in living cells.Nat Rev Mol Cell Biol. 2001; 2: 444-456Crossref PubMed Scopus (967) Google ScholarWe measured the characteristic kinetic parameters of Tat peptide intracellular dynamics and concluded that the molecular mechanism directing Tat peptide movement across the nuclear envelope (NE) is passive diffusion. Important limitations of this route in terms of acceptable cargo size are provided. We believe that the present results can be of great interest for those who exploit Tat peptide as a molecular carrier, to fine-tune its diagnostic and therapeutic efficacy.ResultsEngineering, expression, and localization of constructsTo assess the nucleus/cytoplasm shuttling properties driven by the Tat peptide sequence we engineered three pcDNA3-based constructs encoding differently sized GFP-based cargoes.We constructed a prototypical passive diffusion system composed of a small protein able to diffuse freely between nucleus and cytoplasm (single enhanced GFP, or EGFP) and a larger complex consisting of an enhanced blue fluorescent protein (EBFP) linked to an EGFP (hereafter, GFP2) (Figure 1a). In addition, we studied a third fusion protein, consisting of a higher molecular weight (MW) cargo composed of E1GFP (a mutated EGFP derivative), EGFP, and the dimeric red fluorescent protein tdTomato (hereafter, GFP4) (Figure 1a), whose size makes it unable to diffuse across the NE.20Wei X Henke VG Strubing C Brown EB Clapham DE Real-time imaging of nuclear permeation by EGFP in single intact cells.Biophys J. 2003; 84: 1317-1327Abstract Full Text Full Text PDF PubMed Scopus (81) Google Scholar GFPs were chosen because they are known not to interact with intracellular substrates and, in particular, not to contain an NLS capable of triggering active nuclear transport.19Ormo M Cubitt AB Kallio K Gross LA Tsien RY Remington SJ Crystal structure of the Aequorea victoria green fluorescent protein.Science. 1996; 273: 1392-1395Crossref PubMed Scopus (1902) Google ScholarExpression of the three different proteins and their correct size was verified by western blot analysis. As shown in Figure 1b, the single GFP protein shows an apparent mass of 27 kd, and GFP2 and GFP4 are detected at 56 and 110 kd, respectively. For this last construct, data indicate the presence of a detectable fraction of degraded fragments at lower MW, a result not unexpected in the case of such a complex system.To examine intracellular distribution of these proteins in living cells, Chinese hamster ovary (CHO) K1 cells were transfected with the three constructs. After 20 hours, cells were incubated with the DNA-staining Hoechst dye for 30 minutes and then examined by confocal microscopy (Figure 1c). As expected for a purely diffusive molecule, GFP displayed a uniform distribution throughout the cell with no sub-cellular organelle-specific localization.20Wei X Henke VG Strubing C Brown EB Clapham DE Real-time imaging of nuclear permeation by EGFP in single intact cells.Biophys J. 2003; 84: 1317-1327Abstract Full Text Full Text PDF PubMed Scopus (81) Google Scholar We obtained the same result in the case of GFP2: the cytoplasm and the nucleus of cells were uniformly labeled by this protein. In contrast, GFP4 was detected in the cytoplasm but excluded from the nuclear compartment in most of the cells analyzed, as expected from the expression of a 110-kd protein lacking an NLS. A small (∼10%) fraction of the cells, however, showed detectable nuclear staining (data not shown) that can be linked to degradation fragments of the GFP4 protein, as suggested by immunoblotting analysis (GFP4 lane in Figure 1b).To investigate the ability of Tat11 to mediate nuclear import of these cargoes, we obtained three additional constructs that expressed the three proteins with an additional N-terminal tag corresponding to the Tat11 peptide sequence (YGRKKRRQRRR).Finally, a third set of constructs was produced by replacing the Tat11 sequence with the NLS of the SV40 T antigen (YPKKKRKVEDP). This set provides our active import benchmark. A multiple site-directed mutagenesis was performed to replace the selected single amino acids of the Tat11 sequence with the residues belonging to the NLSSV40 sequence, as indicated in Figure 1a.Cargo-dependent sub-cellular localization of constructsAs a first test, we transfected CHO K1 cells with Tat11-tagged GFP, GFP2, and GFP4 cargoes and analyzed their sub-cellular distribution using a confocal microscope. Independent of the level of transfection, Tat11-GFP sub-cellular distribution was relatively uniform between cytoplasm and nucleoplasm (Keq = nuclear concentration/cytoplasmic concentration ∼ 1; "B" in Table 1), with a concomitant detectable enrichment in nucleolar fluorescence (Figure 2a). In an analogous manner, Tat11-GFP2 preserved the homogeneous distribution between nucleus and cytoplasm (Figure 2b). Remarkably, Tat11-GFP4 was almost exclusively localized within the cytoplasm of expressing cells (Figure 2c), indicating that the Tat11 sequence was not able to mediate the nuclear import of a cargo that is above the limit for passive diffusion across the NPC. In short, the cargo-dependent sub-cellular localization of Tat11-based constructs closely resembles that of their corresponding passive diffusion benchmarks discussed above (Figure 1c). The actual size of the GFP and Tat11-GFP constructs was verified by western blot analysis using an anti-GFP monoclonal antibody. Two lanes were detected at different positions in the gel, consistent with a ∼1.5-kd mass difference (data not shown).Table 1Transport parameters derived from nuclear FRAP measurementsRowProteinKeqτC→N (s)D/W (μm3/s)NAGFP∼177 ± 1913.6 ± 3.724BTat11-GFP∼1410 ± 1252.7 ± 1.539CNLSSV40-GFP2.2–4.460 ± 35.2 ± 0.614DGFP2∼11178 ± 2921.13 ± 0.58ETat11-GFP2∼11165 ± 3051.2 ± 0.45FNLSSV40-GFP28–22240 ± 570.8 ± 0.313GNLSSV40-GFP424–56NDNDNDAbbreviations: D/W passive diffusion parameter (mean ± SD values, defined in Materials and Methods and Results); GFP, green fluorescent protein; Keq, ratio between nucleoplasmic and cytoplasmic fluorescence (range of reported values); N, number of cells examined; ND, no data; NLSSV40, nuclear localization signal of the simian virus 40; τC→N, time constant of nuclear fluorescence recovery (mean ± SD values). Open table in a new tab Figure 2Cargo-dependent sub-cellular localization. Sub-cellular localization of Tat-tagged cargoes imaged by confocal microscopy: (a) Tat11-green fluorescent protein (Tat11-GFP) and (b) Tat11-GFP2 are uniformly distributed between compartments (cargo molecular weight (MW) is under the limit for passive diffusion), whereas (c) Tat11-GFP4 is excluded from the nucleus (cargo MW above the limit for passive diffusion). In contrast, nuclear localization signal of the simian virus 40 (NLSSV40)–tagged cargoes are predominantly localized in the nucleus of expressing cells. In particular, the nuclear accumulation of these constructs is proportional to the increasing MW of the cargo (compare sub-cellular localizations in d–f). In fact, these proteins are actively and rapidly imported by specific carriers, and their nucleus-to-cytoplasm passive diffusion is progressively impaired.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Finally, we analyzed the sub-cellular localization of NLSSV40-tagged constructs. A markedly different behavior was observed (Figure 2, lower panels). In this case, all GFP cargoes were successfully imported and, consequently, under physiological conditions, were predominantly detected in the nucleus (Keq > 1, "C," "F," and "G" in Table 1). It is worth noting that nuclear accumulation of NLSSV40-based constructs was even more marked with increasing cargo MW (compare sub-cellular localizations in Figure 2d–f). This peculiar behavior can be linked to the fact that although both large and small proteins are rapidly imported owing to the NLS-specific active processes, their nucleus-to-cytoplasm passive diffusion is impaired by the increasing cargo size.Nuclear permeation upon energy depletionSelectively to monitor the role of active processes in determining the nuclear permeation of the proteins tested, we analyzed their sub-cellular localization in response to energy depletion. Intracellular adenosine triphosphate/guanosine triphosphate stores were depleted by incubating cells with 2-deoxy-d-glucose and sodium azide in glucose-free medium. Within 30 minutes of this treatment, most cells lost Mitotracker staining (Figure 3k and l), indicating that their mitochondria were no longer active.21Farkas DL Wei M Febbroriello P Carson JH Loew LM Simultaneous imaging of cell and mitochondrial membrane potentials.Biophys J. 1989; 56: 1053-1069Abstract Full Text PDF PubMed Scopus (208) Google Scholar,22Calapez A Pereira HM Calado A Braga J Rino J Carvalho C et al.The intranuclear mobility of messenger RNA binding proteins is ATP dependent and temperature sensitive.J Cell Biol. 2002; 159: 795-805Crossref PubMed Scopus (101) Google Scholar We found that when cells expressing NLSSV40-GFP were incubated in the energy depletion medium, fluorescence rapidly equilibrated between nucleus and cytoplasm (Figure 3b). This behavior is consistent with the dominant NLS-mediated nuclear import: when the latter is blocked by energy depletion, NLSSV40-GFP passive diffusion to the cytoplasm can successfully restore a homogeneous intracellular distribution. Notably, this behavior is reversible: within a few minutes of shifting cells back to physiological conditions, fluorescence was again predominantly localized in the nucleus (Figure 3c). The same treatment showed no effect on Tat11-GFP sub-cellular localization (Figure 3dand e), providing the first evidence that Tat11-driven cargo trafficking is an energy-independent mechanism.Figure 3Nuclear permeation on energy depletion. (a) The functional nuclear localization signal (NLS) of simian virus 40 is able to accumulate green fluorescent protein (GFP) cargo into the nucleus under physiological conditions (growing cells). (b) After 30 minutes of energy depletion treatment, NLSSV40-GFP equilibrated between nucleus and cytoplasm (−ATP). (c) Within 10–15 minutes after shifting the same cell back to physiological conditions, active nuclear import was restored (+ATP). (d, e) Similar energy depletion assays did not alter the sub-cellular localization of Tat11-GFP. Scale bar: 10 μm. (f–h) Accordingly, we observed that NLSSV40-GFP2 localization was reversibly modulated by energy depletion assay, (i, j) While Tat11-GFP2 localization was not altered by the same treatment Scale bar: 10 μm. (k) Under physiological conditions, Mitotracker is selectively sequestered by active mitochondria. (l) Within 30 minutes after treatment with 2-deoxy-d-glucose and sodium azide, a 60–70% decrease in Mitotracker staining is observed, indicating mitochondrial failure. Scale bar: 10 μm. ATP, adenosine triphosphate.View Large Image Figure ViewerDownload Hi-res image Download (PPT)We performed analogous measurements on NLSSV40-GFP2 and Tat11-GFP2. As shown in Figure 3f–h, NLSSV40-GFP2 equilibrated between nucleus and cytoplasm under energy depletion conditions and promptly accumulated in the nucleus when active import was restored. Once again, however, the same treatment showed no effect on Tat11-GFP2 sub-cellular localization (Figure 3i and j). Analogously to Tat peptide constructs, isolated GFP and GFP2 showed no energy-dependent sub-cellular distribution (data not shown), as expected for these passive diffusion benchmark proteins. Comparing the behaviors displayed by Tat11 constructs and the two benchmarks provides a further indication of a dominant passive diffusion mechanism for the Tat peptide across the NE.Dynamic model for nuclear/cytoplasmic exchangeWe addressed the quantitative aspects of the nuclear/cytoplasm exchange by means of a set of time-resolved experiments analyzed using a dynamic model that includes both reversible passive diffusion between the two compartments and active nuclear import of proteins from the cytoplasm (Supplementary Figure S1). The passive diffusion flux plays a role in the case of proteins that are characterized by an MW less than the nuclear pore exclusion limit. Active transport flux must be included when NLS properties need be included. We did not include nucleus-to-cytoplasm active transport processes in our dynamic model. Each contribution is characterized by one transport parameter: D/W for passive diffusion and v0/CC0 for active transport. It is worth noting that D/W is a purely dynamic parameter, specific to the diffusing protein, whereas v0/CC0 depends both on the NLS-induced nuclear import rate (v0) and on the expression level in the cytoplasm (CC0). These parameters can be experimentally determined by studying the FRAP of nuclear and cytoplasmic compartments.FRAP analysis of nucleus/cytoplasm exchange: passive diffusionConstructs encoding for untagged GFP proteins were investigated first to provide the passive diffusion benchmark parameters. The fluorescence of the nuclear compartment of CHO K1 cells transiently transfected with GFP was photobleached by irradiating a single point with high laser power for 8 seconds. The subsequent fluorescence recovery was recorded by time-lapse acquisition at 20-second intervals (Figure 4a). As photobleaching is an irreversible process, the fluorescence recovery in the nucleus is attributable to the influx of unbleached GFP molecules from the cytoplasm, in equilibrium with the efflux of bleached molecules from nucleus to cytoplasm (compare green curves on open and filled circles in Figure 4e). Indeed, the fluorescence in the cytoplasm decreased as GFP diffused from cytoplasm into the nucleus. As expected, after cytoplasm photobleaching, cytoplasm fluorescence recovered concomitantly with a decrease in nuclear fluorescence (Supplementary Figure S2). Consistent with the hypothesis of an effective nucleus/cytoplasm exchange, the kinetic characteristics of cytoplasm fluorescence recovery resembled those of the nucleus (data not shown). Also, as we are dealing with restricted exchange between two well-mixed compartments (the nucleus and the cytoplasm) and the boundary properties (i.e., the permeability properties of the nuclear membrane owing to the finite number of nuclear pores) mostly influence the diffusive recovery kinetics, the post-bleaching concentration dynamics is described by exponential decays.23Svoboda K Tank DW Denk W Direct measurement of coupling between dendritic spines and shafts.Science. 1996; 272: 716-719Crossref PubMed Scopus (395) Google Scholar,24Majewska A Brown E Ross J Yuste R Mechanisms of calcium decay kinetics in hippocampal spines: role of spine calcium pumps and calcium diffusion through the spine neck in biochemical compartmentalization.J Neurosci. 2000; 20: 1722-1734Crossref PubMed Google Scholar Indeed, the kinetics of fluorescence recovery in all measured cells was accurately fitted by a single exponential function (fit errors were always found to be in the range 1–5%) whose time constant (τ) expresses the rate of the protein shuttling across the nuclear membrane.Figure 4In vivo fluorescence recovery after photobleaching (FRAP) analysis of reversible passive diffusion and active nuclear import. (a) Fluorescence recovery after nuclear photobleaching as green fluorescent protein (GFP) diffuses from cytoplasm to nucleus. Nuclear fluorescence (pre-bleaching panel, white arrow) was photobleached by irradiating a single point with high laser power for 8 seconds. Recovery was recorded by time-lapse imaging: selected images are reproduced, with the corresponding time of acquisition. Scale bar: 10 μm. (b) As in a, FRAP analysis of nucleus/cytoplasm exchange of GFP2 (16-second single-point bleaching). Scale bar: 10 μm. (c) FRAP analysis of actively imported nuclear localization signal of the simian virus 40 GFP (NLSSV40-GFP). Nuclear fluorescence (pre-bleaching panel) was photobleached by irradiating a single point with high laser power for 10 seconds, and subsequent recovery was monitored by time-lapse imaging: selected images are reproduced, with the corresponding time of acquisition. Scale bar: 10 μm. (d) As in c, FRAP analysis of actively imported NLSSV40-GFP2 (18-second single-point bleaching). The intensity of post-bleaching images has been adjusted to show the fluorescence recovery signal better. (e) Time course of nucleoplasmic fluorescence recovery (filled green circles) for the cell shown in a. Cytoplasmic fluorescence concomitantly decreases as GFP diffuses from cytoplasm to nucleus (open green circles). This symmetric process shows the same kinetics, yielding a time constant (τ) of approximately 70 seconds for the analyzed cell (single-exponential fits are represented by continuous black lines). Both FRAP curves are normalized by pre-bleaching values: the asymptotic value of 1 indicates that no immobile fraction of molecules can be detected in the bleached compartment (the nucleus). (f) Kinetics of nucleoplasmic fluorescence recovery after photobleaching for cells shown in b–d. τ-values (mean ± SD) are 704 ± 8 seconds (GFP2, green triangles), 230 ± 5 seconds (NLSSV40-GFP2, yellow triangles), 54 ± 2 seconds (NLSSV40-GFP, open yellow circles). As in e, the asymptotic value of 1 of recovery curves indicates that no immobile fraction of molecules is present in the nuclear compartment. Transport parameters for the whole population of analyzed cells are reported in Table 1.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Results from the whole population of analyzed cells yielded a τ-value of 77 ± 19 seconds ("A" in Table 1) for GFP shuttling. All recovery curves were normalized taking into account the pre-bleaching fluorescence values, to estimate the amount of immobile fraction within the bleached compartment. The asymptotic value of 1 in Figure 4e and f indicates that the pool of nuclear proteins was completely exchangeable with the cytoplasm, as expected for a protein that does not interact with any sub-cellular substrates. These findings are in agreement with published data addressing GFP passive bi-directional diffusion across the nuclear membrane.20Wei X Henke VG Strubing C Brown EB Clapham DE Real-time imaging of nuclear permeation by EGFP in single intact cells.Biophys J. 2003; 84: 1317-1327Abstract Full Text Full Text PDF PubMed Scopus (81) Google ScholarWe performed analogous FRAP measurements to evaluate nucleus/cytoplasm shuttling of isolated GFP2 in the same conditions (Figures 4b and f). Nuclear fluorescence was photobleached by irradiating for 16 seconds a single point with high laser power. The subsequent recovery w