Figure 10.

UBAP2L regulates homeostasis of NPCs at the intact NE. (A–D) Representative immunofluorescence images depicting localization of Nups (mAb414) in HeLa cells treated with indicated siRNAs and synchronized in interphase by DTBR at 12 h. Note that CHX was used at a concentration of 0.1 mg/ml for 2 h 40 min prior to sample collection. Nuclei were stained with DAPI. Scale bar, 5 μm (A). UBAP2L protein levels in A were analyzed by western blot (B). The percentage of cells with the cytoplasmic granules of Nups (mAb414) (C) and the NE intensity of Nups (mAb414) (D) shown in A were quantified. At least 100 cells per condition were analyzed (mean ± SD, ns: not significant, *P < 0.05, **P < 0.01, ****P < 0.0001, unpaired two-tailed t test, n = 3 independent experiments). (E and F) Validation of SNAP-Nup85 HeLa cells by western blot (E) and immunofluorescence microscopy (F). SNAP-Cell TMR-Star was used according to the established protocols. Note that SNAP-Nup85 cells were incubated with SNAP-Cell TMR-Star for 30 min, washed extensively, and the medium was exchanged two times to remove any unreacted SNAP-tag substrate before sample collection. Nuclei and chromosomes were stained with DAPI. Scale bar, 5 μm. (G–K) Scheme of the experimental setup of the SNAP-Nup85 experiment (G). Representative immunofluorescence images depicting localization of SNAP-Nup85 in unsynchronized SNAP-Nup85 HeLa cells treated with indicated siRNAs. Nuclei were stained with DAPI. Scale bar, 5 μm (H). UBAP2L and Nup153 protein levels in H were analyzed by western blot (I). The percentage of cells with the cytoplasmic granules of SNAP-Nup85 (J) and the NE intensity of SNAP-Nup85 (K) shown in H were quantified. At least 100 cells per condition were analyzed (mean ± SD, ns: not significant, *P < 0.05, ***P < 0.001, ****P < 0.0001, unpaired two-tailed t test, n = 3 independent experiments). (L) Hypothetical model of how UBAP2L regulates the homeostasis of NPCs at the intact NE. In the proximity of the NE, UBAP2L (dark purple) interacts with cytoplasmic Y-complex Nups (green) and may facilitate the formation of Y-complex. UBAP2L also interacts with the transporting factor of Nups in the cytoplasm, FXR1 (blue), and restricts its localization to NE during early G1 phase, and promotes its interaction with Nups to fuel assembly NPCs. UBAP2L also regulates the interaction of Y-complex Nups with Nup153 (light purple) and POM121 (yellow), which facilitates the assembly of functional and mature NPCs during interphase. At the same time, UBAP2L may exert its repair function to maintain the stability of existing NPC on NE. This dual regulatory mechanism integrates the cytoplasmic and the nuclear NPC assembly as well as the NE NPC repair signals and ensures efficient nuclear transport, adaptation to nutrient stress, and cellular proliferative capacity, highlighting the importance of NPC homeostasis at the intact NE. Source data are available for this figure: SourceData F10.