RanBP10 overexpression in MKs increases MT numbers and thickness and displaces Ran. (A-D) MKs that overexpress RanBP10 show many partially coiled internal MT fibers that are substantially thicker and longer than those in control MKs. MKs infected with GFP-RanBP10 retrovirus were recognized by virtue of green fluorescence (A) and their MT structure examined by β-tubulin IF (B-D; single z-sections are displayed after deconvolution). Panels A and B show the same cell, and nuclei are seen by DAPI staining (B,D). Arrows in panel B point to a bona fide proplatelet, whereas the rest of the cell is filled with thicker MT bundles. (E) Indirect Ran IF (red), coupled with DAPI nuclear stain (blue), contrasts the nuclear confinement of Ran in control EGFP virus-infected MKs (top) with extra-nuclear Ran accumulation in cells that overexpress RanBP10 (bottom). (F,G) The merged fluorescence images were rotated 90° along the axis shown in panel E to visualize x/z planes after 3-dimensional reconstruction. Ran and nuclear material overlap in distribution in control cells (F), whereas in RanBP10-overexpressing MKs, a substantial fraction of Ran is present outside the nucleus. Cross-sectional intensity profiles are displayed immediately below, with pixel position on the abscissa and fluorescence intensity on the ordinate (scale for DAPI signal on the left and for Texas Red on the right); blue and red arrows mark boundaries of the respective fluorescent signals. Scale bars: B-D, 15 μm; E, 10 μm.