In vivo analysis of importin alpha proteins reveals cellular proliferation inhibition and substrate specificity

Abstract

The "classical" nuclear import pathway depends on importins alpha and beta. Humans have only one importin beta, while six alpha importins have been described. Whether or not distinct alpha importins are essential for specific import pathways in living human cells is unclear. We used RNA interference technology to specifically down-regulate the expression of ubiquitously expressed human alpha importins in HeLa cells. Down-regulation of importins alpha3, alpha5, alpha7, and beta strongly inhibited HeLa cell proliferation, while down-regulation of importins alpha1 and alpha4 had only a minor effect or no effect. Nucleoplasmin import was not prevented by down-regulation of any alpha importin, indicating that the importin alpha/beta pathway was generally not affected. In contrast, importin alpha3 or alpha5 down-regulation specifically inhibited the nuclear import of the Ran guanine nucleotide exchange factor, RCC1. Coinjection of recombinant alpha importins and RCC1 into down-regulated cells demonstrated that these transport defects were specifically caused by the limited availability of importin alpha3 in both cases. Thus, importin alpha3 is the only alpha importin responsible for the classical nuclear import of RCC1 in living cells.

FIG. 1.

α importins as well as importin β can be specifically down-regulated in cultured HeLa cells. (A) Regions within cDNA sequences of the importins used to design siRNAs are shown as solid boxes. The two boxes in importin α3 represent the two different siRNAs used. (B) Western blots of HeLa cells transfected with siRNAs directed against importin α1 or α4. HeLa cells were transfected twice with specific siRNAs and analyzed after either 3 or 7 days. Equal amounts of protein lysates derived from siRNA-treated cells were compared to different amounts of protein lysates obtained from control cells (co). The antibody against trap α served as a loading control. (C) Western blots of protein lysates from HeLa cells transfected with different siRNAs. At day 7, protein lysates were prepared and 10 μg of total protein was loaded per lane. Control cells were treated similarly, but without siRNAs. α, importin α; β, importin β; co, control cells. The antibody against trap α served as loading control. (D) Western blots of protein lysates from HeLa cells transfected with two different siRNAs against importin α3 (α3.1- and α3.2-siRNA) are shown. At day 7, protein lysates were prepared and compared to different amounts of protein lysates obtained from control cells.

FIG. 2.

Estimation of transfection efficiency with trap α. (A) Western blots of HeLa cells transfected with siRNAs directed against trap α. HeLa cells were transfected with a specific siRNA directed against trap α exactly as described for the different importins either twice, by using Oligofectamine for up to 7 days, or once, by using electroporation. Equal amounts of protein lysates derived from siRNA-treated cells and control cells were compared to different amounts of protein lysates obtained from untreated HeLa cells (HeLa-std). tr, cells transfected with a siRNA against trap α; co, control cells; β, importin β (loading control). (B) Control of RNAi transfection efficiency using immunofluorescence analysis. (a) HeLa cells 3 days after the start of RNAi by oligofectamine transfection; (b) HeLa cells 7 days after the start of RNAi by oligofectamine transfection; (c) control cells, treated with oligofectamine for 7 days, but without siRNA; (d) HeLa cells 4 days after the start of RNAi by transfection using electroporation; (e) control cells 4 days after electroporation without siRNA. Left section,trap α staining; middle section, Hoechst nuclear DNA staining; right panels, merge of stainings (red, trap α; blue, Hoechst). Quantification analysis revealed that more than 95% of siRNA-treated cells showed strong inhibition of trap α immunofluorescence staining. For this figure, areas displaying both transfected and untransfected HeLa cells were selected.

FIG. 3.

Down-regulation of a single α importin does not affect the subcellular distribution of other α importins. Western blotting was performed with nuclear and cytosolic extracts of HeLa cells transfected with siRNAs directed against importin α1, α3, α4, α5, or α7. HeLa cells were transfected once by using electroporation, and nuclear and cytosolic extracts were prepared after 4 days. The antibody against RCC1 served as a nuclear control as well as a loading control for nuclear extracts, and the antibody against α-tubulin served as a cytosolic control as well as a loading control for cytosolic extracts. Two different exposure times are shown for both control antibodies. α, importin α; co, control cells.

FIG. 4.

The α importins differ in their stability. Western blots of cycloheximide decay experiments for up to 48 h are shown. In addition, control cells were incubated for 6 h with both cycloheximide and the ubiquitin-proteasome inhibitor ALLN. Ten micrograms of total protein was loaded per lane, and blots were incubated with specific antibodies against the individual importins. An antibody against trap α served as a loading control. α, importin α; β, importin β; CHX, cycloheximide.

FIG. 5.

Effect of RNAi-induced importin down-regulation on cellular growth. HeLa cells were transfected twice with specific siRNAs. At day 7, cells were harvested and counted. Data are means ± standard errors of the means derived from three to eight experiments and are expressed as percentages of the growth of untransfected control cells, set at 100%. co, control; α, importin α; β, importin β. α3.1 and α3.2 represent the two different siRNAs used for importin α3 down-regulation.

FIG. 6.

Analysis of apoptosis and cell cycle in importin-deficient cells. (A) Western blot of HeLa cells transfected twice with specific siRNAs. At day 7, protein lysates were prepared, and 10 μg of total protein was loaded per lane and analyzed with an antibody against PARP. Protein lysates derived from staurosporine-treated cells (5 μM; 8 h) served as a positive control. Arrow indicates the apoptosis-specific fragmentation band. α3.1 and α3.2 indicate the two different siRNAs used for down-regulation of importin α3. stauro, staurosporine; α, importin α; β, importin β; co, control cells without siRNA treatment. (B) Absolute and relative amounts of apoptotic cells detected by Hoechst staining after treatment with siRNAs. Cells were treated for 7 days with the siRNAs indicated, fixed, and stained for apoptosis with Hoechst stain. Two independent experiments were performed. About 300 cells were analyzed each time by counting apoptotic and nonapoptotic cells in at least three different areas. From left to right, columns list siRNAs used for down-regulation, the total number of cells counted, the total number of apoptotic cells identified, and the mean percentage of apoptotic cells (and the respective standard deviation) for at least three areas analyzed for each experiment. Asterisks indicate statistical significance (P < 0.05). (C) FACS profiles derived from propidium iodide-stained HeLa cells transfected with siRNAs directed against importin α1, α3, α4, α5, α7, or β. HeLa cells were transfected twice with specific siRNAs by using electroporation and were prepared for FACS analysis after 7 days. Red lines, FACS profiles of control cells; black lines, FACS profiles of cells after importin down-regulation as indicated.

FIG.7.

Analysis of subcellular distribution of microinjected RCC1, nucleoplasmin, and IgG proteins in untreated HeLa cells and cultured HeLa cells after specific down-regulation of the various α importins. ph, phase-contrast images. (A) Analysis of the nuclear import kinetics of microinjected recombinant RCC1 and nucleoplasmin in cultured HeLa cells. Fluorescein-labeled RCC1 (RCC1-fl) and Texas red-labeled nucleoplasmin (NPL-TR) were injected into the cytoplasm of cultured HeLa cells. Representative images of time lapse analyses for both proteins are shown. Images were captured at the indicated time points by using confocal microscopy. Microinjected Alexa 594-labeled IgG served as a control and displayed no translocation into cell nuclei. (B) Analysis of subcellular distribution of microinjected RCC1 and nucleoplasmin in cultured HeLa cells after specific down-regulation of the various α importins. Representative images of two to four experiments for each α importin down-regulation, with a minimum of 10 cells investigated per experiment, are shown. For cells in which importin α1 or α7 was down-regulated, images with two simultaneously injected cells are shown. (C) Summary of the effects of RNAi-induced importin α down-regulation on nuclear import of microinjected RCC1-fl. Subcellular distribution of microinjected RCC1-fl was analyzed after 1 and 3 min by using confocal microscopy, and the percentage of cells in which nuclear staining was stronger than cytoplasmic staining was determined. RCC1 showed nuclear accumulation in less than 1 min in 80, 61, 10, 64, 25, or 49% of mock-transfected or importin α1, α3, α4, α5, or α7 knockdown cells, respectively. After 3 min, RCC1 was imported in 92, 79, 41, 86, 49, or 68% of mock-transfected cells or cells transfected with siRNAs directed against importin α1, α3, α4, α5, or α7, respectively.

FIG. 8.

Coinjection of recombinant importin α3 but not of importin α5 restores nuclear RCC1 accumulation in HeLa cells with decreased expression of importin α3 or α5. (A) RCC1 was microinjected either alone or in combination with recombinant importin α3 or α5, and subcellular distribution was analyzed by confocal microscopy at the time points indicated. Representative images of two independent experiments analyzing at least 10 cells per experiment are shown. Stars indicate no nuclear accumulation, but artifact caused by injection. (B) Summary of the experiments described in the legend to panel A. Subcellular distribution of microinjected RCC1-fl was analyzed after 1 and 3 min by using confocal microscopy, and the percentage of cells with stronger nuclear than cytoplasmic staining was determined. Coinjection of importin α3 caused fast RCC1 import, in less than 1 min, in 100% of importin α3 and α5 knockdown cells. Coinjection of importin α5 resulted in nuclear accumulation after 1 or 3 min in 12 or 28% and 35 or 47% of cells depleted of importin α3 or α5, respectively.