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First published online 30 November 2004
doi: 10.1242/jcs.01569

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Importin ß is transported to spindle poles during mitosis and regulates Ran-dependent spindle assembly factors in mammalian cells

Marilena Ciciarello¹, Rosamaria Mangiacasale¹, Catherine Thibier², Giulia Guarguaglini¹, Enzo Marchetti³, Barbara Di Fiore^1,* and Patrizia Lavia^1,

¹ Institute of Molecular Biology and Pathology, Section of Genetics, CNR Consiglio Nazionale delle Ricerche, Via degli Apuli 4, Rome 00185, Italy
² Laboratory of Developmental Biology, Université Pierre-et-Marie Curie, Paris VI, 4 place Jussieu, 75005, France
³ Department of Genetics and Molecular Biology, University `La Sapienza', Pz. A. Moro 5, Rome 00185, Italy

View larger version (65K): [in a new window]   Fig. 1. A detergent-resistant fraction of importin ß in HeLa cells. (A) IF analysis of importin ß in interphase (a,c) or mitotic (b,d) cells after PFA fixation (a,b) or TritonX-100 pre-extraction (c,d); in each series, importin ß is depicted on the left and DAPI staining on the right. Scale bars, 10 µm. (B) Confocal co-localization of importin ß (FITC, green) and -tubulin (rhodamine, red) at spindle poles of a metaphase HeLa cell. (C) Merged signals colocalize to poles throughout all sections of the metaphase; representative focal planes (0.37 µm sections) are shown.

View larger version (76K): [in a new window]   Fig. 2. Importin-ß localization to spindle poles is temporally regulated during mitosis. (A) Progression from G2, in which the NE is still intact (a) through mitosis (b-f) is revealed by DAPI (top). Centrosomes are visualized by -tubulin staining (red); importin ß is depicted in green. Scale bar, 10 µm. (B) Frequency of mitotic cells with pole-associated importin-ß signals. At least 100 mitotic figures per stage were analysed in three experiments. (C) Western blot of importin ß in extracts from asynchronous cultures (asy) in thymidine-arrested cells (time 0), and at the indicated times after thymidine wash-out, with or without inhibition of protein synthesis by cycloheximide (CHX). For control, cyclin B1 levels peaked 5-10 hours after the block release (G2- to M-phase progression) in a protein-synthesis-dependent manner.

View larger version (94K): [in a new window]   Fig. 3. RanGTP and importins and ß colocalize to spindle poles. (A) The AR-12 antibody was used in unfixed cells to label native RanGTP, followed by FITC- (a) or rhodamine- (b) conjugated secondary antibody. Importin ß was revealed using Texas-Red- (a) or FITC- (b) conjugated secondary antibody. Scale bar, 10 µm. (B) Importin in PFA-fixed metaphase (left) and anaphase (middle) cells; after Triton X-100 extraction (right), a pole-associated fraction is evident at spindle poles. Scale bars, 10 µm.

View larger version (35K): [in a new window]   Fig. 4. Importin-ß localization to asters/spindle poles depends on MT integrity and dynamics. (A) IF analysis of importin-ß/FITC and -tubulin/rhodamine in mitotic cells released from thymidine arrest and exposed to NOC or TAX, or after NOC release. (B) Frequency of mitotic figures in which importin ß did or did not localize to asters/spindle poles in the presence of MT-directed drugs. 200 mitoses per condition were analyzed in four experiments.

View larger version (37K): [in a new window]   Fig. 5. The mitotic localization of importin ß is sensitive to temperature-dependent MT depolymerization and regrowth. (A) Monitoring of MT depolymerization and regrowth by staining with -tubulin/FITC antibody (a-c). In parallel slides, importin ß was visualized by FITC (d-f) and -tubulin by rhodamine (visualized together with DAPI in g-i). Scale bar, 10 µm. (B) Frequency of mitoses in which importin ß did or did not localize at spindle poles during the assay. At least 100 mitoses per condition were analysed (two experiments). (C) Western blot of importin ß in interphase (I) and mitosis (M), with (37°C) or without (0°C) polymerized MTs.

View larger version (27K): [in a new window]   Fig. 6. Importins and ß sediment with polymerized mitotic MTs. (A) Fractions of importin and ß sediment with the MT pellet (P) after sucrose centrifugation of mitotic HeLa extracts treated with Taxol, and are enriched in the presence of AMP-PnP, but not with the addition of exogenous MTs. S, supernatant. (B) The incubation of mitotic extract with anti-DIC antibody prevents dynein association with the P fraction. Arrows indicate the position of dynein intermediate chain (DIC), antibody heavy (IgM-h) and light (IgM-l) chains in the S fraction. (C) Western-blot analysis of the indicated proteins in the MT pellet pre-incubated with anti-dynein antibody (anti-DIC lane), non-specific antibody (IgM lane) or buffer alone (BRB80 lane).

View larger version (48K): [in a new window]   Fig. 7. Importin ß overexpression induces spindle abnormalities. (A) Localization of exogenous importin-ß/GFP (green) in transfected HeLa cells, showing stable association with the NE in interphase (left) and with spindle poles in mitosis (right). Merged signals with DAPI (blue) are shown. Scale bar, 10 µm. (B) A typical abnormal spindle in a mitotic cell expressing importin-ß/GFP: notice the chromosome misalignment (DAPI, left) and a small `extrapole' in addition to the two main poles (-tubulin, middle). Scale bar, 10 µm. (C) Induction of mitotic abnormalities (left) and delay (right) in importin-ß-expressing human and murine cell lines. Abnormalities were counted among 500 mitoses in HeLa cultures, 250 in MRC5 and 400 in NIH-3T3 (two independent experiments) and their frequency in importin-ß- (pIB-GFP) and vector- (pGFP) transfected populations was statistically evaluated using the 2 test (P<<0.001 in HeLa cells, P<0.02 in MRC5 cells and P<0.001 in NIH-3T3 cells). The mitotic index was calculated in 1000 transfected cells in HeLa cultures, 800 in MRC5 cultures and 1200 in NIH-3T3 cultures (three experiments per cell line) and analysed using the 2 test (P<<0.001 in all cell lines). (D) Localization of mitotic regulators to poles of importin-ß-induced abnormal spindles (GFP emission not depicted). Markers in the leftmost column are revealed by AMCA-conjugated secondary antibody (blue) and in the central column by a redemitting secondary antibody (Cy3 or Texas Red). Merged images are shown on the right. Scale bar, 10 µm.

View larger version (32K): [in a new window]   Fig. 8. Rescue of importin-ß-dependent mitotic abnormalities by TPX2- and NLS-expressing constructs. (A) Examples of mitotic abnormalities induced by exogenous TPX2 in HeLa cells. Scale bar, 10 µm. (B) Frequency of mitotic abnormalities in HeLa cells transfected with importin ß (pIB) or TPX2 constructs, alone or together (between 150 and 250 mitoses per group were counted in two experiments). Using pGFP vector transfection as the reference group, P<0.01 for TPX2 or pIB alone, and non-significant for TPX2 plus pIB. Using pIB transfection as the reference group, P was non-significant for TPX2 alone and P<0.02 for TPX2 plus pIB. (C) Frequency of mitotic abnormalities in HeLa cells transfected with importin ß (+) or NLS constructs; symbols indicate the molar ratio of NLS to importin-ß sequence used in transfection (between 170 and 260 mitoses per group were counted in two experiments). Using pGFP as the reference group, P<0.001 for pIB; P<0.01 for pIB coexpression with equimolar NLS; P<0.05 or at the limit of significance with higher NLS amount.

View larger version (30K): [in a new window]   Fig. 9. Targeting of importin ß at spindle poles is TPX2 dependent. (A) IF to importin ß (green) in mitotic cells (revealed by DAPI, blue) from cultures treated with siRNA oligonucleotides targeting luciferase (GL2) (top) or TPX2 (bottom); TPX2 is stained in red and images are taken under identical exposure conditions. Scale bar, 10 µm. (B) NuMA recruitment (green) at spindle poles (top) in TPX2-interfered cultures (red); in the lower-magnification field (bottom), NuMA is correctly localized to poles of normal and abnormal spindles. Scale bars, 10 µm. (C) Frequency of mitotic cells showing importin ß (left) or NuMA (right) signals at spindle poles in TPX2- or GL2-interfered cultures (n=150 mitoses per group). (D) Frequency of prometaphases (identified by DAPI staining) showing pole-associated or delocalized importin-ß signals in spindles with normal or abnormal centrosomes, visualized by centrin-2 staining (n=100 mitoses per group).

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