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First published online 16 October 2007
doi: 10.1242/jcs.009308

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RANBP1 localizes a subset of mitotic regulatory factors on spindle microtubules and regulates chromosome segregation in human cells

Antonio Tedeschi^*, Marilena Ciciarello, Rosamaria Mangiacasale, Emanuele Roscioli, Wilhelmina M. Rensen and Patrizia Lavia

IBPM Institute of Molecular Biology and Pathology, CNR National Research Council, Via degli Apuli 4, 00185 Rome, Italy

View larger version (41K): [in this window] [in a new window]   Fig. 1. RANBP1 downregulation in human U20S cells. (A) Western blotting of the indicated proteins in asynchronously cycling U2OS cultures interfered with RANBP1-specific 202 (+) or GL2 (–) siRNA. OF, oligofectamine only; NT, non-transfected cells; RanBP1(i), RANBP1-interfered. (B) Representative RANBP1(i) cells (lower panels) with virtually no RANBP1 signal (green) and elevated RAN-GTP content (red). Bars, 10 µm. The graph (right) shows the distribution of RAN-GTP signal intensity in RANBP1(i) and in GL2-interfered cells. A total of 30 cells per group were randomly selected; the signal was quantified by densitometry and normalized to the cell area; cells were grouped in classes of fluorescence intensity expressed in arbitrary units (a.u.); mean values in the RANBP1(i) (red arrow) and GL2 control (blue arrow) groups are shown. (C) Representative fields from U2OS cultures (40x objective) show morphological changes in cell shape in RANBP1(i) compared with GL2-interfered cultures. Bar, 20 µm. (D) The MI is consistently lower in RANBP1(i) cultures compared with samples incubated with non-specific siRNAs. P values indicate statistically significant (*) or highly significant (**) differences between control versus RANBP1(i) cultures using the 2 test. (E) FACS analysis of apoptosis in RANBP1(i) and control cultures after 64 hours of RNAi: RANBP1(i) cells accumulate in the <2C region after propidium iodide (PI) staining (upper panels) and show an increased fluorescence intensity after incubation with annexin V (lower panels; viable and apoptotic cells are distributed, respectively, before and after the threshold value of 10 in the logarithmic scale). (F) A representative apoptotic cell from RANBP1(i) cultures (bottom panel); compare to the control cell in the top panel. The graph shows that the percentage of apoptotic cells increases over time in RANBP1(i) cultures and is consistently higher compared with samples incubated with non-specific siRNAs at all time-points studied. P values indicate statistically significant (*) or highly significant (**) differences between control versus RANBP1(i) cultures using the 2 test. Quantitative results in D and F were obtained from three experiments using two RANBP1-specific (202 and 459) and two control (GL2 and 116) siRNAs. A total of 1000-1500 cells were scored for each time-point.

View larger version (31K): [in this window] [in a new window]   Fig. 2. RANBP1 depletion hinders mitotic entry and progression in synchronously cycling cells. (A) Cell cycle progression in synchronized cultures. Cells were arrested at the G1/S boundary by double thymidine (thym) block, treated with siRNA during the second block, then released to resume cell cycle progression. RANBP1-interfered [RANBP1(i)] and control samples were harvested at regular intervals after thymidine release (time 0) and analyzed by FACS. (B) Histogram representing the proportion of cells in different phases at each time. (C) Western blot analysis of the indicated proteins in RANBP1-interfered (+) and control (–, GL2-interfered) synchronized cultures. (D) Histograms represent the MI scored in RANBP1-interfered and control cultures. *Significant (P<0.01) and **highly significant (P<0.001) differences for each time-point were evaluated using the 2 test.

View larger version (23K): [in this window] [in a new window]   Fig. 3. The mitotic distribution of RAN-GTP is abnormal in RANBP1-interfered mitoses. (A) Co-sedimentation of RAN and its effectors with MTs from mitotic cells treated with buffer (–), NOC or taxol (indicated with +). HURP and TPX2 are shown for comparison. WCE, whole cell extract; sup, supernatant. (B) Localization of AR12-reactive RAN-GTP in control and RANBP1-interfered [RANBP1(i)] prometaphases (a,c) and metaphases (b,d,e). Bars, 10 µm. Graph represents the frequency of RAN-GTP distribution phenotypes in 215 RANBP1- and 210 GL2-interfered cells. The differences were statistically significant (*P<0.02) or highly significant (**P<0.001) using the 2 test.

View larger version (70K): [in this window] [in a new window]   Fig. 4. Impaired association of cyclin B1 with spindle MTs in RANBP1-interfered cells. (A) Cyclin B1 (green) localization in prometaphase (a,c) and metaphase (b,d) cells, identified by DAPI (schematics on the left, in which DNA is blue and MTs red), transfected with GL2 (no colour; a,b) or Cy3-conjugated RANBP1-specific siRNA (red; c,d). (B) Cyclin B1 localization in GL2-(a,b) and RANBP1-(c,d) interfered prometaphase (a,c) and metaphase (b,d) cells. RANBP1 depletion can be appreciated in the green channel. (C) Cyclin B1 localization in GL2-(a) and RANBP1-(b) interfered metaphases with morphologically normal spindles, revealed by -tubulin. (D) Cyclin B1 localization in mitotic cells exposed to taxol (b) or to a dynamics-suppressing NOC dose (c). (a) An untreated metaphase is shown. (E) Frequency of mitotic (M) cells showing cyclin B1 misrecruitment to MTs (200-250 counted mitoses for each condition). **Highly significant differences compared with controls (P<0.001). BP1(i), RANBP1-interfered. Bars, 10 µm.

View larger version (45K): [in this window] [in a new window]   Fig. 5. Mislocalization of HURP, but not TPX2 or TOGp, in RANBP1-depleted mitoses. (A) Unaltered localization of TOGp in RANBP1-depleted mitotic cells. (B) Unaltered localization of TPX2 in RANBP1-depleted mitotic cells. (C) Deconvolution images showing the abnormal organization of HURP (red) over MTs (green) in RANBP1-interfered [RANBP1(i)] mitoses (c,d,g,h), compared with the plus-end-restricted distribution seen in control cells (a,b,e,f). Bars, 10 µm.

View larger version (34K): [in this window] [in a new window]   Fig. 6. RANBP1-depleted mitoses harbour hyperstable MTs. (A) Graph represents the frequency of mitotic cells with resistant MTs (MTs shown in green in images on right) after 12 or 20 hours of incubation in 50 ng/ml NOC in RANBP1-interfered [RANBP1(i)] cultures and in controls interfered with non-specific siRNAs. Examples of mitotic cells at similar stages that did assemble MTs (indicated as resistant, a) or displaying depolymerized MTs (b,c) are shown on the right. A total of 300 mitotic cells were examined in two independent experiments. *Differences between RANBP1-depleted and controls cells were statistically significant (P<0.01) using the 2 test. (B) Cold-resistant MTs in GL2- and RANBP1-interfered cells. Representative MT phenotypes are shown (-tubulin, green; CREST, red); the frequency of each phenotype is quantified in the table below. Statistical differences were calculated using the 2 test. ns, non significant. Bars, 10 µm.

View larger version (30K): [in this window] [in a new window]   Fig. 7. RANBP1-interfered cells show chromosome segregation defects. (A) Examples of mitotic abnormalities (arrows) in RANBP1-interfered [RANBP1(i)] U20S cells. Bar, 10 µm. (B) Time-course analysis of mitotic abnormalities. The efficiency of RANBP1-interference during synchronization was monitored by western blot. We began to collect mitotic cells 9 hours after release from G1/S arrest; thereafter, samples were harvested every 20 minutes and were examined for mitotic abnormalities. Graph shows the frequency of abnormalities at the indicated times; at least 300 mitotic cells were counted for each time point in two experiments. thym, thymidine; meta, metaphase; ana, anaphase; telo, telophase. (C) Representative metaphase (a-d) showing CREST-stained KTs (red); the left panels show merged pictures with MTs (-tubulin, green). Anaphase images (e-j) show extending MTs between separating chromosomes in RANBP1-(h-j) but not in GL2-(e-g) interfered cells.