The nucleoporin gp210/Nup210 controls muscle differentiation by regulating nuclear envelope/ER homeostasis.
Bottom Line: Here, we show that gp210/Nup210 mediates muscle cell differentiation in vitro via its conserved N-terminal domain that extends into the perinuclear space.Unexpectedly, a gp210/Nup210 mutant lacking the NPC-targeting transmembrane and C-terminal domains is sufficient for C2C12 myoblast differentiation.Our results suggest that the role of gp210/Nup210 in cell differentiation is mediated by its large luminal domain, which can act independently of NPC association and appears to play a pivotal role in the maintenance of nuclear envelope/ER homeostasis.
Affiliation: Molecular and Cell Biology Laboratory, Salk Institute for Biological Studies, La Jolla, CA 92037 Division of Biological Sciences, University of California, San Diego, La Jolla, CA 92093.Show MeSH
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Mentions: We have previously reported Nup210 to be exclusively located at NPCs in differentiated C2C12 cells (D’Angelo and Gomez-Cavazos et al., 2012). However, it remained unclear whether NPC association is important for Nup210’s function in cell differentiation. Targeting of Nup210 to the nuclear pore membrane has been shown to be dependent on its single transmembrane segment and its C-terminal tail (Wozniak and Blobel, 1992). To determine the localization of Nup210 fragments to the NE and NPCs in C2C12 cells, stable cell lines expressing Nup210-GFP, Nup210ΔLUMEN-GFP, and Nup210ΔCT-GFP were fixed and stained with mAb414 to visualize nuclear pores. Stable cell lines expressing Sec61-β–GFP and NDC1-GFP were used as experimental controls for ER and NE/NPC localization, respectively (Fig. 3, A–D). Using confocal microscopy and structured illumination microscopy, we were able to confirm that Nup210ΔLUMEN-GFP, which contains both previously characterized NE sorting signals, properly localized at the NE and NPCs similar to full-length Nup210 (60.2 ± 3.7% vs. 75.6 ± 6.7%; Fig. 3, A–D). In contrast, Nup210-ΔCT-GFP lost its ability to concentrate at the NE and NPCs (36.3 ± 8.3% vs. 75.6 ± 6.7%), being predominantly distributed throughout the ER (Fig. 3, A–D). These results confirm that the C-terminal domain, which faces the nuclear pore, plays a critical role in targeting Nup210 to the NPC.
Affiliation: Molecular and Cell Biology Laboratory, Salk Institute for Biological Studies, La Jolla, CA 92037 Division of Biological Sciences, University of California, San Diego, La Jolla, CA 92093.