Ndc1 drives nuclear pore complex assembly independent of membrane biogenesis to promote nuclear formation and growth

Abstract

The nuclear envelope (NE) assembles and grows from bilayer lipids produced at the endoplasmic reticulum (ER). How ER membrane incorporation coordinates with assembly of nuclear pore complexes (NPCs) to generate a functional NE is not well understood. Here, we use the stereotypical first division of the early C. elegans embryo to test the role of the membrane-associated nucleoporin Ndc1 in coupling NPC assembly to NE formation and growth. 3D-EM tomography of reforming and expanded NEs establishes that Ndc1 determines NPC density. Loss of ndc1 results in faster turnover of the outer scaffold nucleoporin Nup160 at the NE, providing an explanation for how Ndc1 controls NPC number. NE formation fails in the absence of both Ndc1 and the inner ring component Nup53, suggesting partially redundant roles in NPC assembly. Importantly, upregulation of membrane synthesis restored the slow rate of nuclear growth resulting from loss of ndc1 but not from loss of nup53. Thus, membrane biogenesis can be decoupled from Ndc1-mediated NPC assembly to promote nuclear growth. Together, our data suggest that Ndc1 functions in parallel with Nup53 and membrane biogenesis to control NPC density and nuclear size.

Keywords: C. elegans; C. elegans embryos; NDC1; Nup107-160 complex; cell biology; lipid synthesis; nuclear envelope; nuclear pore complex.

Figure 1.. Smaller pronuclear size resulting from loss of Ndc1 corresponds to reduced nuclear import.

(A) Schematic of a nuclear pore complex (left). Schematic and domain organization of Ce Ndc1 (right, bottom). (B) Plot of percentage embryonic lethality for indicated conditions. N = # of worms. n = # of embryos. (C) Left: fixed overview and magnified images of C. elegans embryos immunostained for lamin for indicated conditions. Scale bars, 10 μm and 5 μm for magnified images. Right: plot of nuclear size for indicated conditions. Mean ± S.D. N = # of slides, n = # of nuclei. (D) Schematic of stereotypical nuclear events relative to nuclear import in the C. elegans zygote, with pseudocleavage (PC) regression used as a reference time point. Time is in seconds. (E) Confocal overview and magnified images of embryo from a time lapse series of GFP:NLS-LacI in indicated conditions. Scale bars, 10 μm for overview image and 5 μm for magnified images. (F) Plot of nuclear to cytoplasmic ratio of GFP:NLS-LacI for indicated conditions. n = # of embryos. Average ± S.D. is shown. (G) Pronuclear diameter for indicated conditions at indicated time point. Average ± S.D. is shown. n = # of embryos. A two-way ANOVA was used to determine statistical significance between control and each RNAi condition. p-Values all <0.0001.

Figure 1—figure supplement 1.. Generation and characterization of mutant ndc1 alleles and RNAi depletion of ndc1.

Related to Figure 1. (A) Schematic of CRISPR guides to generate ndc1 null allele. (B) Plot of ndc1 mRNA fold change for control and ndc1Δ worms. Mean ± S.D. (C) Confocal overview and magnified images of one-cell stage embryo from a time lapse series of indicated markers for indicated conditions. Scale bars, 10 μm for overview image and 5 μm for magnified images. (D) Schematic of ndc1 genomic locus (top) and tm1845 allele (below). (E–F) Plots of percentage embryonic lethality for indicated conditions. N = # of worms. n = # of embryos. (G) Schematic for equation to calculate nuclear to cytoplasmic ratio of GFP:NLS-LacI.

Figure 2.. Ndc1 is necessary for timely formation of a transport competent nucleus after mitosis.

(A) Schematic of the first mitotic division in C. elegans embryos relative to anaphase onset and initiation of furrow ingression. All measurements of one- to two-cell stage embryos are done on the AB cell/nucleus (nuclear envelope [NE] of the AB nucleus is highlighted in magenta). (B) (Left) Confocal images from time series of mitotic nuclear formation relative to furrow ingression with indicated markers. (Right) Three-pixel wide line scan for the ‘core’ and ‘non-core’ region of the NE. Scale bars, 10 μm for overview image and 5 μm for magnified images. (C–D) (Above) Confocal images from time series mitotic nuclear formation with indicated markers for indicated conditions. (Below) Line scans measuring background-corrected fluorescence intensities as indicated in schematic for each time point and fluorescent marker (LEM-2:mCH is magenta and NLS-LacI:GFP is green). Scale bars, 10 μm and 5 μm for magnified images. (E) Confocal images of chromosome region from time series relative to anaphase onset with indicated markers and in indicated conditions. Scale bar, 5 μm. (F) Plot of nuclear to cytoplasmic ratio of GFP:NLS-LacI for indicated conditions. n = # of embryos. Average ± S.D. is shown.

Figure 2—figure supplement 1.. Ndc1^en:mNG is recruited early to the nuclear rim and localizes to the endoplasmic reticulum (ER) and cytoplasmic puncta, and nuclear import is delayed in post-mitotic nuclei without Ndc1.

Related to Figure 2. (A) Schematic of ndc1 genomic locus and CRISPR guide to generate ndc1^en:mNG and ndc1^en:mRuby at its endogenous locus. (B) Confocal images from time series of one-cell stage embryo expressing Ndc1^en:mNG and mCh:H2B. Scale bar, 10 μm. (C) Confocal images from time series of one-cell stage embryo expressing Ndc1^en:mNG and SP12:mCh to mark the ER. Scale bars, 10 μm and 5 μm for magnified images. (D) Confocal images from time series of two-cell stage embryo expressing Ndc1^en:mNG and SP12:mCh during mitotic nuclear formation. Scale bars, 10 μm for overview image and 5 μm for magnified images. (E) Plot of normalized nuclear to cytoplasmic ratio of GFP:NLS-LacI for indicated conditions. n = # of embryos. (F) Equation and plot of rate of change in the nuclear to cytoplasmic ratio of GFP:NLS-LacI between each time point in the first 200 s after anaphase for the indicated conditions. Average is shown.

Figure 3.. Fewer nuclear pore complexes (NPCs) assembled on nascent nuclear envelopes (NEs) and a higher mobile pool of Nup160:GFP result from loss of ndc1.

(A, B) Overview images: z-slice from electron tomogram of nuclear formation timed relative to initiation of furrow ingression. 3D model: traced membranes (magenta) and NE holes <100 nm (blue) for region shown as single z-slice in overview image. Magnified representative z-slice traced and untraced from electron tomogram is shown. To calculate the density of ‘NPC’ holes during NE reformation, we segmented and quantified four areas from a single control embryo (0.14 μm², 0.08 μm², 0.05 μm², and 0.04 μm²) and four areas from a single ndc1Δ embryo (0.22 μm², 0.19 μm², 0.18 μm², and 0.17 μm²). Scale bars indicated in figure. (C) Plot of diameters of NE holes <100 nm. n = # of ‘NPC’ holes analyzed. (D) Schematic of Y-complex (also known as Nup107/160 complex) with vertebrate (gray) and C. elegans (black) names shown, adapted from Figure 1B in Hattersley et al., 2016 (note that NPP-23/Nup43 not included). Representative magnified images of AB nucleus from confocal series for each indicated marker (above) and line scans measuring background-corrected fluorescence intensities (below) for each condition. Scale bars, 5 μm. (E) (Top) Schematic of C. elegans gonad and oocytes. (Middle) Confocal images from time lapse series of fluorescence recovery after photobleaching (FRAP) of Ndc1^en:mNG the NE of an oocyte is shown. (Bottom) Representative plot of fluorescence intensities of bleached region over time normalized to the prebleach intensity for each condition is shown. Scale bars, 5 μm. (F) Confocal images from time lapse series of FRAP of Nup160:GFP at the NE of an embryo for indicated conditions are shown (above). Representative plot of fluorescence intensity of bleached region over time normalized to the prebleach intensity for each condition is shown (below). Scale bars, 5 μm.

Figure 3—figure supplement 1.. Reduced nuclear pore complex density in expanded nuclei, levels of outer ring scaffold nucleoporins at the nuclear rim and cytoplasmic puncta resulti from loss of Ndc1.

Related to Figure 3. (A, B) 3D model of tomograms of expanded nuclei showing membranes (magenta) and NE holes ~100 nm (blue) for indicated conditions. To calculate the density of ‘nuclear pore complex’ holes during interphase, we segmented and quantified six regions (1.52 μm²,1.20 μm², 0.99 μm², 0.95 μm², 0.31 μm², and 0.19 μm²) from two different control nuclei and nine regions (1.03 μm², 1.02 μm², 0.87 μm², 0.65 μm², 0.58 μm², 0.55 μm², 0.52 μm², 0.41 μm², and 0.23 μm²) from ndc1Δ nuclei. Scale bars indicated in figure. (C) Confocal overview and magnified images of embryo from a time lapse series of Nup160:GFP and Ndc1^en:mRuby (left). Yellow arrows indicate example puncta. The 5 μm line scans measuring background-corrected fluorescence intensities for each marker drawn across two representative cytoplasmic puncta with co-localized Ndc1 and Nup160 signal (right). Scale bars, 10 μm for overview image and 2.5 μm for magnified images. (D) Max projection confocal images of Nup160:GFP for indicated conditions (left). Plot of average number of cytoplasmic Nup160:GFP structures under indicated conditions at time points relative to pseudocleavage (PC) regression (right). Scale bar, 10 μm. Student’s T-test was used to determine statistical significance. (E) Fixed overview and magnified images of C. elegans embryos immunostained with antibodies that recognize Nup107 for indicated conditions (left). Plot of NE to cytoplasmic ratio for normalized Nup107 fluorescence intensity (right). Scale bars, 10 μm for overview image and 5 μm for magnified images. (F) Fixed overview and magnified images of C. elegans embryos immunostained for antibodies against Elys^Mel-28 for indicated conditions (right). Plot of NE to nucleoplasmic ratio for normalized Elys^Mel-28 fluorescence intensity (right). Scale bars, 10 μm and 5 μm for magnified images. (G) Immunoblot of whole worm lysates probed for antibodies that recognize Nup107 and α-tubulin for indicated conditions.

Figure 3—figure supplement 2.. Characterization of protein levels and localization of nucleoporins in ndc1 deletion mutant and fluorescence recovery after photobleaching (FRAP) analysis of Ndc1^en:mNG in nuclear envelope (NE) of embryos.

Related to Figure 3. (A) Fixed overview and magnified images of C. elegans embryos immunostained for antibodies that recognize Nup53 for indicated conditions (left). Scale bars, 10 μm for overview image and 5 μm for magnified images. Plot of NE to cytoplasmic ratio for normalized Nup53 fluorescence intensity (right). (B) Immunoblot of whole worm lysates probed for antibodies that recognize Nup53 and α-tubulin for indicated conditions. (C) (Left) Fixed overview images of C. elegans oocytes immunostained for mAb414 for indicated conditions. Scale bars, 10 μm for overview image and 5 μm for magnified images. Scale bars, 10 μm. (Right) Average line scan quantification for mAb414 signal under indicated conditions. (D) Immunoblot of whole worm lysates probed for mAb414 and α-tubulin for indicated conditions. (E) Immunoblot of whole worm lysates probed for Nup96 and α-tubulin for indicated conditions. (F) Immunoblot of whole worm lysates probed for IMA-3 and α-tubulin for indicated conditions. (G) Confocal images from time lapse series of fluorescence recovery after photobleaching (FRAP) of Ndc1^en:mNG are shown (above). Scale bars, 5 μm. Representative plot of average fluorescence intensities of bleached region over time normalized to the prebleach intensity for each condition is shown (below).

Figure 4.. Increasing membrane biogenesis restores the slow rate of nuclear expansion and small nuclear size resulting from loss of ndc1.

(A) Dual-view inverted light sheet microscopy 3D projection of wild-type one-cell stage embryo. Scale bar, 10 μm. (B) Theoretical and calculated pronuclear volume for indicated time points. Each color represents a distinct embryo. (C) Magnified images of AB nucleus from confocal series for mCh:H2B for indicated conditions. Scale bar, 5 μm. (D) AB nuclear volume for indicated conditions at indicated time points. Average ± S.D. is shown. (E) Magnified images of AB nucleus from confocal series for mCh:H2B for indicated conditions. Scale bar, 5 μm. (F) AB nuclear volume for indicated conditions at indicated time points. Average ± S.D. is shown. (G) Confocal images of chromosome region from time series relative to anaphase onset with indicated markers and in indicated conditions. Scale bar, 5 μm. (H) Plot of nuclear to cytoplasmic ratio of GFP:NLS-LacI for indicated conditions. n = # of embryos. Average ± S.D. is shown. (I) Magnified images of AB nucleus from confocal series for Nup160:GFP (above) and line scans measuring background-corrected fluorescence intensities (below) for each condition. Scale bar, 5 μm. (J) (Left) Magnified images of paternal pronucleus at –100 s relative to pseudocleavage (PC) regression expressing Nup160:GFP under indicated conditions. Scale bar, 5 μm. (Right) Plot of average pronuclear volume for indicated conditions at –100 s relative to PC regression. n = # of embryos.

Figure 4—figure supplement 1.. Characterization of nuclear size in embryos lacking ndc1 and cnep-1.

Related to Figure 4. (A) (Left) Confocal overview images of embryo from a time lapse series of indicated markers for indicated time points. Scale bar, 10 μm. (Right) Same images as in left, but with nuclear perimeter traced based off MATLAB nuclear tracking script. (B) Confocal images from time lapse series of nuclear expansion for indicated conditions relative to expansion start. Scale bar, 5 μm. (C) Plot of pronuclear volume for indicated conditions at indicated time points. Average ± S.D. is shown. (D) Magnified images of paternal pronucleus from confocal series for GFP:H2B for indicated conditions. Scale bar, 5 μm. (E) Plot of pronuclear volume for indicated conditions at indicated time points. Average ± S.D. is shown. (F) Max projection confocal images of Nup160:GFP for indicated conditions. Scale bar, 10 μm. (G) Plot of average number of cytoplasmic Nup160:GFP structures under indicated conditions at time points relative to pseudocleavage regression. (H) Plot of nuclear to cytoplasmic ratio of GFP:NLS-LacI for indicated conditions. The ndc1(RNAi) data is duplicated from Figure 2F. n = # of embryos. Average ± S.D. is shown.

Figure 5.. Parallel functions for Ndc1 and Nup53 in nuclear assembly.

(A) Schematic of nuclear pore complex (NPC) (left) and NPC subcomplex organization (right) is shown. (B) Plot of percentage embryonic lethality for indicated conditions. N = # of worms. n = # of embryos. (C) Confocal overview and magnified images of embryo from a time lapse series of indicated markers for indicated conditions. Scale bars, 10 μm for overview image and 5 μm for magnified images. (D) Confocal overview and magnified images of embryo from a time lapse series of Nup160:GFP for indicated conditions. Scale bars, 10 μm for overview image and 5 μm for magnified images. (E) Plot of pronuclear diameter for indicated conditions at indicated time point. Average ± S.D. is shown. n = # of embryos. cntrl n=9, nup53(RNAi) n=11, nup53^tm2886 n=7, and nup53^tm2886;ndc1(RNAi) n=7. (F) Fixed overview and magnified images of C. elegans embryos immunostained for mAb414 and DAPI in indicated conditions. Scale bars, 10 μm for overview image and 5 μm for magnified images. (G) Plot of pronuclear diameter for indicated conditions at indicated time point. Average ± S.D. is shown. n = # of embryos. ndc1Δ n=20, ndc1Δ;nup53(RNAi) n=18, and nup155(RNAi) n=30. (H) Magnified images of paternal pronucleus from fixed one-cell stage embryos immunostained with mAb414 for indicated conditions (top). Scale bar, 5 μm. Plot of mAb414 appearance surrounding chromatin under indicated conditions (bottom). A two-way ANOVA was used to determine statistical significance between indicated conditions. n = # of embryos. Scale bars, 10 μm for overview image and 5 μm for magnified images.

Figure 5—figure supplement 1.. Characterization of nup53 RNAi depletion and a nup53 mutant allele.

Related to Figure 5. (A) Schematic of Nup53 domain architecture and binding interactions based on human Nup53 binding sites. (B) Schematic of C. elegans Nup53 domain architecture and the resulting mutant protein resulting from the nup53^tm2886 allele. (C) Fixed overview and magnified images of C. elegans embryos immunostained for mAb414 and Nup53 for indicated conditions. Scale bars, 10 μm for overview image and 5 μm for magnified images. (D) Confocal overview and magnified images of one-cell stage embryo from a time lapse series expressing Nup160:GFP and mCh:H2B for indicated conditions. Scale bars, 10 μm for overview image and 5 μm for magnified images.

Figure 6.. Independent requirements for Ndc1 and lipid synthesis in nuclear formation and expansion.

(A) In the absence of Ndc1, the nuclear envelope (NE) is more continuous, and outer ring scaffold components in the NE are highly dynamic. Some NPCs still assemble and so nuclear transport is eventually established. Ndc1 functions at least in part redundantly to Nup53 in post-mitotic NPC assembly. Both Ndc1 and Nup53 may function through the shared factor Nup155. Loss of Ndc1 in combination with either cnep-1 or chmp-7 leads to additional sealing defects (represented by holes in the NE) that further delay nuclear formation. (B) Endoplasmic reticulum (ER) membranes feed surface area expansion and nuclear import accumulates macromolecules inside the nucleus to increase nuclear volume. (C) Independent requirements for Ndc1 and membrane biogenesis promote nuclear growth.

Author response image 1.

Author response image 2.