A change in nuclear pore complex composition regulates cell differentiation

Abstract

Nuclear pore complexes (NPCs) are built from ∼30 different proteins called nucleoporins or Nups. Previous studies have shown that several Nups exhibit cell-type-specific expression and that mutations in NPC components result in tissue-specific diseases. Here we show that a specific change in NPC composition is required for both myogenic and neuronal differentiation. The transmembrane nucleoporin Nup210 is absent in proliferating myoblasts and embryonic stem cells (ESCs) but becomes expressed and incorporated into NPCs during cell differentiation. Preventing Nup210 production by RNAi blocks myogenesis and the differentiation of ESCs into neuroprogenitors. We found that the addition of Nup210 to NPCs does not affect nuclear transport but is required for the induction of genes that are essential for cell differentiation. Our results identify a single change in NPC composition as an essential step in cell differentiation and establish a role for Nup210 in gene expression regulation and cell fate determination.

Figure 1. Nup210 expression is induced during myogenic differentiation

(A) mRNA and protein levels of Nup210 during C2C12 differentiation were analyzed by qPCR and western blot respectively (n=3). Nup210 mRNA levels in differentiating cells were normalized to the expression of dividing cells (Day 0). Myosin Heavy Chain (MHC) was used as a differentiation marker and Histone H3 (His H3) as loading control. Data are presented as average values ± SD. (B) Nup210 mRNA and protein levels in dividing myoblasts (M), differentiated C2C12 cells (D), myotubes (T) and quiescent myoblasts (Q) were analyzed by semi-quantitative PCR and western blot respectively. (C) Immunofluorescence against Nup210 and Pom121 in dividing and differentiated (Day 4) C2C12 cells. MHC was used as a maker for differentiated myotubes and nuclei were stained with Hoechst. (D) Higher magnification of immunofluorescence against Nup210 and Pom121. Arrows indicate quiescent myoblasts. See also Figure S1.

Figure 2. Nup210 is required for myoblast differentiation

(A) C2C12 cells were infected with lentivirus carrying control or Nup210-specific shRNAs. Infected cells were selected and induced to differentiate. Differentiated myotubes were stained against MHC at 48, 72, and 96 hours after differentiation. Nuclei were stained with Hoechst. (B) The percentage of nuclei in multinucleated cells (>2 nuclei) to the total number of nuclei in the field (Fusion Index) in panel (A) was quantified. Values represent the average of 4 different independent experiments ± SD. (C) Quantification of MHC positive cells shown in panel (A). Percentage of cells containing 1 (mononuclated), 2 (binucleated), or more nuclei (multinucleated) were determined at different times of differentiation. Data are presented as average values of 3 independent experiments. (D) C2C12 cell lines carrying control or Nup210 shRNAs were transfected with GFP or an RNAi resistant Nup210-GFP. Cells were reselected, induced to differentiate and stained against MHC on Day 4. Inlets show a higher magnification of GFP and Nup210-GFP expressing cells. (E) Fusion Index for the experiment in panel (E) was quantified as described. See also Figure S2.

Figure 3. NPC-associated Nup210 does not affect INMP targeting or global nucleo-cytoplasmic transport

(A) Post-mitotic myotubes were fractionated by sequential centrifugation and Nup210 protein leveles were analyzed in the different subcellular fractions by western blot (C, Cytoplasm; N, nucleus; E/G, Endoplasmic Reticulum/Golgi; PM, Plasma Membrane). (B) Nup210 localization in post-mitotic myotubes was analyzed by immunofluorescence. MHC was used as a marker of differentiation and mAb414 as a marker for NPCs. (C) Co-localization of Nup210 and mAb414 signals at the nuclear envelope was determined using Image J. (D) C2C12 myoblasts were transfected with Emerin-V5, V5-Lap2B, Lem2-V5 and V5-Man1 and induced to differentiate. Cells were infected with lentivirus carrying control or Nup210 shRNAs at 36 hours after differentiation and stained for Nup210 and V5 at 96h post-infection. Inlets show higher magnification of myotube nuclei. (E) C2C12 myoblasts were transfected with NES-Tomato-NLS and Lamin A-EGFP and induced to differentiate. Cells were infected with lentivirus carrying control or Nup210 shRNAs at 36 hours after differentiation and nuclear transport was analyzed by FRAP of NES-Tomato-NLS 96h post-infection. Values represent the average of 3 different independent experiments ± SD. See also Figures S3 and S4.

Figure 4. Nup210 regulates gene expression in post-mitotic myotubes

(A) C2C12 cells were infected with lentivirus carrying control or Nup210 shRNAs at 36 hours after differentiation and whole genome expression was analyzed by microarray. Heat map shows the microarray expression profile of altered expression in Nup210 knock-down myotubes. See also Table 1 and S1. (B) The expression of Nup210, GDF5, Clic5, Asb2, Neu2, Cand2, Igfbp4, Stra13 and NDRG2 during myoblast differentiation was analyzed by qPCR. mRNA levels in differentiating cells were normalized to the levels of dividing cells. Values represent average ± SD of 3 different independent experiments. (C) C2C12 myoblasts were infected with lentivirus carrying control, Clic5, GDF5, Neu2 or Nup210 shRNAs and induced to differentiate. Immunofluorescence against MHC was performed at 48, 72, and 96h post differentiation. See also Figure S5.

Figure 5. Ectopic expression of Nup210 in myoblasts increases the expression levels of differentiation genes and accelerates myotube formation

(A) The expression levels of Asb2, Clic5, GDF5, Neu2 and Ndrg2 in GFP and Nup210-GFP expressing myoblasts were analyzed by qPCR. mRNA levels for each gene was normalized to the levels of GFP expressing cells. Values represent average ± SD of 3 different independent experiments. (B) GFP and Nup210-GFP C2C12 expressing myoblasts were induced to differentiate. Immunofluorescence against MHC was performed at 48, 72, and 96 hours post differentiation. (C) Fusion Index (percentage of nuclei in multinucleated cells (>2 nuclei) to the total number of nuclei in the field) for experiment in panel B. Values represent the average of 4 different independent experiments ± SD. (D) Percentage of cells containing 1–4, 5–10, 11–20 or >20 nuclei in panel B were determined at different times of differentiation. Data are presented as average values of 3 independent experiments. (E) C2C12 myoblasts ectopically expressing Nup210-GFP were infected with lentivirus carrying control, Clic5, GDF5 or Neu2 shRNAs and induced to differentiate. Immunofluorescence against MHC was performed at 24, 48 and 72h post differentiation. GFP overlay shows the expression of Nup210-GFP. See also Figure S6.

Figure 6. Nup210 is essential for neuronal differentiation

(A) Embryonic stem (ES) cells were induced to differentiate into neuroprogenitor (NP) cells and stained for Nup210. Oct4 and Nestin were used as specific markers for ES and NP cells respectively. Hoechst was used to stain nuclei. (B) ES cells were infected with lentivirus carrying control or Nup210 shRNAs and induced to differentiate into NP cells. Differentiated cells were stained against Nup210 and Nestin. Hoechst was used to stain nuclei. (C) Differentiated NP cells were infected with lentivirus carrying control or Nup210 shRNA and stained against Nup210 and Nestin. (D) Control or Nup210 knockdown NP cells were stained with Nestin and Cleaved Caspase-3 (Caspase 3) antibodies. (E) Schematic model of Nup210 regulation of cell differentiation. In undifferentiated myoblast the expression of Nup210 is repressed. Early differentiation signals activate Nup210 gene expression. In myoblast, Nup210 induction is likely carried out by Myogenin/MyoD binding to its promoter E-boxes. Nup210 protein is then recruited to the NPC where it regulates the expression of genes required for myogenic and neuronal differentiation. Prevention of Nup210 addition to the NPC by shRNAs prevents the activation of Nup210 regulated genes and leads to the death of the differentiation-committed cell by apoptosis.