Research ArticleGENETICS

The NEMP family supports metazoan fertility and nuclear envelope stiffness

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Science Advances  28 Aug 2020:
Vol. 6, no. 35, eabb4591
DOI: 10.1126/sciadv.abb4591
  • Fig. 1 Drosophila Nemp is a NE protein required for fertility.

    (A) Predicted membrane topology of dNemp. (B) dNemp colocalizes with LaminB. (C) dNemp localizes at the INM of S2 cells. OMX microscopy of dNemp localization relative to RanGap and GP210. (D) Schematic of nuclear markers used in (C). (E) (i) Experimental schematic and (ii) results of sperm exhaustion assay to test fertility of dNemp mutant males rescued with tubulin-Gal4-driven dNemp or hNEMP1 cDNA and decreased fertility as ote−/− (otePK/B279G) mutant males age. (F) Phase-contrast imaging of adult testes from (i) WT and (ii) dNemp−/− (dNempRR1/y) mutants reveals drastic morphological changes. (G) Phase-contrast imaging of (i) WT ovary and (ii) pair of dNemp−/− (dNempRR1/RR1) ovaries reveals lack of mature oocytes (yellow arrowheads). (H) dNemp and hNEMP1 rescue the eclosure defects of dNemp−/− flies. Eclosure rates of dNemp−/− adults alone or rescued with tubulinGal4-driven dNemp or hNEMP1. Eclosure rates are normalized to dNemp−/− rescue by dNemp transgene, and balancer effects are taken into account. (I) Armadillo (Arm) staining of (i) dNemp−/− and (ii) dNemp−/− flies rescued with the BAC construct showing significant rescue of testis morphology. The hub of the testis is marked by an asterisk. (J and K) (i) WT and (ii) nos>dNemp RNAi testes (J) and ovaries (K) show germline defects. P-values are denoted as follows; *0.05 > P > 0.01; **0.01 > P >0.001; ***: 0.001 > P > 0.0001; **** P < 0.0001.

  • Fig. 2 Drosophila Nemp supports NE shape.

    (A and B) Larval testes. The hub of the testis is marked by an asterisk. LaminB staining of germ cells from (A) WT and (B) dNemp−/− testes shows blebbing of the NE in (i) and at higher magnification in (ii) and (iii). (C and D) Otefin staining of somatic cyst cells examined from (C) WT and (D) dNemp−/− testes. Otefin mislocalization in a subset of cyst cells (yellow arrowheads). (E to G) Transmission EM analysis of early germ cells in (E) WT and (F) dNemp−/− larval testes. Red arrows point to the NE. C, cytoplasm; N, nucleus. (G) Quantification of WT and dNemp−/− germ cell NE circularity. (H and I) Larval testes in (H) WT reveal a LaminC gradient, while (I) dNemp−/− mutants show increases of LaminC in late cysts (yellow arrowheads).

  • Fig. 3 Nemp defects in C. elegans and Danio rerio.

    (A) Brood sizes of control (nemp-1oz534/+, n = 24) and KO (nemp-1 oz534/535, n = 14) C. elegans mutants bred over 5 days. (B) Dead egg counts per broods in (A). (C) Quantification of sterility defects in nemp-1 homozygous oz534 and oz535 animals compared to WT control. Mutants show increased sterility when aging. (D and E) Lamin and DAPI staining of (D) WT and (E) KO (oz534 homozygous) C. elegans. Insets depict single nuclei from (i) WT and (ii) KO animals. (F) Fertility of female zNemp1hsc98;zNemp2hsc100 double mutants declines with repeated breeding trials compared to zNemp1hsc98/+;zNemp2hsc100/+ double heterozygous over a 12-week period. (G) Fertility loss of male double mutants compared to double heterozygous siblings. ns, not significant.

  • Fig. 4 Loss of human NEMP leads to defects in nuclear shape and survival, and EMERIN (EMD) forms a complex with NEMP1.

    (A to C) Lentivirus knockdown of hNEMP1 in HT1080 cells causes convoluted nuclei and increased LaminA/C. (A) Quantification of knockdown efficiency and (B) corresponding nuclear shape. (C) Example of nuclei from respective short hairpin RNA (shRNA) transfections. (D) Knockdown of hNEMP1 and hNEMP2 using different combinations of siRNAs (combos A and B) causes a reduction in cell number in various cell types. Cell number remains unchanged in SHP77 cells. (E) Level of hNEMP1 and hNEMP2 knockdown measured by quantitative polymerase chain reaction. (F) Apoptosis of RPE-1 cells assessed by annexin V/PI (propidium iodide) staining at 6 days following combined knockdown of hNEMP1 and hNEMP2. (G) Top 5 hNEMP1 BioID interactors identified by spectral counts. Yellow indicates LEM domain proteins. P value of 2.0 × 10−9 and a Benjamini of 6.2 × 10−8; The database for annotation, visualization and integrated discovery (DAVID) Bioinformatics Resource. (H) Top 5 hNEMP1 AP-MS interactors identified by spectral counts. All proteins listed for BioID and AP-MS are high-confidence <1% false discovery rate. (I) Endogenous hNEMP1 coimmunoprecipitates with EMD-GFP (green fluorescent protein). HEK293 cells transiently transfected with GFP or EMD-GFP were subjected to GFP pull-down. (J) Endogenous EMERIN coimmunoprecipitation with hNEMP1-BirA*-Flag. HEK293 cells stably expressing either hNEMP1-BirA*-Flag or BirA*-Flag were subjected to Flag immunoprecipitation (IP).

  • Fig. 5 Human NEMP and EMERIN (EMD) cause changes to NE stiffness.

    (A) (i) SEM image of a FIB-modified AFM probe. Representative AFM force-displacement data measured from (ii) sg-Control nontargeted CRISPR clone 1 cells and (iii) hNEMP1/2 KO clone cells. (B) Apparent Young’s modulus of T24 cell nuclei treated with siControl, sihNEMP1, sihEMD alone, or sihNEMP1/sihEMD together. (C) Apparent Young’s modulus of cell nuclei of SHP77 hNEMP1 and hNEMP1/2 CRISPR KO cells. (D and E) Apparent Young’s modulus of WT (D) and NEMP1/2KO (E) SHP77 cells transfected with GFP-LMNA. An increase in both WT and NEMP1/2KO NE stiffness indicates that LMNA acts independently of NEMP to provide stiffness to the NE. (F and G) WT (F) and NEMP1/2KO (G) SHP77 cells stained with phalloidin to mark the actin cytoskeleton under DMSO- or latrunculin B–treated (200 nM) condition in (i). Inhibiting actin polymerization with latrunculin B treatment causes the apparent Young’s modulus to decrease in WT and not in NEMP1/2KO cells in (ii). (H) Apparent Young’s modulus of WT or NEMP1/2KO SHP77 cells treated with or without siRNA against EMERIN (siEMD). (I) EMERIN (EMD) knockdown with siRNA shows decreased protein levels by immunoblot. Actin was used as a loading control.

  • Fig. 6 Loss of mNemp1 in mice causes fertility defects and reduced NE stiffness in oocytes.

    (A) Litter sizes of mNemp1+/+, mNemp1+/−, and mNemp1−/− females. (B) Follicle count of 4-week-old mNemp and EMD mutant ovaries showing early and highly significant depletion of primordial (Prd) follicles [n = 5 WT, 6 heterozygous (HET) and 5 KO, and 3 EMD KO], but not primary (Prim) or secondary (Sec) follicles. M1, metaphase 1. (C) (i) mNemp1 is required for spindle organization of ovulated oocytes (oocyte number analyzed: WT = 101, HET = 89, and KO = 87 at 4 weeks; WT = 57, HET = 100, and KO = 123 at 7 months). (ii) Illustration of spindles in ovulated oocytes. (D) Immunostaining of mNemp1 and LaminA/C in primordial, primary, secondary, and antral follicles of a WT ovary. Orange arrowheads mark the NE of each cell type. (E) Immunostaining of EMD in primordial, primary, and secondary follicles of a WT ovary. (F) mNemp1 is required for early mouse embryonic development (WT = 59, HET = 90, and KO = 53 embryos examined). Genotypes refer to females mated with WT males. dpc, days post-coitus. (G) (i) Micropipette aspiration of germinal vesicles (GV) measures stiffness of the NE in (ii) WT and mNemp1 mutant oocytes. (H) Schematic model of a mammalian ovary. A gradient of stiffness occurs with high stiffness in the cortex and decreasing stiffness in the medulla. Primordial follicles in the cortex have high levels of Nemp/EMD (red) and low levels of LaminA/C (green). As the oocytes mature, Nemp/EMD levels drop and LaminA/C levels increase.

Supplementary Materials

  • Supplementary Materials

    The NEMP family supports metazoan fertility and nuclear envelope stiffness

    Yonit Tsatskis, Robyn Rosenfeld, Joel D. Pearson, Curtis Boswell, Yi Qu, Kyunga Kim, Lacramioara Fabian, Ariz Mohammad, Xian Wang, Michael I. Robson, Karen Krchma, Jun Wu, João Gonçalves, Didier Hodzic, Shu Wu, Daniel Potter, Laurence Pelletier, Wade H. Dunham, Anne-Claude Gingras, Yu Sun, Jin Meng, Dorothea Godt, Tim Schedl, Brian Ciruna, Kyunghee Choi, John R. B. Perry, Rod Bremner, Eric C. Schirmer, Julie A. Brill, Andrea Jurisicova, Helen McNeill

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