A, Immunoblot shows the expression of RanBPM in the E14 rat brain lysate. α-tubulin is used on the same blot as an internal control. B, Evaluation of RanBPM shRNA constructs by immunoblotting. RanBPM-shRNA1 (targeting 3’UTR region of RanBPM) is more effective than RanBPM shRNA2 (targeting coding region of RanBPM) in knocking down RanBPM expression in B104 cells. A four-base mutation construct of RanBPM-shRNA1 (RanBPM-shRNA1M4) is used as the control shRNA. α-tubulin is shown as control. C, A single confocal z section shows that RanBPM shRNA1 transfected dividing cells (left panels) decrease RanBPM on the membrane compared to the non-transfected cells (right panels). Arrowhead indicates high RanBPM expression on the cell membrane. Scale bar, 50 µm. D, RanBPM is polarized at the ventricular zone surface in E15 rat neocortex. Scale bar, 100 µm. E, Endogenous RanBPM and ZO-1 expression. E15 brain sections were immunostained with anti-RanBPM antibody and anti-ZO-1 antibody. RanBPM expression is shown as green and ZO-1 as red. F, Endogenous RanBPM (green) and β-catenin (red) expression in E15 rat brain. Scale bars, 10 µm. Insets show the dotted boxes with higher magnification. The orthogonal projected panels of single-z images confirm the co-localization of RanBPM and junctional proteins, ZO-1 and β-catenin.

A, Endogenous localization of Citron kinase (red) and RanBPM (green) is shown. E15 brain sections were immunostained with anti-Citron antibody and anti-RanBPM antibody. Arrows represent adjacent expression of both proteins in dividing cells at the VZ surface. Scale bar, 10 µm. Higher magnifications of labeled cells are shown in B–D (B:early telophase (left pair) and early anaphase (right pair), C:cytokinesis, D:metaphase). E–F, Transmission electron micrographs show that midbodies (MB) have prominent bundles of microtubules (MT). Note that the midbodies are nearly adjacent to adherens junctions (AJ). G–I, Citron kinase (red) localizes adjacent to RanBPM (G, green) similar to the pattern of localization between Citron kinase (green) and other junctional markers (red), ZO-1 (H) and β-Catenin (I). Scale bars, 10 µm. J, RanBPM-flag co-immunoprecipitates with CITK-myc in transfected Cos7 cells. Lysates of Cos7 cells transfected with CITK-myc were immunoprecipitated with either an anti-myc antibody (top panel) or a rabbit anti-IgG antibody (middle panel) followed by detection with an anti-RanBPM antibody. Endogenous RanBPM (90 kDa, arrowhead) in Cos7 cells co-immunoprecipitates with CITK-myc (lane 1); Lysates of Cos7 cells transfected with only RanBPM-flag were subjected to IP with an anti-myc antibody and detected with an anti-RanBPM antibody. Anti-myc antibodies did not immunoprecipitate RanBPM-flag (lane 2); Lysates of Cos7 cells co-transfected with CITK-myc and RanBPM-flag were immunoprecipitated with an anti-myc antibody followed by detection with an anti-RanBPM antibody. RanBPM-flag co-immunoprecipitates with CITK-myc, but not with rabbit IgG (lane 3). K, CITK-myc co-immunoprecipitates with RanBPM-T7 in transfected Cos7 cells. Lysates of Cos7 cells transfected with CITK-myc were immunoprecipitated with either an anti-T7 antibody (top panel) or a mouse anti-IgG antibody (middle panel) followed by detection with an anti-myc antibody. Anti-T7 antibodies did not immunoprecipitate CITK-myc (lane 1); Lysates of Cos7 cells transfected with RanBPM-T7 were subjected to IP with an anti-T7 antibody followed by detection with an anti-myc antibody. Anti-T7 antibodies did not co-immunoprecipitate CITK-myc (lane 2); Lysates of Cos7 cells co-transfected with CITK-myc and RanBPM-T7 were immunoprecipitated with an anti-T7 antibody followed by detection with an anti-myc antibody. CITK-myc (220 kDa, arrow) co-immunoprecipitates with RanBPM-T7, but not with a mouse IgG (lane 3). L, Interaction between endogenous RanBPM and CITK in rat developing brain. In E12 brain, anti-citron antibody (CT295) immunoprecipitated CITK and RanBPM co-immunoprecipitated with it (top panel). RanBPM does not co-immuoprecipitate with a rabbit IgG (second panel). The third panel shows that CT295 immunoprecipitates CITK in E12 brain lysate. Arrowhead:RanBPM, arrow:CITK. M, Protein overlay assay shows direct interaction between the N-terminal region of CITK (NTCITK, a.a.1–448) and RanBPM. NTCITK-His (65 kDa), MCPH-His (57 kDa), and BSA (67 kDa) were subjected to immunoblotting and overlayed with RanBPM-GST (left) and GST alone (right). The bound protein was probed with anti-GST antibodies and detected by chemiluminescence. RanBPM-GST bound to NTCITK-His, but to neither MCPH-His nor BSA. GST alone did not bind to NTCITK-His.

A, CITK localization in cells at different stages of cell cycles in E15 rat brain. a, In metaphase cells, CITK aggregates apically towards the VZ surface. b, Then CITK dynamically moves to both the basal and apical sides of the cleavage furrow during telophase. c, In cytokinesis, CITK localizes to the midbody adjacent to the VZ surface. Scale bar, 5 µm. Diagrams depicting CITK localization (green) at the VZ surface during cell cycle. a, metaphase; b, telophase; c, cytokinesis. B, RanBPM-shRNA1 (shRNA1) results in mislocalization of CITK at the VZ surface 2 days and 3 days after transfection. Control (shRNA1m4) and shRNA1 were co-transfected with GFP in E13 rat embryonic brains by in utero electroporation. Phospho-vimentin (phVim, not shown) was used for labeling mitotic cells. RanBPM-shRNA1 disrupts the polarization of CITK (red, arrowheads) in metaphase cells (phVim positive) by 2 days after in utero electroporation and shows diffuse distribution of CITK 3 days after in utero electroporation, while control transfected cells maintained the polarization of CITK (arrows). Scale bars, 10 µm. The transfected metaphase cells are outlined, and ‘a’ stands for apical side and ‘b’ stands for basal side of mitotic cells. The dotted lines represent the apico-basal axis and perpendicular half lines. The graph in D shows the ratio of CITK expression in the apical half of metaphase cells relative to CITK expression in the basal half of metaphase cells as depicted in the schematic shown in C. The significance was tested by two-sample Student’s t-tests in two independent groups and by ANOVA in all three groups followed by post hoc Tukey-Kramer HSD and Wilcoxon/Kruskal-Wallis tests (**, p<0.01). Error bars represent ± s.e.m.

left panels, Endogenous RanBPM co-localizes with β-catenin at the VZ surface of wildtype (A) and CITK^fh/fh mutant littermates (B). E17 brain sections from both were immunostained with anti-RanBPM antibody (green) and anti-β-catenin antibody (red). Co-localization of RanBPM and β-catenin is shown at exposed junctional complexes. Orthogonal projections confirming their co-localization show a very similar intensity pattern for the two proteins. Scale bars, 50 µm. right panels, Co-localization analysis with JACoP. Intensity correlation analysis (ICA) was performed for co-localization of RanBPM and β-catenin. A (CITK^wt/wt) and B (CITK^fh/fh) show intensity correlation plots of the images with the respective plots of the pixel intensities of RanBPM staining (y axis) against their (Ai-a)(Bi-b) values (Ai: individual green (RanBPM) pixel intensity, a: mean of green (RanBPM) pixel intensity, Bi: corresponding red (β-catenin) pixel intensity, and b: mean of red (β-catenin) pixel intensity). Both plots show different intensity co-localization, indicating that absence of CITK does not produce a change in RanBPM localization at the apical VZ junction. ICQ, Intensity correlation quotient value; PC, Pearson’s coefficient.

RanBPM-shRNA1 (shRNA1, A) and control shRNA1m4 (B) were co-transfected with GFP in E13 rat embryonic brains by in utero electroporation and analyzed with immunostaining 3 days after transfection. The boxed regions in the upper panels are shown at higher magnifications in the bottom panels (A and B). Note that larger numbers of both phosH3-positive and GFP-positive cells appear in RanBPM-shRNA1-transfected VZ than in control shRNA1m4-transfected VZ. Scale bars, 50 µm. C, The analysis of mitotic marker, phosH3, in RanBPM-shRNA1 and control shRNA1m4 treated VZ. Both phosH3-positive and GFP-positive cells at the VZ were counted and normalized to total GFP-positive cells at the VZ, and then normalized to control transfected condition (n=3 brains, 6 sections from each brain). The control value is shown as the black dotted line. Note that higher numbers of mitotic cells (phosH3+ GFP+/total GFP+ cells) are observed with shRNA1 and shRNA3 transfection than with shRNA4 transfection compared to control shRNA1m4 transfection. In contrast, by co-expression of RanBPM-myc with shRNA1 the number of mitotic cells was similar to that for cells transfected with GFP alone. **, p<0.01, determined by Student’s t-test. Error bars represent ± s.e.m.

A, Both the diagram (left) and confocal image (middle) show the markers, CITK and AuroraB, used to assess the RanBPM RNAi effects on cytokinesis. CITK (green) and AuroraB (red) label midbody structures in E15 brain. Citron kinase localizes to the midbody ring and AuroraB marks the flanking regions in U-shaped midbodies at the VZ surface. Scale bar, 5 µm. The diagram in right represents the regions that were analyzed in this experiment. B and C, RanBPM RNAi decreases the midbody formations at the VZ surface. RanBPM-shRNA1-transfected brains (bottom 2 panels) labeled with CITK (red, left panels) or AuroraB (red, right panels) display fewer midbodies than do control shRNA1m4-transfected brains (upper 2 panels). Scale bar, 50 µm. CITK-positive and AuroraB-positive midbodies were counted within 200 µm of the VZ surface. C, n=4 brains, 6 sections from each. *, p<0.01 and **, p<0.05, determined by Student’s t-test. Error bars represent ± s.e.m. D, The increase in mitotic cells by RanBPM-shRNA1 is correlated to the decreased numbers of cytokinesis showing the delay of M-phase progression.

A, RanBPM RNAi does not cause accumulation of mitotic cells in CITK^fh/fh, as it does in CITK^wt/wt. RanBPM-shRNA1 or control shRNA1m4 constructs were co-transfected with GFP into E13 embryonic CITK mutant brains by in utero electroporation. Double-immunopositive cells (GFP and phosH3) were quantified. Note that RanBPM-shRNA1 does not increase the numbers of mitotic cells, indicating that RanBPM RNAi function on mitosis through CITK. In addition, RanBPM may have a separate pathway by which it affects the VZ cell cycle. Arrowheads indicate both GFP- and phosH3-positive cells. Scale bar, 50 µm. Quantification is shown in B (n=3 brains, 4 sections from each brain). Two-way ANOVA was used to validate the significance between genotypes (p<0.01), RNAi and control (p<0.01), and their interaction (p<0.01).