Fig 1: XPO1-mediated nuclear export of RanBP1 cargo protein. (A) Endogenous RanBP1 cargo protein (green) localized in the cytoplasm of untreated wild-type and mutant XPO1C528S Jurkat cells; (B) 3 h after treatment with 1 µM KPT-330 (Selinexor), RanBP1 accumulates in the nucleus of wild-type cells (left) but remains localized in the cytoplasm of mutant XPO1C528S cells (right); (C,D) 3 h after treatment with 4 µM of prototype compounds 9 or 10, respectively, nuclear accumulation of RanBP1 is found in wild-type Jurkat cells. In contrast, no nuclear retention of the cargo was induced in the mutant XPO1C528S cells. Scale bar 25 µm, and nuclei were counterstained with DAPI (blue).
Fig 2: RanBP1 Regulates Neuronal Migration In Vivo(A) E13.5 mice were electroporated with shCTL, shranBP1 (shranBP1_1), or shranBP1 + hranBP1 and analyzed at E16.5. Electroporated cells are GFP+ (green).(B) Quantification of the distribution of electroporated cells in indicated cortical areas represented as the percentage of GFP+ cells. Embryos were obtained from three independent electroporation experiments (shCTL [n = 14], shRanBP1 [n = 17], shranBP1+hranBP1 [n = 10]). Neuronal migration was analyzed using two-way ANOVA with Tukey’s post-hoc test. p values are as follows: for VZ: shCTL versus sh1 0.0003, shCTL versus sh1+Rescue 0.9757, sh1 versus sh1+Rescue 0.1526; for SVZ: shCTL versus sh1 > 0.9999, shCTL versus sh1+Rescue > 0.9999, sh1 versus sh1+Rescue 0.9999; for IZ: shCTL versus sh1 > 0.9999, shCTL versus sh1+Rescue 0.6250, sh1 versus sh1+Rescue 0.8169; for CP: shCTL versus sh1 0.0040, shCTL versus sh1+Rescue > 0.9999, sh1 versus sh1+Rescue 0.0063. CP, cortical plate; IZ, intermediate zone; SVZ, subventricular zone; VZ, ventricular zone.(C) E13.5 embryos were subjected to in utero electroporation and analyzed at E16.5. Representative images of coronal mouse embryonic sections stained for GFP and Ki67 antibodies. Scale bar, 100 μm.(D) Quantification of cells expressing Ki67. Data are from 3 independent experiments and 10 embryos per condition.(E) Representative images of coronal mouse embryonic sections stained for GFP, Edu, and Ki67 antibodies.(F) The proliferation index was measured as GFP+Ki67+/GFP+ cells. No significant difference was detected when comparing shCTL (10.96 ± 1.24) to shranBP1 (9.72 ± 1.45). The rate of cell-cycle exit was measured as the ratio of Edu+Ki67−/Edu+ cells. No significant difference was detected when comparing shCTL (67.13 ± 7.91) to shranBP1 (77.14 ± 5.18).
Fig 3: RanBP1 Is Required for LKB1-Dependent Axonogenesis(A) LKB1 localization in a representative 72 hr rat cortical neuron overexpressing RanBP1::Myc, compared to non-transfected neurons (open triangles). Scale bar, 10 μm.(B) Quantification of the LKB1 signal intensity in the nucleus and cytosol of neurons overexpressing RanBP1::Myc (n = 23 neurons), compared to neighboring non-transfected neurons (n = 29 neurons). Wild-type nuclear: 30.55 ± 1.69; RanBP1::Myc nuclear: 32.32 ± 1.94. Error bars represent SEMs (p = 0.50, two-tailed unpaired t test. Wild-type cytosol: 34.37 ± 1.72; RanBP1::Myc cytosol: 58.03 ± 1.70. Error bars represent SEMs (p < 0.0001, two-tailed unpaired t test).(C) LKB1 localization (white arrow) in representative RanBP1 deficient 72 hr neurons compared to non-GFP neurons. Scale bar, 10 μm.(D) Quantification of LKB1 signal intensity in the nucleus and cytosol of neurons 72 hr after siRNA-mediated knock down of ranBP1 (n = 20) compared to non-GFP neurons (n = 19). Wild-type nuclear: 37.25 ± 1.81; ranBP1-siRNA nuclear: 57.98 ± 1.61. Error bars represent SEMs (p = 0.0001, two-tailed unpaired t test). Wild-type cytosol: 34.13 ± 1.35; ranBP1-siRNA cytosol: 32.97 ± 1.71. Error bars represent SEMs (p = 0.60, two-tailed unpaired t test).(E) Co-expression of LKB1 and STRADα together with an eGFP reporter plasmid in rat cortical neurons. White arrows indicate axons.(F) ranBP1-siRNA neurons overexpressing LKB1 and STRADα.(G) The mean percentage of 72 hr neurons with 0, 1, or ≥2 axons over three independent replicates was determined for each condition: overexpression of LKB1+STRADα (n = 32 neurons) and overexpression of LKB1+STRADα with knock down of ranBP1 (n = 34 neurons). After converting percentages to arcsin values, two-tailed unpaired t tests were performed. Comparing LKB1+STRADα against LKB1+STRADα for the proportion of neurons with 1 axon (p = 0.00187, ∗∗) and 2 axons (p = 0.00790, ††).
Fig 4: Subcellular Localization of Ran, RanGAP, and RanBP1 in Cortical Neurons(A) Successive stages of rat cortical neuron polarization in vitro. Neurons transfected with the control eGFP-reporter plasmid were imaged at different stages of development, and z stacks of individual cells were skeletonized using ImageJ (NIH).(B–D) Rat cortical neurons stained for Tuji-1, Ran, and Tau-1 at Stage 2 (B), Stage 3 (C), and Stage 4 (D). The right column shows a heat histogram of Ran staining intensity. Scale bar, 10 μm.(E–G) Neurons stained for eGFP, RanGAP, Tau-1, and DAPI at Stage 2 (E), Stage 3 (F), and Stage 4 (G). Scale bar, 10 μm.(H–J) Neurons stained for eGFP, Tuji-1, Tau-1, and RanBP1 at Stage 2 (H), Stage 3 (I), and Stage 4 (J) using a previously validated RanBP1 antibody (Yudin et al., 2008). A white arrow points to RanBP1 staining in the axon and the arrowhead points to RanBP1 staining in a dendritic process.
Fig 5: (A) Overall structure of the XPO1 protein (in deep purple), Ran (olive) and RanBP1 (sky blue). The NES-binding cleft is shown in grey. (B) Surface representation of the NES-binding cleft (grey) and the Φ0–Φ4 pockets. ScCys539 is shown in yellow and labelled. (C) Detailed view of KPT-8602 (yellow spheres, pdb id: 5jlj) inside of the NES-binding cleft.
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