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. 2023 Dec 1;12(23):2757. doi: 10.3390/cells12232757

Figure 1.

Figure 1

Translocated promoter region (Tpr) phosphorylation determines subcellular localization in neural stem/precursor cells (NSPCs). (A) Scheme illustrating the localization of Tpr and phenylalanine-glycine repeat nucleoporins (FG-Nups) in nuclear pore complexes (NPCs). (B) Comparative imaging of NPCs in subventricular zone- (SVZ-) derived NSPCs immunolabeled with Tpr (red) and FG-Nups (green) using confocal laser scanning microscopy (CLSM) (left) and SR Airyscan microscopy (right). Enlargements at the right of each panel indicate representative resolutions of the Tpr and FG-Nup signals, and white arrowheads indicate the distinct localizations of TPR and FG-Nups resolved with SR Airyscan microscopy. Nuclei are stained with DAPI (blue). Scale bars: 2 µm, 500 nm (enlargement). Representative images from three independent experiments (10 nuclei were analyzed in total). (C) Scheme illustrating Tpr localization relative to FG-Nups in an NPC after SR Airyscan microscopy and Imaris 3-D processing. (D) Representative SR Airyscan microscopy and Imaris-processed image of an SVZ-derived NSPC nucleus immunolabeled for Tpr (red) and FG-Nups (green). Representative image from three independent experiments (10 nuclei were analyzed in total). Scale bar: 2 µm. (E) 3-D visualization revealing distinct localizations for Tpr (red) and FG-Nups (green) in the NPC of SVZ-derived NSPCs. Representative image from three independent experiments (10 nuclei were analyzed in total). Scale bar: 1 µm. (F) 3-D visualization of confocal images revealing nuclear phospho-Tpr (P-Tpr, red) localization in NSPCs in vitro. Representative images from three independent experiments (10 nuclei were analyzed in total). Scale bars: 2 µm, 500 nm (enlargement). (G) Immunolabeling for P-Tpr (red) in combination with doublecortin (DCX) (green, marker for neuroblasts) in the subgranular zone (SGZ) of the hippocampus in adult wild-type (WT) mice. Enlargements indicate a DCX+ cell with nuclear P-Tpr and a DCX−cell with no nuclear P-Tpr. Quantification of nuclear or nuclear envelope P-Tpr localizations in Ki-67+, DCX+, and NeuN+ cells in the SGZ of the hippocampus in adult WT mice (n = 4 mice, 79–98 single cells were analyzed per Ki-67, DCX, or NeuN condition). Scale bars: 3 µm, 5 µm (enlargement). Values are the mean ± SEM (p-values calculated by one-way ANOVA with Bonferroni’s multiple comparisons test, * p < 0.05, ** p < 0.01) (H) Electron microscopy determining the localization of Tpr and P-Tpr in comparison to FG-Nups in NSPCs of the hippocampal SGZ in adult WT mice. Quantification of nuclear or nuclear envelope Tpr- and P-Tpr-IR in NSPCs of the hippocampal SGZ in adult WT mice (n = 3 mice, 27–30 single cells were analyzed per Tpr or P-Tpr localization). Scale bar: 100 nm. Values are the mean ± SEM (p-values calculated by unpaired Student’s t-test, * p < 0.05, ** p < 0.01).