Subcellular localization of p57kip2 in the CC of healthy and diseased adult mouse brain. Immunostaining of p57kip2+/PDGFRα+ OPCs in healthy adult wild-type C57BL/6 mouse CC. OPCs either displayed nuclear (A–A′′′′; arrowheads) or cytoplasmic p57kip2 localization (B–B′′′′; arrows). Immunostaining of p57kip2+/PDGFRα+ OPCs in the CC of cuprizone-challenged mice infused with vehicle (C–C′′), BMP4 (D–D′′), or noggin (E–E′′). Arrows indicate PDGFRα+ OPCs with nuclear p57kip2. F, G, Numbers of OPCs displaying a nuclear p57kip2 signal in the CC of mice treated with cuprizone for 4 weeks after infusion of vehicle, BMP4 (400 ng/d) or noggin (400 ng/d) for 5 d. F, Upon BMP4 infusion, an increase in the number of OPCs with nuclear p57kip2 signals was observed, whereas noggin infusion led to a decreased number of OPCs with strong nuclear p57kip2 signals (G). Scale bars: A–B′′′′ 10 μm; C–E′′ 50 μm. Data are means ± SEM (n = 3, 4 in F, n = 5, 4 in G; total number of counted cells: 8750; t test: *p < 0.05).
Distribution of p57kip2 during spontaneous differentiation of cultured OPCs. A, Percentage of oligodendroglial cells displaying nuclear (white bars) or cytoplasmic (dashed bars) p57kip2 localization over the course of 9 d. The number of oligodendroglial cells with p57kip2 expelled from the nucleus steadily increased (B–D′). Double staining for p57kip2/PDGFRα after 1 d (K–K′′′), p57kip2/CNPase after 1 d (L–L′′′′), p57kip2/MBP (M–M′′′), and p57kip2/CC1 (N–N′′′) after 3 d and p57kip2/MOG (O–O′′′) after 6 d in culture. p57kip2 translocation from the nucleus (arrowheads) to the cytoplasm (arrows) could be observed in some precursor cells (K–K′′′) and during differentiation in cells having acquired the expression of maturation markers (L–O′′′). E, Significantly fewer PDGFRα-positive OPCs display cytoplasmic p57kip2 signals as opposed to nuclear signals (white bars: cells with nuclear p57kip2; dashed bars: cells with cytoplasmic p57kip2), whereas the percentage of oligodendroglial cells with CNPase, MBP, MOG, or CC1 expression was significantly increased in cells with translocated p57kip2 protein (F–I). J, Morphological classification of oligodendroglial cells cultured for 6 d and determination of the percentage of cells with nuclear (white bars) and translocated/cytoplasmic p57kip2 signals (dashed bars). P–Q′′′′, Representative citrine (yellow fluorescent protein) expressing oligodendroglial cells with “simple” and “high” morphological complexity, respectively, and corresponding p57kip2 localizations (red). Data are means derived from n = 4 experiments ± SEM (total number of counted cells: 27,937 in A; 12,997 in E; 2450 in F; 2291 in G; 3653 in H; 2828 in I; 425 in J; t test: **p < 0.01, ***p < 0.001). Scale bars, 20 μm.
In vitro myelinating cocultures. A–J, Representative pictures of neuron–oligodendrocyte cocultures fixed and stained at DIV26, displaying active myelination of βIII-tubulin-positive axons (C,I). B, MBP-positive oligodendrocyte (arrow) and MBP-positive myelinated segments (asterisks). Myelin was formed by oligodendrocytes with cytoplasmic p57kip2 signals (arrow in D,F–F′′′), but not by cells with nuclear accumulation of p57kip2 (arrowhead in D,E–E′′′). G–J, Moreover, in this coculture p57kip2 expression could be confined to oligodendroglial cells and CC1-positive oligodendrocytes also exhibited cytoplasmic p57kip2 localization (J, arrow, L–L′′′). Scale bars, 30 μm.
Pharmacological blockade of CRM1-mediated nuclear export. Immunofluorescent staining of cultured oligodendroglial cells for p57kip2 and CNPase (A–D) and p57kip2 and CC1 (F–I) after 1 d in culture, revealing a correlation between p57kip2 translocation from the nucleus (arrowheads) to the cytoplasm (arrows) and the abundance of CNPase and CC1 expression. E, J, Determination of the percentage of CNPase- or CC1-positive cells upon CXCL12 exposure in the absence or presence of ratjadone. This demonstrated a CXCL12-dependent promotion of cellular maturation and of cytoplasmic localization of p57kip2 [compare white areas (nucleus) with dashed areas (cytoplasm) within the bar], which was abolished in the presence of ratjadone. In the presence of ratjadone, most oligodendroglial cells expressing marker proteins exhibited a nuclear accumulation of p57kip2. K, Determination of the percentage of oligodendroglial cells with “medium” and “high” morphological complexity after 3 d in culture in response to CXCL12 stimulation (gray bars), or (co)application of ratjadone (brown bars), revealing that nuclear export blockade efficiently interfered with morphological maturation. Data are means derived from n = 3 experiments ± SEM (total number of counted cells: 15,649 in E; 12656 in J; 625 in K; t test: *p < 0.05, **p < 0.01, ***p < 0.001). Scale bars, 20 μm.
Colocalization of p57kip2 with oligodendroglial binding proteins. A–C, Evaluation of the percentage of oligodendroglial cells displaying different subcellular distribution pattern of p57kip2 in combination with nuclear and/or cytoplasmic accumulation of LIMK-1 (A), CDK2 (B), and Mash1 (C), respectively, after 6 d in culture. The following distribution patterns of p57kip2 and binding partner (bp) were examined: p57kip2nuclear/bpnuclear; p57kip2cytoplasm/bpcytoplasm; p57kip2nuclear/bpcytoplasm; p57kip2cytoplasm/bpnuclear. D–I′′′, Representative images illustrating nuclear (arrowheads) and cytoplasmic p57kip2 localization (arrows) within oligodendroglial cells after 6 d under differentiation conditions. Colocalization with both LIMK-1 (D–D′′′,E–E′′′) and CDK2 (F–F′′′,G–G′′′) was observed, whereas only nuclear coexpression of Mash1 and p57kip2 in immature oligodendroglial cells (H–H′′′, I–I′′′) was found. Scale bars, 20 μm. Data are means derived from n = 6 experiments ± SEM (total number of counted cells: 3353 in A; 3858 in B; 2378 in C; t test: ***p < 0.001).
PLA-confirmed interactions among p57kip2 and CDK2, LIMK-1, Mash1, and Hes5. PLA of cultured oligodendroglial cells after 1 d (Mash1) or 3 d (CDK2, LIMK-1, Hes5) under differentiating culture conditions. Positive PLA events (red dots) are indicated by arrows and could be detected for CDK2/p57kip2 (A–A′′), LIMK-1/p57kip2 (B–B′′), Mash1/p57kip2 (C–C′′), and Hes5/p57kip2 (D–D′′). In negative antibody controls (E–E′′), no PLA events could be detected. Phalloidin was used to visualize cell bodies and processes. Scale bars, 20 μm.
Functional evaluation of Mash1 transactivation properties upon overexpression of p57kip2. A, Determination of the percentage of CNPase-positive oligodendroglial cells upon combinatorial overexpression of an empty vector as control (pIRES), overexpression of Mash1 (pIRES_Mash1), and overexpression of p57kip2 (pIRES_p57) after 3 d in culture. B–D′′, Representative immunofluorescent CNPase staining of cultured cells upon combinatorial gene overexpression. E, Quantitative real-time RT-PCR illustrating the relative expression levels of the Mash1 reporter plasmid tdTomato upon combinatorial transfection of expression vectors pIRES (empty control), pIRES_Mash1, and pIRES_p57kip2. Data are means derived from n = 3 experiments ± SEM (total number of counted cells: 1177 in A; t test: *p < 0.05, **p < 0.01). Scale bars, 30 μm.
p57kip2 overexpression during OPC differentiation and in vitro myelination. A, Cytoplasmic p57kip2 localization in oligodendroglial cells either transfected with the wild-type overexpression construct for p57kip2 (pIRES_p57; B–B′′) or with the NLS mutant p57kip2 construct (pIRES_NLS; C–C′′) after 24 h. D, G, Determination of CNPase- and MBP-positive cells after transfection of control, wild-type, and NLS mutant p57kip2 constructs after 24 h (CNPase) and 72 h (MBP), respectively. Representative cells double stained for p57kip2/CNPase (E–F′) and p57kip2/MBP (H–I′); negative (arrowheads) and positive (arrows) for myelin expression. J, Immunostaining revealed that overexpression of the p57kip2 NLS mutant protein increased the number of oligodendrocytes generating MBP-positive internodes (asterisks) when seeded on neuron–oligodendrocyte cocultures compared with control or wild-type p57kip2 transfected cells. (K–L′′′) Representative oligodendroglial cells without (pIRES_p57) and with myelin formation (pIRES_NLS) capacity. Data are means derived from n = 3 experiments ± SEM (total number of counted cells: 475 in A; 1471 in D; 630 in G; 2024 in J; t test: *p < 0.05, **p < 0.01, ***p < 0.001). Scale bars, 20 μm. OL, Oligodendrocyte.
Colocalization of binding proteins upon overexpression of wild-type or NLS mutant p57kip2 proteins. Examination of cytoplasmic localization of LIMK-1 (A–C′′), CDK2 (D–F′′), and transcription factor Hes5 (M–O′′), and percentage of cells positive for Sox10 (J–L′′) and Mash1 (G–I′′). LIMK-1, CDK2, and Hes5 follow subcellular translocation of p57kip2, whereas Sox10 and Mash1 remained in the nucleus, the latter of which was downregulated and upregulated upon wild-type and NLS mutant p57kip2 overexpression, respectively. Data are means derived from n = 4 experiments ± SEM (total number of counted cells: 677 in A; 569 in D; 541 in G; 546 in J; 415 in M; t test: *p < 0.05, **p < 0.01, ***p < 0.001). Scale bars, 20 μm.
p57kip2 subcellular localization in primary human oligodendroglial cells. A, Quantification of the percentage of GalC- (C) and CC1-positive cells in absence or presence of stimuli such as CXCL12 and/or tri-iodo-thyronine. The fraction of oligodendroglial cells with cytoplasmic p57kip2 localization is represented by the dashed areas. Data are means derived from n = 3 experiments ± SEM (total number of counted cells: 3553 in A; 6612 in C; t test: **p < 0.01). B–B′′′, D–D′′′, Representative cells displaying the association between maturation marker expression and nuclear export of p57kip2. Arrowheads mark immature and arrows mark mature oligodendroglial cells. Scale bars, 20 μm. E–F′′, PLA of cultured oligodendroglial cells after 3 d in culture. Positive PLA events (red dots) are indicated by arrows and could be detected for Hes5/p57kip2 (E–E′′) and Mash1/p57kip2 (F–F′′). Phalloidin was used to visualize cell bodies and processes. Scale bars, 10 μm.