A role for Nup153 in nuclear assembly reveals differential requirements for targeting of nuclear envelope constituents

Abstract

Assembly of the nucleus following mitosis requires rapid and coordinate recruitment of diverse constituents to the inner nuclear membrane. We have identified an unexpected role for the nucleoporin Nup153 in promoting the continued addition of a subset of nuclear envelope (NE) proteins during initial expansion of nascent nuclei. Specifically, disrupting the function of Nup153 interferes with ongoing addition of B-type lamins, lamin B receptor, and SUN1 early in telophase, after the NE has initially enclosed chromatin. In contrast, effects on lamin A and SUN2 were minimal, pointing to differential requirements for the ongoing targeting of NE proteins. Further, distinct mistargeting phenotypes arose among the proteins that require Nup153 for NE targeting. Thus, disrupting the function of Nup153 in nuclear formation reveals several previously undescribed features important for establishing nuclear architecture: 1) a role for a nuclear basket constituent in ongoing recruitment of nuclear envelope components, 2) two functionally separable phases of NE formation in mammalian cells, and 3) distinct requirements of individual NE residents for continued targeting during the expansion phase of NE reformation.

FIGURE 1:

Lamins B1/B2 accumulate at ectopic sites in telophase when Nup153 is depleted. (A) Confocal microscopy of HeLa cells expressing LEM2-mCherry, costained to detect Nup153, tubulin, and DNA. Images represent distinct stages of nuclear assembly, from initial membrane recruitment in early anaphase, establishment of core/noncore regions in late anaphase, and dissolution of core/noncore regions along with nuclear envelope expansion (early telophase, late telophase). Early telophase cells are identified by a compact midbody structure connecting cells that have not fully flattened, while late-telophase cells remain connected by a thin midbody structure and are considerably flatter. Scale bar, 10 µm. (B) Widefield microscopy showing representative images of lamins A/C, B1, and B2 in late-telophase stage cells, after treatment with siControl (siCon) or siNup153 (si153) oligos for 48 h. Yellow arrows indicate sites of lamin ectopic targeting. Scale bar, 20 µm. (C) Quantification of the percent of cells with mislocalized endogenous lamin B2 at metaphase/anaphase (Meta/Ana), early telophase (Early Telo), and late telophase (Late Telo) after 48-h treatment with siCon or si153 oligos. The phenotype observed in early telophase cells was notably weaker. Data from three experiments plotted as mean ± SD; siCon: meta/ana 0 ± 0%, early telo 0 ± 0%, late telo 0 ± 0%; si153: meta/ana 0 ± 0%, early telo 38 ± 15%, late telo 80 ± 13%. *, P < 0.05; ***, P < 0.001. Ø indicates raw values were equal to 0. (D) Montage of cells expressing GFP-lamin B2 and H2B-mCherry progressing through telophase captured by live spinning disk microscopy after treatment with siCon or si153 for 48 h, with t = 0′ being the time of complete cleavage furrow ingression observed by time-lapse video. Images were adjusted independently to optimize visibility of the markers. Scale bar, 10 µm.

FIGURE 2:

Lamin B2 mistargeting is not the result of disrupting the pore basket. (A) Immunoblot of Nup153, Nup50, Tpr, Nup155, and tubulin protein levels after depletion with indicated siRNAs for 48 h. Nup155 and tubulin were probed as nucleoporin and loading controls, respectively. (B) Quantification of the percent of late-telophase cells with mistargeted lamin B2 after treatment with the indicated siRNAs. Data from three experiments plotted as mean ± SD; siCon: 0 ± 0%; si153: 90 ± 10%; si50: 0 ± 0%; siTpr: 0 ± 0%. ***, P < 0.001. Ø indicates raw values were equal to 0. (C) Widefield microscopy of lamin B2 for each depletion condition; brackets indicate late-telophase cells determined by tubulin costain (not shown). Scale bar, 20 µm.

FIGURE 3:

SUN1, but not SUN2, enriches at ectopic sites in late-telophase cells when Nup153 is depleted. (A) Widefield microscopy of SUN1 and tubulin and SUN2 and tubulin (B) in late-telophase cells following treatment with siCon or si153 under thymidine-synchronization conditions. Scale bar, 20 µm. (C) Quantification of the percent late-telophase cells with ectopic SUN1 targeting. Data from three experiments plotted as mean ± SD; siCon: 1 ± 1%; si153: 73 ± 13%; ***, P < 0.001. (D) Relative levels of SUN1 and SUN2 at ectopic foci compared with nuclear levels under si153 conditions. Data collected from three independent biological replicates are depicted in different colors, N = 25, 26, 20; the mean of each experiment was used to calculate the mean ± SD, indicated in black; SUN1: 2.0 ± 0.3%; SUN2: 0.5 ± 0.1%. (E) Graphed arbitrary fluorescence of SUN1 and SUN2 along plot line profile indicated on the spinning disk confocal micrographs shown in F. Scale bar in F, 10 µm.

FIGURE 4:

Mistargeted nuclear envelope proteins coenrich at ectopic sites that are membranous but distinct from micronuclei. (A) Representative widefield images of lamin B1 and B2 in Nup153-depleted cells after thymidine synchronization. Enlargements are 2×. Scale bar, 20 µm. (B) Spinning disk confocal micrographs showing lamin B2 and SUN1 after Nup153 depletion. Scale bar, 20 µm. (C) Arbitrary fluorescence units corresponding to the plot line shown in B are graphed. (D) Spinning disk confocal micrographs showing Lap2β and SUN1 after Nup153 depletion. Scale bar, 20 µm. (E) Arbitrary fluorescence units corresponding to the plot line shown in D are graphed. (F) Costain of SUN1 and Lap2α in Nup153-depleted cells by widefield microscopy, distinguish micronucleus from sites of SUN1 accumulation. Scale bar, 20 µm. (G) Live spinning disk confocal microscopy of BFP-Sec61β and GFP-lamin B2 in si153-treated cells, showing continuity of ER membrane with ectopic site of lamin B2 localization. Enlargements are 2.5×. Scale bar, 10 µm.

FIGURE 5:

LBR accumulates ectopically in late-telophase stage cells depleted of Nup153. (A) Widefield images of LBR and lamin B2 detection in late-telophase cells, determined by tubulin staining (unpublished data), following treatment with siCon and si153 oligos. Scale bar, 20 μm. Enlargements are 2X. (B) Quantification of cytoplasmic to nuclear ratio for LBR. Data collected from three biological replicates are depicted in different colors, N = 71, 68, 36 (siCon) and 72, 69, 36 (si153). The mean of each experiment was used to calculate the mean ± SD, indicated in black; siCon: 0.39 ± 0.03; si153: 0.67 ± 0.04. (C) Confocal image of LBR and lamin B2 codetection in siNup153-treated cells. Scale bar, 20 µm. (D) Arbitrary fluorescence units corresponding to the plot line shown in C are graphed.

FIGURE 6:

Nup153 plays a critical role specific to NE formation. (A) Immunoblot of DLD1-Nup153^AID cells growing asynchronously or in the presence of thymidine to prevent progression into mitosis. Cells were treated with auxin for 15 h, as indicated. Following detection of Nup153 and lamin B2, blots were reprobed for vinculin as a loading control. (B) Widefield microscopy of cells treated as in A tracking the appearance of endogenous lamin B2 with DNA counterstained. Images were adjusted independently to best illustrate lamin B2 distribution pattern. Enlargements are 2×. Scale bar, 20 μM. (C) Quantification of nuclei with associated lamin B2 foci. Data collected from three biological replicates are graphed using the mean from each experiment to calculate the overall mean ± SD; no thymidine, no auxin 8.5 ± 0.9% (N = 1078, 771, 784); no thymidine, plus auxin 53.6 ± 5.4% (N = 664, 538, 465); thymidine, no auxin 1.6 ± 0.4% (N = 486, 404, 389); thymidine, plus auxin 6.8 ± 2.5% (N = 460, 353, 509). *, P < 0.05; ***, P < 0.001.

FIGURE 7:

Summary model of observations. After the genome segregates in early anaphase, nuclear envelope proteins dispersed throughout the mitotic ER seed the formation of the nascent nuclear envelope at the chromatin surface. By late anaphase, INM proteins form distinct core and noncore domains at NE. As cells exit late anaphase and enter telophase, these transient domains redistribute as midzone microtubules condense into a midbody structure. During the progression from late anaphase to telophase, Nup153 is required for the continued targeting of primarily noncore INM proteins. At interphase, INM protein targeting occurs independently of Nup153 with the exception to-date of LBR.