Glycobiologie, Vectorologie et Trafic Intracellulaire, Centre de Biophysique Moléculaire, Centre National de la Recherche Scientifique, F-45071 Orléans Cedex 2, France
Received on August 6, 2002; revised on February 27, 2003; accepted on March 4, 2003
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Abstract |
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Key words: cell synchronization / karyophilic sugars / microinjection / nuclear import
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Introduction |
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The mechanism of the NLS-mediated nuclear import and the import factors involved were discovered using bovine serum albumin (BSA), a nonnuclear protein of Mr 67,000. The unsubstituted protein did not pass through the nuclear pores, whereas BSA substituted with either the simian virus 40 (SV40) large T antigen NLS (simple NLS: PKKKRK) (Goldfarb et al., 1986; Kalderon et al., 1984
) or the nucleoplasmin NLS (bipartite NLS: KRPAATKKAGQAKKKK) (Robbins et al., 1991
) rapidly migrated from the cytosol to the nucleus.
In addition to the various nucleocytosolic transport mechanisms involving the stretches of basic amino acids and the superfamily of transport receptors importin/karyopherin ß, there are several other routes that do not fit this model (for a recent review, see Kuersten et al., 2001). For instance, the human RNA-editing enzyme ADAR1 has an atypical NLS, MMPNVKRKIGELVRYLNTNPVG, and displays the characteristics of a shuttling protein (Eckmann et al., 2001
). The heterogeneous nuclear RNP (hnRNP) A1 protein, mainly localized in the nucleoplasm, also shuttles between the nucleus and the cytoplasm. Unlike many nuclear proteins, hnRNP A1 does not contain a renowned NLS but a segment of
40 amino acids near the carboxyl end of the protein (designated M9); M9, which does not contain any cluster of basic residues, acts as a quite efficient NLS (Siomi and Dreyfuss, 1995
). Interestingly, sequences similar to M9 are also found in other nuclear RNA-binding proteins, such as hnRNP A2. The M9-dependent protein import pathway involves a 90-kDa protein termed transportin (Nakielny et al., 1996
). Protein kinase C does not have any known NLS, but nevertheless, it is redistributed from the cytoplasm to the nucleus on various stimuli, such as phorbol ester (Schmalz et al., 1998
); this transport is independent of importin/karyopherin ß. The transcription factor STAT1, in which NLS is composed of positively charged and hydrophobic residues (Meyer et al., 2002
), and ß-catenin, which has no NLS (Fagotto et al., 1998
), are other examples of proteins that are imported into the nucleus via an importin/karyopherin ßindependent mechanism.
Besides the mechanisms that involve peptide-based NLS-mediated nuclear import, another one makes use of sugar signals. Indeed, NLS-free neoglycoproteins (BSA substituted with sugars such as -glucopyranosides,
-mannopyranosides,
-fucopyranosides) (Duverger et al., 1993
, 1995
) or ß-di-N-acetylchitobiosides (Duverger et al., 1996
), on introduction into the cytosol of HeLa cells either by electroporation (Duverger et al., 1993
) or on digitonin-permeabilization (Duverger et al., 1995
), are transported into the nucleus. This discovery brought an experimental evidence supporting a previous hypothesis suggesting that sugar residues could act as a nuclear targeting signal (Hubert et al., 1989
; Schindler et al., 1987
).
In preliminary experiments on microinjection of fluorescent karyophilic neoglycoproteins into the cytoplasm, it appeared that the nuclear import varied from one cell to another. These results induced the following question: Is this variation related to the cell cycle? Furthermore, dealing with dividing cells, another question was: Given that the nucleus breaks down at each cell cycle, why does the cell set up sophisticated mechanisms for nuclear import?
The aim of this study was (1) to compare the intracellular fate of Glc-BSA (BSA substituted with -glucoside moieties) with that of SV40 large T antigen NLS-BSA on microinjection, that is, under nondisruptive (physiological) conditions; (2) to determine if glycosylated proteins are imported into the nucleus by a mechanism different from that involving the importin/karyopherin ß import factors; and (3) to study the regulation of the sugar-mediated import pathway through the cell cycle.
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Results |
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The highest fluorescence intensity in the nucleus of most microinjected cells was obtained with a fluorescent probe-labeled neoglycoprotein bearing -glucoside residues (either R-,Glc-BSA or F-,Glc-BSA); based on this result, this neoglycoprotein was then used in the following experiments.
To show that this import activity was sugar-specific, we performed competitive inhibition experiments by coinjecting a 10-fold molar excess of the unlabeled neoglycoprotein (Glc-BSA). The presence of an excess of the unlabeled neoglycoprotein strongly inhibited the nuclear import of the fluorescent glycoconjugate (Figure 1, row 2) but did not inhibit the import of the NLS-BSA (Figure 1, row 6). In addition, the presence of 100 µg/ml wheat germ agglutinin (WGA), a lectin known to block the traffic of macromolecules through the nuclear pores (Davis and Blobel, 1987) and specially to inhibit the nuclear import of NLS-BSA (Dabauvalle et al., 1988
; Newmeyer and Forbes, 1988
; Wolff et al., 1988
; Yoneda et al., 1987
), also abolished the nuclear import of R-,Glc-BSA (Figure 1, row 3). This result confirms that the nuclear import of glucosylated BSA involves functional nuclear pores.
Lack of nuclear export of Glc-BSA
The fact that part of the Glc-BSA microinjected in the cytosol was still present in the cytosol after 2 h incubation at 37°C could be due either to (1) a cytosolic retention mechanism or (2) a shuttling activity between the two compartments, suggesting that karyophilic sugars could also act as nuclear export signals.
To look for a putative role of karyophilic sugars as nuclear export signals, HeLa cells were injected into the nucleus (Figure 2A, row 1). After incubation for 2 h at 37°C (Figure 2A, row 2), R-,Glc-BSA as well as sugar-free F-BSA remained in the nucleus. Therefore, glucose does not behave as a nuclear export signal, and the localization of R-,Glc-BSA into the cytosol, on microinjection in the cytosol, is probably related to a retention mechanism. Similarly, NLS-BSA injected into the nucleus also remained in the nucleus after 2 h incubation (not shown).
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The nuclear import of glycoconjugates is cell cycledependent
On microinjection of fluorescein- or rhodamine-labeled neoglycoproteins in the cytosol, the fluorescence detected in the nucleus of dividing HeLa cells was variable in intensity. In some cells, a large part of R-,Glc-BSA accumulated in the nucleus; in other cells an important fraction stayed in the cytosol.
To determine if this cellcell variation in nuclear import was related to the cell cycle phase of individual cells, cells were synchronized and injected at various stages of the cell cycle. HeLa cells were synchronized at the G1/S boundary by inhibition of DNA synthesis either on a double thymidine block procedure or on a hydroxyurea treatment. Propidium iodide staining was used to check the phases of the cell cycle by flow cytometry (Figure 3).
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Concerning R-,NLS-BSA, its nuclear entry was efficient at each phase of the cell cycle with a mean N/C ratio larger than 1 (85% of the cell population) (Figure 4, rows 4 and 5), except in G2 phase, in which the mean ratio was close to 1 (Figure 4, row 6) (86% of the cells).
Nuclear exclusion of sugar-free proteins on mitosis
During mitosis, the nuclear envelope dissociates, allowing large molecules to be in contact with the nucleoplasm. Thus both neoglycoproteins and sugar-free proteins coinjected before mitosis were expected to be trapped in the nucleoplasm when the nuclear envelope is resealed. When both F-BSA and R-,Glc-BSA were injected into the cytosol of synchronized HeLa cells just before mitosis (14 h after the second thymidine block) and fixed 2 h later, surprisingly, after the reassembly of the nuclear envelope (as evidenced by the presence of two nuclei side by side), the sugar-free BSA was excluded from the nuclei (Figure 5, row 1a) but the neoglycoprotein was present inside the nucleus (Figure 5, rows 1b and 1c).
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The presence of R-,Glc-BSA into the nucleus after nuclear envelope resealing is either due to (1) an exclusion from the nucleus during mitosis, followed by an import after the reformation of the nuclear pores; (2) a nuclear retention mechanism; or (3) both mechanisms.
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Discussion |
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In this article, we used fluorescent neoglycoproteins. Neoglycoproteins are synthetic glycosylated proteins and present three interesting properties: (1) they have an Mr above 40,000, preventing a passive diffusion through nuclear pores and therefore requiring an active process to enter the nucleus; (2) their specificity is linked to the nature of the sugar borne; and (3) they are recognized by lectins with a high binding constant deriving from the presence of a large number (about 25) of the same sugar and related to avidity (Monsigny et al., 2000), also known as cluster effect (Lee and Lee, 2000
).
Both BSA substituted with sugars moieties (Mr75,000) and sugar-free BSA (Mr
67,000) used as a nonnuclear nonkaryophilic protein were microinjected into the cytosol. BSA bearing
-D-glucosyl,
-D-fucosyl, ß-di-N-acetylchitobiosyl, or
-D-mannosyl residues entered the nucleus of HeLa cells; under the same conditions, sugar-free BSA or BSA bearing ß-D-lactosyl moieties remained in the cytosol. The specificity of the import system was demonstrated by competition experiments using a 10-fold excess of unlabeled neoglycoproteins. The sugar-dependent nuclear import occurs across nuclear pores, as suggested by the efficient inhibition induced by WGA on the injection of this lectin together with a neoglycoprotein.
This alternate nuclear import pathway based on karyophilic sugars was shown to also occur in various cell lines such as A549 lung carcinoma cells, MDA-MB 231 breast cancer epithelial cells, Cos-7 monkey kidney cells, HepG2 hepatoma cells, as well as in cells in primary culture, such as peritoneal macrophages and rat hepatocytes. With the last cells, Glc-BSA was found to be very efficiently imported into the nuclei.
The sugar-mediated nuclear import was less efficient than the NLS-mediated nuclear import in HeLa cells. Indeed, whereas NLS-BSA was almost quantitatively imported into the nucleus, only a part of the neoglycoprotein passed through the nuclear pores and a part remained in the cytosol, even after a long (up to 6 h) incubation time. The presence of Glc-BSA in the cytosol is not simply related to a saturation of the import machinery because a similar cytosolic retention was observed when a 0.2 mg/ml neoglycoprotein solution was used instead of a 1 mg/ml solution (with an estimated injection volume of 50 fl, these quantities correspond to 10 and 50 fg, respectively). Moreover, this cytosolic retention is not due to a putative export activity of glucose moieties because glucose does not act as a nuclear export signal, as shown on injection of the neoglycoprotein into the nucleus, but is more probably associated to interactions with cytosolic components, such as cytosolic lectins. Cytosolic retention could be a method to regulate the nuclear uptake of glycosylated proteins and, consequently, their activity inside the nucleus.
The sugar- and the NLS-dependent nuclear import pathways were previously shown to have distinct properties. Neoglycoprotein-based nuclear import does not depend on N-ethylmaleimide sensitive cytosolic factors and is not inhibited by a large excess of protein bearing NLS peptide (Duverger et al., 1995), whereas the NLS-dependent process is (Adam et al., 1990
). Our present data demonstrated that peptide NLS-based nuclear import is not inhibited by a large excess of protein bearing karyophilic sugars, contrary to the sugar-dependent process. Those results suggest that the nuclear import mediated by sugars was independent from the NLS-mediated pathway and probably involved different transport factors. In this study, we demonstrate that the nuclear import factor (importin/karyopherin ß), which is required for the transport of NLS-BSA, is not involved in the nuclear uptake of Glc-BSA. These results confirm that the nuclear import of glycosylated proteins occurs by a mechanism distinct from that used by classical NLS-bearing proteins.
The rate of nuclear import of Glc-BSA varied from one to another in unsynchronized dividing cells, suggesting that it could depend on the cell cycle. On synchronization of HeLa cells by a double thymidine block or by hydroxyurea, Glc-BSA appeared to be more efficiently imported in the nucleus during the G1/S transition and S phases, whereas the entry of NLS-BSA was not changed but a slight decrease during the G2 phase noted; accordingly, glycosylated proteins could play a role in cellular replication processes, DNA synthesis, and/or transcriptional regulation. The lower nuclear import efficiency of Glc-BSA and NLS-BSA in G2 phase could be related to the slower physiological activity of cells during this phase. Feldherr and Akin (1990) did not find any significant differences in the transport of colloidal gold particles coated with NLS-BSA in relation with the cell cycle of HeLa cells. This discrepancy could be related to both the differences in technical approaches and to the size of the probes used. A cell cycledependent NLS (CDN) was described for the transcription factor SWI5, the nuclear entry of which occurs only in G1 phase (Jans et al., 1995
; Moll et al., 1991
).
Other examples of cell cycle-dependent nuclear import have been described: p53 (Ryan et al., 1994; Shaulsky et al., 1990
) and the Saccharomyces cerevisiae CDC6 (Jong et al., 1996
) localizing in the nucleus during the G1/S transition and S phases; the novel human HUEL protein, which is translocated specifically during S phase (del Sim et al., 2002
); cyclin B1 entering only at the beginning of mitosis (Pines and Hunter, 1991
, 1994
); and v-Jun (Chida and Vogt, 1992
; Tagawa et al., 1995
), which enters most rapidly in the nucleus during G2 phase and only slowly during G1 and S phases. Those CDNs are basic peptide sequences that are mostly regulated by phosphorylation/dephosphorylation events. Two proteins, which have no known NLS-based import mechanism, also enter the nucleus in a cell cycledependent manner: heat shock protein 70 (Hsp70) (Milarski and Morimoto, 1986
) and the heat shock cognate protein 70 (Hsc70) (Lamian et al., 1996
; Zeise et al., 1998
), are more efficiently transported during the S phase than during the G1 and G2/M phases.
Furthermore, on mitosis, the sugar-free BSA was excluded from the nucleus, while the neoglycoprotein Glc-BSA was entrapped in the nucleus because of an import mechanism, a retention mechanism, or both.
By using neoglycoproteins bearing ß-di-N-acetylchitobioside (GlcNAcß4GlcNAc) moieties, Duverger et al. (1996) suggested a role of O-GlcNAc residues in the nuclear import of naturally occurring O-glycosylated cytosolic and nuclear proteins: those which contain GlcNAc-ß-Ser and/or GlcNAc-ß-Thr (for a review, see Snow and Hart, 1998
). In support of a physiological significance of O-GlcNAc-mediated nuclear import, Lefebvre et al. (2003)
recently established a direct relationship between O-GlcNAc glycosylation, phosphorylation, and nuclear import of human microtubuleassociated Tau proteins. Indeed, hyperphosphorylation of Tau proteins induced by okadaic acid treatment was found to be correlated with a reduced incorporation of O-GlcNAc residues into Tau proteins and a diminished Tau transfer into the nucleus. These findings are in agreement with previous data showing that O-GlcNAc glycosylation and phosphorylation can compete for a same or a closely related site, leading to the hypothesis that one function of O-GlcNAc glycosylation is to transiently block phosphorylation (for a review, see Wells et al., 2001
). The hypothesis that O-GlcNAc is a signal for nuclear import is also supported by the presence of GlcNAc-binding lectins both into the cytosol and the nucleus (Facy et al., 1990
), such as CBP70 and CBP22 (Félin et al., 1994
) or Hsp70 proteins (Lefebvre et al., 2001
). These GlcNAc-specific lectins could act in the transport of O-GlcNAc-bearing glycoproteins from the cytosol to the nucleus.
The sugar-dependent nuclear import of neoglycoproteins bearing -glucoside (or
-mannoside) moieties is possibly related to that of endogenous glycoproteins containing oligomannose-type N-glycans (for a review, see Hubert et al., 1989
). Cytosolic and nuclear glycoproteins containing oligomannose-type N-glycans could come from the endoplasmic reticulum by retrotranslocation through membrane protein complexes, allowing the export of glycoproteins from the endoplasmic reticulum to the cytosol (for reviews, see Plemper and Wolf, 1999
; Tsai et al., 2002
; for a recent study on glycoproteins translocation from the endoplasmic reticulum to the cytosol, see Yoshida et al., 2002
). Glycoproteins that follow this pathway usually bear high-mannose oligosaccharides, but in addition, they could bear an
-glucose residue (for a review, see Parodi, 2000
). Such glycoproteins could interact with nucleocytosolic Glc-specific lectins, such as CBP67 (Schröder et al., 1992
), CBP70 (Sève et al., 1993
), or CBP33 (Lauc et al., 1994
). The lack of nuclear import of neoglycoproteins containing lactose moieties means that the nucleocytosolic lactose-binding lectins, such as CBP35/galectin-3 (Roff and Wang, 1983
) or CBP14/galectin-1 (Cuperlovic et al., 1995
), are not involved in this intracellular traffic. This conclusion may also be related to the fact that so far there is no evidence that mature glycoproteins with terminal galactose residues added in the Golgi apparatus could leave the secretory pathway to reach the cytosol.
The mechanism by which, on injection into the cytosol, a large glycoprotein may reach the nucleus is unknown. It could be related either to (1) the presence of a constitutive peptide nuclear localization signal in itself; (2) the interaction of one of its protein domains with a cytosolic protein containing a peptide NLS; (3) in the case of dividing cells, the trapping in the nucleus on mitosis, due to the breakdown and reassociation of the nuclear envelope; (4) the interaction with a sugar-binding protein, a lectin that may act as a nuclear import carrier; or (5) the direct interaction with nuclear pores proteins, without the need of a carrier. The nuclear import of endogenous glycoproteins could be related to one or several of these five mechanisms, and the import of neoglycoproteins should be limited to the two latter ones. Indeed, (1) a neoglycoprotein, made of serum albumin, has no NLS-type amino acid stretch; (2) the lack of inhibition of the neoglycoprotein nuclear entry by an excess of NLS-BSA invalidates the second hypothesis; (3) the nuclear trapping on mitosis is unlikely because BSA is completely excluded from the nucleus, because the concentration of the neoglycoprotein in the nucleus on mitosis is much lower than in the cytoplasm, and because the sugar-mediated nuclear import also occurred within cells in primary culture, such as liver parenchymal cells or macrophages, that are nondividing cells. Therefore, the glyco-dependent nuclear import does not require a mitotic activity; (4) the fourth is possible because nuclear and cytosolic lectins eliciting an affinity for mannose, glucose, and N-acetylglucosamine have been evidenced; and (5) finally the fifth mechanism is attractive because, with digitonin-permeabilized cells, neoglycoproteins did not require the presence of cytosolic factors to enter the nucleus (Duverger et al., 1995), whereas NLS-BSA did require the addition of cytosolic factors including importin.
In conclusion, macromolecules can be imported into the nucleus by either a sugar-based mechanism or by a peptide-based mechanism; moreover, the properties of these two pathways, summarized in Table I, have some major differences. The sugar-dependent nuclear import of glycoconjugates is importin/karyopherin ßindependent. The nuclear import of glycosylated proteins is more efficient during the G1/S transition and S phases than in G1 and G2 phases, whereas the nuclear import of proteins bearing a peptide NLS was efficient during all stages of the cell cycle, but during the G2 phase it was reduced. In addition, NLS-free and sugar-free BSA are excluded from the nucleus on mitosis, whereas glycosylated BSA as well as NLS-BSA is either excluded and reimported, partially retained in the nucleoplasm, or both. Furthermore, the sugar-dependent nuclear import was found to be very efficient in nondividing cells (liver cells in primary culture), suggesting that this nuclear import could be positively associated with environmental stimuli. The results, concerning the sugar-dependent nuclear import, were obtained with model compounds (neoglycoproteins). As long as such findings are obtained with artificial compounds, their physiological significance is not granted, and they urgently call for further studies on the nucleocytosolic traffic of endogenous cytosolic and nuclear glycoproteins and on the mechanism and the regulation of this nuclear transport pathway. These aspects are currently under investigation in our laboratory.
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Materials and methods |
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Preparation of fluorescent (neoglyco)proteins
Synthetic glycoproteins (neoglycoproteins) were prepared by adding 25±3 -D-glucopyranosyl-phenylthiocarbamyl units to BSA (Glc-BSA) or ß-D-lactosyl-phenylthiocarbamyl units (Galß4Glc-BSA), as previously described (Monsigny et al., 1984
; Roche et al., 1983
). BSA substituted with 10±3 SV40 large T antigen NLS peptides (YPKKKKRKVEDPR) was synthesized as previously described (Meunier et al., 1999
).
Fluorescein-labeled (neoglyco)proteins were obtained by using fluoresceinyl isothiocyanate (Monsigny et al., 1984; Roche et al., 1983
). Rhodamine-labeled (neoglyco)proteins were obtained by using an isothiocyanate derivative of lissamine-rhodamine obtained in two steps: lissamine-rhodamine B sulfonyl chloride was converted in lissamine-rhodamine-phenylenediamine and then in lissamine-rhodamine-phenylisothiocyanate. Lissamine-rhodamine B sulfonyl chloride (500 mg/0.865 mmol) (Molecular Probes, Leiden, Netherlands) was added to 1,4-phenylenediamine (1 g/9.25 mmol) dissolved in 10 ml N-methylpyrrolidinone (NMP; Perkin Elmer, Warrington, UK). After addition of 2 mmoles N,N-diisopropylethylamine (PE Applied Biosystems, Warrington, UK) in NMP, the mixture was left at room temperature for 30 min; the reaction was monitored by thin-layer chromatography using the solvent chloroform/methanol/10% trifluoroacetic acid (20/8/1); Rf=0.77 and 0.80 (corresponding to two isomeric forms of the dye). The conjugate lissamine-rhodamine-phenylenediamine was purified by chromatography on a silica gel column (4.5x40 cm) using the same solvent. The fractions were pooled and concentrated under reduced pressure.
According to electrospray ionization mass spectrometry, the mass of the molecular ion was 649.3, in agreement with a calculated mass of 649.2 for C33H36O6N4S2 (M+1)+. Then, lissamine-rhodamine-phenylenediamine (0.29 mg/0.45 µmol) dissolved in 20 µl of a 0.1 M sodium carbonate buffer, pH 9.6, containing 0.3 M NaCl, was converted into a phenylisothiocyanate derivative by adding N,N'-thiocarbamylbisimidazole (Aldrich, Milwaukee, WI) (0.10 mg/0.56 µmol) dissolved in 20 µl chloroform. The mixture was stirred for 30 min at room temperature. The aqueous phase was removed, and the organic phase containing the lissamine-rhodamine-phenylisothiocyanate was evaporated under reduced pressure. It was added to 10 mg BSA or neoglycoproteins (0.15 µmol) dissolved in 2 ml 0.1 M sodium carbonate buffer, pH 9.6, containing 0.3 M NaCl. After stirring for 5 h at room temperature, rhodamine-labeled (neoglyco)proteins were purified by chromatography on a GF 05 Trisacryl column (Sepracor, Villeneuve-la-Garenne, France) (2x50 cm) using an n-butanol/water (5:95) mixture as an eluent at a flow rate of 10 ml/h and lyophilized.
To remove any trace of the free label, fluorescent (neoglyco)proteins were precipitated by adding 10 volumes of absolute ethanol. The neutral sugar content of glycosylated proteins was determined by the resorcinol sulfuric acid micromethod (Monsigny et al., 1988). The fluorescent tag content was determined by spectrometry measurement on pronase digestion and found to be 2.5±1 fluorescent molecules by molecule of (neoglyco)protein (Midoux et al., 1987
).
Microinjection of glycoconjugates and confocal microscopy analysis
Cells were plated on CELLocate microgrid cover slips (Eppendorf, Hamburg, Germany) (7x104 HeLa cells) and cultured for 24 h in complete medium. Proteins or neoglycoproteins were suspended in transport buffer: 20 mM N-(2-hydroxyethyl)-piperazine-N'-(2-ethanesulfonic acid) (HEPES), pH 7.3, 110 mM potassium acetate, 5 mM sodium acetate, 2 mM magnesium acetate, 2 mM dithiothreitol, 1 mM ethylene glycol-bis(ß-aminoethyl ether)-N,N,N',N'-tetraacetic acid, and 1 µg/ml each of aprotinin, leupeptin, and pepstatin (Adam et al., 1990). Any particular material was spun down by centrifugation for 5 min at 60,000xg.
Rhodamine-labeled Glc-BSA (or NLS-BSA) and sugar-free F-BSA were coinjected at a concentration of 1 mg/ml (injection time 0.5 s) into the cytosol or the nucleus with glass micropipettes having a tip diameter ranging from 0.3 to 0.7 µm (Femtotips, Eppendorf). Injections were carried out under visual control on a monitor using a Micromanipulator 5170 and Microinjector 5242 (Eppendorf). Following injection, cells were incubated at 37°C for 5 min to 6 h, in complete medium, with 5% CO2 in a humid atmosphere and then washed and fixed at 37°C in phosphate buffered saline (PBS) containing 2% (w/v) paraformaldehyde (Merck, Darmstadt, Germany) for 20 min.
For inhibition experiments, WGA (100 µg/ml) was injected together with the fluorescent proteins or neoglycoproteins. For competitive inhibition experiments, nonlabeled glycoproteins were coinjected in a 10-fold molar excess with fluorescent proteins. To monitor the inhibitory effect of anti-importin/karyopherin ß/p97 monoclonal antibody, the antibody solution (Affinity BioReagents, Golden, CO) was microinjected together with the Glc-BSA or the NLS-BSA solution in a 1:1 ratio. Cover slips were mounted on slides in a PBS/glycerol mixture (1:1, v/v) containing 1% 1,4-diazabicyclo(2-2-2) octane as an antifading agent (Johnson et al., 1982) and observed with an MRC 1024 confocal microscope (Bio-Rad, Oxfordshire, UK) equipped with a Nikon Optiphot epifluorescence microscope (Nikon, Tokyo) and 60x Planapo objective (numerical aperture 1.4). A krypton/argon laser tuned to produce both 488 nm and 568 nm wavelength beams was used for fluorescein and lissamine-rhodamine excitation, respectively. The images were recorded with the double labeling method and with a Kalman filter (average of 10 images) and treated with Adobe Photoshop software (Adobe Systems, San Jose, CA). For each figure, all images were collected with the same setting (power, gain, iris). In each case, about 50 injected cells were analyzed; the selected cells presented in a figure are representative examples. In a quantitative approach, the intensity profile along a line drawn across a cell was determined for both a karyophilic neoglycoprotein and the sugar-free protein used as a control; the intensity profiles are displayed in a graph.
Cell synchronization by a double thymidine block
Synchronization of HeLa cells at the G1/S boundary was achieved by inhibition of DNA synthesis using the double thymidine block procedure (Stein et al., 1994). HeLa cells were seeded on CELLocate microgrid cover slips or directly on culture plates at a density of 8 x 104 cells/well (4 wells, diameter 15.5 mm) and incubated for 16 h in the presence of 2 mM thymidine (Sigma, St. Louis, MO) in complete medium. Cells were then released from the first block by washing twice with serum-free medium; they were further incubated for 9 h in thymidine-free complete medium containing 24 µM deoxycytidine (Sigma). A second thymidine block was imposed by adding serum-free medium containing 2 mM thymidine. After 16 h, cells were washed twice and released into complete fresh medium. Samples were taken at the indicated time points.
Cell synchronization by hydroxyurea
Hydroxyurea, a known inhibitor of DNA synthesis, was used to synchronize cells at the G1/S boundary, as described in Trimboli et al. (1999). Briefly, cells were maintained at confluence for 24 h in a serum-depleted medium to produce a G0G1 cell cycle arrest. Cells were replated and maintained in a fresh medium for 12 h. Cells were then incubated for 12 h in a medium containing hydroxyurea (2 mM); finally, they were incubated in a fresh medium, allowing them to progress from the G1/S phase through the cell cycle.
Cell synchronization by nocodazole
Cells were arrested in M phase (prophase state) using nocodazole, a drug that disrupts microtubules of cells entering mitosis (Zieve et al., 1980). HeLa cells were seeded on CELLocate microgrid cover slips or directly on culture plates at a density of 1 x 105 cells/well (4 wells, diameter 15.5 mm) and incubated for 12 h in complete medium. Cells were then incubated for 15 h with nocodazole (Sigma) at a final concentration of 200 ng/ml. Round cells were microinjected and released into complete fresh medium for 2 h before fixation with paraformaldehyde (see previous procedure).
Analysis of DNA content by flow cytometry
Analysis of cellular DNA content allows discrimination between cells that differ in DNA content. Cells in G1 phase versus those in G2/M that have replicated their DNA. Flow cytometry DNA quantification was done on nuclei of permeabilized cells. After trypsinization, cells were transferred into 5-ml polystyrene tubes (Becton Dickinson, Franklin Lakes, NJ). Approximately 106 cells were suspended in 0.5 ml PBS and fixed by incubation for 2 h in 4.5 ml 70% ethanol at 4°C. Samples were then centrifuged at 200xg for 5 min and, after washing with PBS, were stained for 30 min at room temperature in the dark, with 1 ml 20 µg/ml propidium iodide (Molecular Probes, Eugene, OR) in PBS containing 200 µg/ml ribonuclease A (Sigma) and 0.1% (v/v) Triton X-100. Propidium iodide intercalates into the DNA. On excitation by an argon laser light (excitation=488 nm), the dye fluorescence was collected in the FL2 channel (
emission=617 nm) of a BD-LSR flow cytometer (Becton Dickinson). Data were analyzed using CellQuest Pro software (Becton Dickinson).
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Acknowledgements |
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1 To whom correspondence should be addressed; e-mail: rondanin{at}cnrs-orleans.fr
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Abbreviations |
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References |
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