2 Cell and Molecular Biology Program, Michigan State University, East Lansing, MI 48824 USA; 3 Department of Microbiology, Michigan State University, East Lansing, MI 48824 USA; 4 INSERM 257, ICGM, Paris, France, 5 Institut Jacques Monod, 75251 Paris Cedex 05, France; and 6 Department of Biochemistry, Michigan State University, East Lansing, MI 48824 USA
Received on November 27, 2001; revised on January 28, 2002; accepted on February 6, 2002.
![]() |
Abstract |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Key words: galectins/nucleocytoplasmic transport/nuclear import/nuclear export/splicing factor
![]() |
Introduction |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Nucleocytoplasmic shuttling is typically defined as the repeated bidirectional movement of a protein across the nuclear membranes (Borer et al., 1989). The association of galectin-3 with the SMN complex raises the possibility that galectin-3 might perform related functions in both the nucleus and the cytoplasm and that it might shuttle between the two compartments. To test directly whether galectin-3 can shuttle, we monitored the localization of galectin-3 in two different types of heterokaryon systems: (1) human-mouse heterodikaryons, and (2) mousemouse heterodikaryons, in which one of the cell types contained a null mutation in the galectin-3 gene (Colnot et al., 1998
). In the human-mouse heterodikaryons, we monitored the localization of human galectin-3 using a mouse monoclonal antibody that recognized human galectin-3 but not the mouse homolog. In the mousemouse heterodikaryons, we monitored the localization of mouse galectin-3 using a rat monoclonal antibody directed against galectin-3. The results obtained from both systems suggest that galectin-3 does shuttle between the nucleus and the cytoplasm.
![]() |
Results |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
|
|
|
Monokaryons were immediately distinguished from di- or polykaryons by the number of nuclei as observed by PI fluorescence (not shown). As shown in Figure 4AC, humanhuman homodikaryons were indicated by the presence of only nonfluorescent beads in the cytoplasm. Similarly, mousemouse homodikaryons were indicated by the presence of only fluorescent beads in the cytoplasm (Figure 4DF). In contrast, the presence of both fluorescent and nonfluorescent beads within a dinucleated cell indicated a humanmouse heterodikaryon (Figure 4GI). Thus, the bead-tagging method proved to be a reliable technique for recognizing heterodikaryons.
|
To assess the contribution of newly synthesized human galectin-3 in supplying the mouse nucleus, cycloheximide (CHX) was employed to block de novo protein synthesis (Agrwal et al., 1989; Borer et al., 1989
). CHX appeared to have two effects on the humanmouse heterdikaryons. First, it decreased the overall fluorescence intensity due to NCL-GAL3 staining, presumably because the drug inhibited de novo synthesis of human galectin-3. For example, in humanmouse heterodikaryons treated with CHX (Figure 5E), the values of fluorescence intensity for the two nuclei were 450 and 530, reflecting a further decrease in fluorescence beyond the dilution of human galectin-3 into the two nuclei of the heterodikaryon (described for Figure 4H, Figure 5B). Nevertheless, both of the nuclei of this heterodikayron (Figure 5E) were above the threshold value of 300 and therefore were scored positive for human galectin-3, indicating that there was still transport into the mouse nucleus.
|
Localization of human galectin-3 to both nuclei of humanmouse heterodikaryons was dependent on nuclear export
Using digitonin-permeabilized 3T3 fibroblasts, we had previously shown that galectin-3 is rapidly and selectively exported from the nucleus and that this export was sensitive to inhibition by leptomycin B (LMB) (Tsay et al., 1999). Therefore, to distinguish between the contributions of the nuclear pool and the cytoplasmic pool of human galectin-3, LMB was employed to block nuclear export of galectin-3. LMB binds and inactivates the chromosome region maintenance (CRM1) nuclear export receptor (Nishi et al., 1994
), which recognizes the leucine-rich nuclear export signal. In Figure 5, panels G and H show a representative humanmouse heterodikaryon treated with CHX and LMB.
Nine hours after fusion, approximately 50% of heterodikaryons treated with CHX alone exhibited human galectin-3 in both nuclei (59 out of 119 heterodikaryons counted, Table I). In the presence of CHX and LMB, the corresponding value was 19% (23 out of 120 heterodikaryons counted, Table I). These results suggest that the cytoplasmic pool of human galectin-3 was not the only source of the protein for the mouse nucleus. Rather, human galectin-3 in the human nucleus must also contribute to supplying the mouse nucleus, because inhibition of export of human galectin-3 from the human nucleus concomitantly reduced the proportion of heterodikaryons exhibiting human galectin-3 in both nuclei. On this basis, we conclude that at least some of the human galectin-3 appearing in the mouse nucleus of a heterodikaryon had to first exit the human nucleus into the common cytoplasm followed by entry into the mouse nucleus, thus fulfilling the definition of shuttling.
|
To confirm the absence of the galectin-3 polypeptide in MEF Gal-3 / cells, we blotted cell lysate from MEF Gal-3 / cells with the anti-Mac-2 antibody. We did not detect any polypeptide in the lane containing MEF Gal-3 / lysate (Figure 6A, lane 2). In contrast, immunoblotting with anti-Mac-2 yielded a band of the same mobility as recombinant galectin-3 (Figure 6A, lane 1) in lysates from MEF Gal-1 / cells (MEF cells containing a null mutation in the Gal-1 gene) (Poirier and Robertson, 1993), MEF wild-type (WT) cells (without any mutations), and mouse 3T3 cells (Figure 6A, lanes 35).
|
The experimental scheme of Figure 1 was used to study galectin-3 shuttling in 3T3-MEF Gal-3 /heterodikaryons. In this case, 3T3 cells served as the source of galectin-3 (cell X) and were tagged with nonfluorescent (black) beads. The MEF Gal-3 / cells served as the recipient of galectin-3 (cell Y) and were tagged with fluorescent green beads. The localization of galectin-3 in these heterodikaryons was monitored by immunofluorescence using anti-Mac-2. The results of shuttling assays conducted in 3T3-MEF Gal-3 / heterodikaryons were generally similar to those obtained from the assays in humanmouse heterodikaryons. Four hours after fusion, 97% (47 out of 48) of heterodikaryons treated with CHX prior to fusion exhibited galectin-3 in both nuclei. In the presence of CHX and LMB, the corresponding value was 18% (16 out of 89 heterodikaryons counted, Table I). In Figure 7, panels AC show a typical 3T3-MEF Gal-3 / heterodikaryon treated with CHX, and panels DF show a typical 3T3-MEF Gal-3 / heterodikaryon treated with CHX and LMB. These results suggest that the localization of galectin-3 to both nuclei of 3T3-MEF Gal-3 / heterodikaryons was independent of protein synthesis but dependent on nuclear export of galectin-3.
|
![]() |
Discussion |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Interspecies heterokaryons have been used to demonstrate nucleocytoplasmic shuttling of several other proteins, including nucleolin and B23/No38 (Borer et al., 1989), hnRNP A1 (Piñol-Roma and Dreyfuss, 1992
), the hdm2 oncoprotein (Roth et al., 1998
), and the 2A7 antigen (Levasseur-Paulin and Julien, 1999
). However, these proteins differ from galectin-3 in that they are typically most prominent in the nucleus, whereas galectin-3 typically exhibits both nuclear and cytoplasmic localization. In the case of a predominantly nuclear protein, two pools of protein could supply a recipient nucleus in a heterokaryon assay: the nuclear pool and the newly synthesized pool. In contrast, when the protein is also present in the cytoplasm, as is the case for galectin-3, then the cytoplasmic pool represents a third pool that can also supply the recipient nucleus.
The presence of a cytoplasmic pool of galectin-3 complicated our analysis of galectin-3 shuttling in that we had to differentiate between the contributions of cytoplasmic galectin-3 and galectin-3 that was exported from the nucleus. Therefore, we employed the antibiotic LMB to block nuclear export of galectin-3. By conducting shuttling assays in the presence of CHX (inhibiting protein synthesis), as well as in the presence of CHX and LMB (inhibiting protein synthesis and nuclear export), we were able to assess the contribution of each of the three pools of galectin-3 to the recipient nuclei in our heterokaryons. Indeed, our data from the humanmouse heterodikaryon assays suggest that all three pools did in fact contribute to supplying the mouse nucleus in roughly equal proportions.
The effect of LMB on the nuclear versus cytoplasmic distribution of galectin-3 has been documented for both of the cell types used in our humanmouse heterokaryon fusions. Incubation of live, intact mouse 3T3 fibroblasts in the presence of LMB (3.8 nM) resulted in the accumulation of galectin-3 in the nucleus, as revealed by accentuation of the nuclear staining (Tsay et al., 1999). At a higher concentration of LMB (15.2 nM), galectin-3 was found predominantly (if not exclusively) in the nucleus. Similar results were obtained for human LG-1 cells; incubation with LMB (15.2 nM) resulted in the accumulation of galectin-3 in the nucleus, as revealed by the almost exclusively nuclear staining (Openo et al., 2000
). These results indicate that the effect of LMB on decreasing the appearance of galectin-3 in both nuclei of heterokaryons must be due to its inhibitory effect on the export of galectin-3 from the donor nucleus, rather than any secondary effects of the drug on nuclear import.
Other shuttling proteins that appear to bear functional similarity to galectin-3 are nucleolin and hnRNP A1. Both of these proteins are associated with RNA processing, and both are believed to leave the nucleus in association with their substrate RNA molecule. Nucleolin imports ribosomal proteins from the cytoplasm into the nucleus and then coordinates binding of the ribosomal proteins to the nascent rRNA transcript (Ghisolfi-Nieto et al., 1996; Ginisty et al., 1998
). The resulting complex may mediate processing of the transcript or stimulate cleavage reactions, after which nucleolin exports assembled ribosomal subunits out of the nucleus and deposits them in the cytoplasm (Ghisolfi-Nieto et al., 1996
). hnRNP A1 mediates nuclear export of mRNA from the nucleus (Piñol-Roma and Dreyfuss, 1992
). Pre-mRNAs are bound by hnRNP A1 in the nucleus, and on completion of splicing, the nuclear export signal in hnRNP A1 directs the export of the hnRNP A1-mRNA complex to the cytoplasm (Michael et al., 1995
; Pollard et al., 1996
). Once in the cytoplasm, hnRNP A1 dissociates from the mRNA and returns to the nucleus to repeat the cycle.
The significance of trafficking of galectin-3 between the nuclear and cytoplasmic compartments is not fully understood. We have recently identified galectin-3 as a component of a macromolecular complex, designated as the SMN complex (Park et al., 2001). Like previous immunofluorescence and ultrastructural studies on galectin-3 (Moutsatsos et al., 1986
; Hubert et al., 1995
), the SMN complex is found in both the cytoplasm and the nucleus (Dietz, 1998
; Matera and Frey, 1998
). In the cytoplasm, the SMN complex is associated with the core proteins of snRNPs and is involved in the biogenesis of the snRNP particles (Fischer et al., 1997
; Mattaj, 1998
). In the nucleus, the SMN complex is found in discrete nuclear bodies called gems (gemini of coiled bodies) and it is required for supplying snRNPs to the H-complex during spliceosome assembly (Pellizzoni et al., 1998
; Meister et al., 2000
). This H-complex juncture is also where galectin-3 appears to be required for splicing, as demonstrated by the effect of galectin depletion on the accumulation of intermediates during the assembly of splicing complexes (Dagher et al., 1995
).
We propose that galectin-3 might initially associate with Gemin4 in the cytoplasm, possibly during the course of snRNP biogenesis. When the SMN complex is imported into the nucleus, galectin-3 may be taken along by way of its interaction with Gemin4. Once in the nucleus, galectin-3 may participate with the SMN complex in the assembly of the spliceosome. Last, galectin-3 may be exported from the nucleus, in association with Gemin4 and possibly other members of the SMN complex, to repeat the cycle of snRNP biogenesis and delivery.
![]() |
Materials and methods |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
The human fibroblast strain, designated LG-1 (Morgan et al., 1991), was a gift from Drs. J.J. McCormick and V.M. Maher at Michigan State University. LG-1 cells were used through passage 20 and were serially passaged at a split ratio of 1:4 as described previously (Openo et al., 2000
) in MEM-ASP supplemented with 10% fetal calf serum at 37°C and 5% CO2. Primary MEFs were derived from 129 WT strain (MEF WT), galectin-1 null mutant strain (MEF Gal-1 /) (Poirier and Robertson, 1993
) and galectin-3 null mutant stain (MEF Gal-3 /) (Colnot et al., 1998
) using the procedure described by Hogan et al. (1994)
. MEF WT, MEF Gal-1 /, and MEF Gal-3 / cells were cultured in Dulbeccos modified Eagle medium with 4.5 g/L glucose, supplemented with 44 mM sodium bicarbonate, 100 U/ml penicillin, 0.1µg/ml streptomycin, 50 µg/ml gentamicin sulfate, plus 10% calf serum at 37°C and 5% CO2. Cells were passaged serially at split ratios of 1:5 or 1:10.
The rat monoclonal antibody anti-Mac-2 recognizes an epitope in the amino-terminus of galectin-3 (Ho and Springer, 1982). The mouse monoclonal antibody NCL-GAL3 was purchased from Novocastra Laboratories (UK). FITC-conjugated goat anti-rat IgG was obtained from Boehringer Mannheim, and FITC-conjugated goat anti-mouse IgG was obtained from Santa Cruz Biotech. Microsphere styrene beads were obtained from Polysciences.
Bead tagging and polyethylene glycolmediated cell fusion
Human LG-1 and mouse 3T3 fibroblasts were seeded separately in 100 x 20 mm tissue culture plates at 5 x 103 cells/cm2, and allowed to attach for approximately 6 h. Microsphere styrene beads were then added to the culture medium at a concentration of 250 beads/cell and incubated overnight. Plates were then rinsed with Versene (140 mM NaCl, 2.68 mM KCl, 10 mM Na2HPO4, 1.47 mM KH2PO4, 0.68 mM ethylenediamine tetra-acetic acid, 0.15% phenol red, pH 7.2), trypsinized, and resuspended separately. The concentration of each cell suspension was determined, and both cell types were seeded together, each at a density of 3.9 x 103 cells/cm2, into 35-mm dishes containing sterile glass coverslips. Cocultured cells were then incubated overnight. CHX (Sigma) was added at 10 µg/ml in MEM-ASP 1 h prior to fusion, removed during fusion, and returned after fusion. LMB was a gift from Dr. Minoru Yoshida (University of Tokyo), and was added at 2 ng/ml (3.8 nM) in MEM-ASP 10 h prior to fusion, removed during fusion, and returned after fusion.
Polyethylene glycol-1000 (Fluka Chemical) was melted and diluted to 45% (v/v) with warmed serum-free MEM-ASP media, then filtered through a 0.22-µm Millex syringe driven filter unit (Millipore). Cocultured LG-1 and NIH 3T3 cells were rinsed in warmed phosphate buffered saline (PBS; 140 mM NaCl, 2.68 mM KCl, 10 mM Na2HPO4, 1.47 mM KH2PO4, pH 7.4), and treated with 45% polyethylene glycol-1000 for 55 s. The medium was promptly removed, and the cells were rinsed gently three times in warmed PBS. Cells were then incubated with MEM-ASP containing 10% fetal calf serum until immunostaining.
Immunostaining and fluorescence microscopy
Cells were prepared for fluorescence microscopy following fixation with 4% paraformaldehyde and permeabilization with 0.2% Triton X-100 (Tsay et al., 1999). Human-mouse fusions were stained with NCL-GAL3 (2.1 µg/ml in PBS containing 0.2% gelatin) for 1 h at room temperature, then washed three times with T-TBS (10 mM Tris, pH 7.5, 0.5 M NaCl, 0.05% Tween 20) on an orbital shaker at room temperature. The coverslips were then stained with FITC-goat anti-mouse IgG (in PBS containing 0.2% gelatin) for 30 min at room temperature. This and all subsequent steps were carried out in the dimmest light possible. The coverslips were washed with T-TBS, stained with PI (32 µg/ml in PBS containing 0.2% gelatin) for 30 min at room temperature, and rinsed again in T-TBS. The coverslips were then inverted onto clean microscope slides with a drop of Perma-Fluor (Lipshaw Immunon) and allowed to dry in a darkened environment overnight at room temperature. 3T3-MEF Gal-3 / fusions were treated similarly except they were stained with anti-Mac-2 (25 µg/ml), followed by FITC-conjugated goat anti-rat IgG.
Fluorescent cells were viewed using an Insight Plus laser scanning confocal microscope (Meridian Instruments). The maximum and minimum fluorescence intensities of each nucleus were determined using the image analysis subroutine. By viewing over 100 images, including human and mouse monokaryons as well as homodikaryons and heterodikaryons, we found that a nucleus devoid of human galectin-3 (and therefore, not stained by NCL-GAL3) yielded a maximum fluorescence of 250 (arbitrary units). In contrast, a nucleus containing human galectin-3 always yielded a fluorescence intensity of 300 or greater. On this basis, (1) a nucleus whose maximum fluorescence intensity was 250 or lower was scored negative for the presence of human galectin-3, and (2) a nucleus whose minimum fluorescence intensity was 300 or higher was scored positive for the presence of human galectin-3.
Preparation of cell lysates and immunoblotting
Cells were grown to near confluence in 100 x 20 mm tissue culture plates, rinsed once with ice-cold PBS, and scraped in 4 ml PBS with a rubber policeman. Scraped cells were pooled into 15-ml tubes, centrifuged at 1470 x g for 10 min at 4°C, and the supernatant was decanted. The cells were resuspended in 1 ml ice-cold PBS, transferred to Eppendorf tubes, and pelleted in a microfuge at 3406 x g for 2 min at 4°C. The supernatant was aspirated and the cells were resuspended in 150 µl of 10 mM Tris, pH 7.4, then incubated on ice for 10 min. The cell suspension was then sonicated eight times (15 s each) and stored at 20°C.
The amount of protein in each sample was quantitated by the Bradford method (Bradford, 1976) using Coomassie protein assay reagent (Pierce). Equal amounts of protein from each lysate were separated on a 12.5% sodium dodecyl sulfatepolyacrylamide gel and then transferred electrophoretically to a nitrocellulose membrane in a buffer containing 25 mM Tris, 193 mM glycine, and 10% methanol (pH 8.3). The membrane was blocked overnight at room temperature in T-TBS containing 10% nonfat dehydrated milk, then rinsed three times briefly in T-TBS and blocked with a 1:1000 dilution of goat anti-rabbit IgG in T-TBS containing 1% nonfat dehydrated milk. Following blocking, the membrane was rinsed with T-TBS.
In immunoblots employing NCL-GAL3, the membrane was incubated with NCL-GAL3 (21 ng/ml) in T-TBS containing 1% nonfat dehydrated milk for 1 h at room temperature. Following incubation with NCL-GAL3, the membrane was rinsed with T-TBS and incubated with horseradish peroxidase (HRP)conjugated goat anti-mouse IgG in T-TBS containing 1% non-fat dehydrated milk for 1 h at room temperature. The membrane was then rinsed with T-TBS, and the proteins were visualized using the Renaissance western blot chemiluminescence reagents (New England Nuclear Life Science Products, Boston, MA).
Immunoblots employing anti-Mac-2 were carried out in a similar fashion except that anti-Mac-2 was used at 125 ng/ml, and HRP-conjugated goat anti-rat IgG was used as the secondary antibody.
![]() |
Acknowledgments |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
![]() |
Abbreviations |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
![]() |
Footnotes |
---|
![]() |
References |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Barondes, S.H., Castronovo, V., Cooper, D.N.W., Cummings, R.D., Drickamer, K., Feizi, T., Gitt, M.A., Hirabayashi, J., Hughes, C., Kasai, K., and others. (1994) Galectins: a family of animal ß-galactoside-binding lectins. Cell, 76, 597598.[ISI][Medline]
Borer, R.A., Lehner, C.F., Eppenberger, H.M., and Nigg, E.A. (1989) Major nucleolar proteins shuttle between nucleus and cytoplasm. Cell, 56, 379390.[ISI][Medline]
Bradford, M.M. (1976) A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal. Biochem., 72, 248254.[CrossRef][ISI][Medline]
Charroux, B., Pellizzoni, L., Perkinson, R.A., Yong, J., Shevchenko, A., Mann, M., and Dreyfuss, G. (2000) Gemin4: a novel component of the SMN complex that is found in both gems and nucleoli. J. Cell Biol., 148, 11771186.
Colnot, C., Fowlis, D., Ripoche, M.-A., Bouchaert, I., and Poirier, F. (1998) Embryonic implantation in galectin 1/galectin 3 double mutant mice. Dev. Dyn., 211, 306313.[CrossRef][ISI][Medline]
Craig, S.S., Krishnaswamy, P., Irani, A.-M.A., Kepley, C.L., Liu, F.-T., and Schwartz, L.B. (1995) Immunoelectron microscopic localization of galectin-3, an IgE binding protein, in human mast cells and basophils. Anat. Rec., 242, 211219.[ISI][Medline]
Dagher, S.F., Wang, J.L., and Patterson, R.J. (1995) Identification of galectin-3 as a factor in pre-mRNA splicing. Proc. Natl Acad. Sci. USA, 92, 12131217.[Abstract]
Dietz, H. (1998) Polishing the cutting edge of gems. Nature Genet., 20, 321322.[CrossRef][ISI][Medline]
Fischer, U., Qing, L., and Dreyfuss, G. (1997) The SMN-SIP1 complex has an essential role in spliceosomal snRNP biogenesis. Cell, 90, 10231029.[ISI][Medline]
Ghisolfi-Nieto, L., Joseph, G., Dutilleul, F.-P., Amalric, F., and Bouvet, P. (1996) Nucleolin is a sequence-specific RNA-binding protein: characterization of targets on pre-ribosomal RNA. J. Mol. Biol., 260, 3453.[CrossRef][ISI][Medline]
Ginisty, H., Amalric, F., and Bouvet, P. (1998) Nucleolin functions in the first step of ribosomal RNA processing. EMBO J., 17, 14761486.
Ho, M.K. and Springer, T.A. (1982) Mac-2, a novel 32, 000 Mr mouse macrophage subpopulation-specific antigen defined by monoclonal antibodies. J. Immunol., 128, 12211228.
Hogan, B., Beddington, R., Costantini, F., and Lacy, E. (1994) Manipulating the mouse embryo, 2d ed. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY.
Hubert, M., Wang, S.-Y., Wang, J.L., Sève, A.-P., and Hubert, J. (1995) Intranuclear distribution of galectin-3 in mouse 3T3 fibroblasts: comparative analyses by immunofluorescence and immunoelectron microscopy. Exp. Cell Res., 220, 397406.[CrossRef][ISI][Medline]
Levasseur-Paulin, M. and Julien, M. (1999) Characterization of the 2A7 antigen as a 85-kDa human nucleocytoplasmic shuttling protein. Exp. Cell Res., 250, 439451.[CrossRef][ISI][Medline]
Matera, A.G. and Frey, M.R. (1998) Nuclear structure 98. Coiled bodies and gems: janus or gemini? Am. J. Hum. Genet., 63, 317321.[CrossRef][ISI][Medline]
Mattaj, I.W. (1998) Ribonucleoprotein assembly: clues from spinal muscular atrophy. Curr. Biol., 8, 9395.
Meister, G., Bühler, D., Laggerbauer, B., Zobawa, M., Lottspeich, F., and Fischer, U. (2000) Characterization of a nuclear 20S complex containing the survival of motor neurons (SMN) protein and a specific subset of spliceosomal Sm proteins. Hum. Mol. Genet., 9, 19771986.
Michael, W.M., Choi, M., and Dreyfuss, G. (1995) A nuclear export signal in hnRNP A1: a signal-mediated, temperature-dependent nuclear protein export pathway. Cell, 83, 415422.[ISI][Medline]
Morgan, T.L., Yang, D., Fry, D.G., Hurlin, P.F., Kohler, S.K., Maher, V.M., and McCormick, J.J. (1991) Characteristics of an infinite life span diploid human fibroblast cell strain and a near-diploid strain arising from a clone of cells expressing a transfected v-myc oncogene. Exp. Cell Res., 197, 125136.[ISI][Medline]
Moutsatsos, I.K., Davis, J.M., and Wang, J.L. (1986) Endogenous lectins from cultured cells: subcellular localization of carbohydrate-binding protein 35 in 3T3 fibroblasts. J. Cell Biol., 102, 477483.[Abstract]
Nishi, K., Yoshida, M., Fujiwara, D., Nishikawa, M., Horinuchi, S., and Beppu, T. (1994) Leptomycin B targets a regulatory cascade of crm1, a fission yeast nuclear protein, involved in control of higher order chromosome structure and gene expression. J. Biol. Chem., 269, 63206324.
Openo, K.P., Kadrofske, M.M., Patterson, R.J., and Wang, J.L. (2000) Galectin-3 expression and subcellular localization in senescent human fibroblasts. Exp. Cell Res., 255, 278290.[CrossRef][ISI][Medline]
Park, J.W., Voss, P.G., Grabski, S., Wang, J.L., and Patterson, R.J. (2001) Association of galectin-1 and galectin-3 with Gemin4 in complexes containing the SMN protein. Nucleic Acids Res., 27, 35953602.[CrossRef]
Pellizzoni, L., Naoyuki, K., Charroux, B., and Dreyfuss, G. (1998) A novel function for SMN, the spinal muscular atrophy disease gene product, in pre-mRNA splicing. Cell, 95, 615624.[ISI][Medline]
Piñol-Roma, S. and Dreyfuss, G. (1992) Shuttling of pre-mRNA binding proteins between nucleus and cytoplasm. Nature, 355, 730732.[CrossRef][ISI][Medline]
Poirier, F. and Robertson, E.J. (1993) Normal development of mice carrying a null mutation in the gene encoding the L14 S-type lectin. Development, 119, 12291236.
Pollard, V.W., Michael, W.M., Nakielny, S., Siomi, M.C., Wang, F., and Dreyfuss, G. (1996) A novel receptor-mediated nuclear protein import pathway. Cell, 86, 985994.[ISI][Medline]
Roth, J., Dobbelstein, M., Freedman, D.A., Shenk, T., and Levine, A.J. (1998) Nucleo-cytoplasmic shuttling of the hdm2 oncoprotein regulates the levels of the p53 protein via a pathway used by the human immunodeficiency virus rev protein. EMBO J., 17, 554564.
Tsay, Y.G., Lin, N.Y., Voss, P.G., Patterson, R.J., and Wang, J.L. (1999) Export of galectin-3 from nuclei of digitonin-permeabilized mouse 3T3 fibroblasts. Exp. Cell Res., 252, 250261.[CrossRef][ISI][Medline]