NLRP4E regulates actin cap formation through SRC and CDC42 during oocyte meiosis

Abstract

Background: Members of the nucleotide-binding oligomerization domain, leucine rich repeat and pyrin domain containing (NLRP) family regulate various physiological and pathological processes. However, none have been shown to regulate actin cap formation or spindle translocation during the asymmetric division of oocyte meiosis I. NLRP4E has been reported as a candidate protein in female fertility, but its function is unknown.

Methods: Immunofluorescence, reverse transcription polymerase chain reaction (RT-PCR), and western blotting were employed to examine the localization and expression levels of NLRP4E and related proteins in mouse oocytes. small interfering RNA (siRNA) and antibody transfection were used to knock down NLRP4E and other proteins. Immunoprecipitation (IP)-mass spectrometry was used to identify the potential proteins interacting with NLRP4E. Coimmunoprecipitation (Co-IP) was used to verify the protein interactions. Wild type (WT) or mutant NLRP4E messenger RNA (mRNA) was injected into oocytes for rescue experiments. In vitro phosphorylation was employed to examine the activation of steroid receptor coactivator (SRC) by NLRP4E.

Results: NLRP4E was more predominant within oocytes compared with other NLRP4 members. NLRP4E knockdown significantly inhibited actin cap formation and spindle translocation toward the cap region, resulting in the failure of polar body extrusion at the end of meiosis I. Mechanistically, GRIN1, and GANO1 activated NLRP4E by phosphorylation at Ser429 and Thr430; p-NLRP4E is translocated and is accumulated in the actin cap region during spindle translocation. Next, we found that p-NLRP4E directly phosphorylated SRC at Tyr418, while p-SRC negatively regulated p-CDC42-S71, an inactive form of CDC42 that promotes actin cap formation and spindle translocation in the GTP-bound form.

Conclusions: NLRP4E activated by GRIN1 and GANO1 regulates actin cap formation and spindle translocation toward the cap region through upregulation of p-SRC-Tyr418 and downregulation of p-CDC42-S71 during meiosis I.

Keywords: Actin cap; CDC42; Meiosis; NLRP4E; SRC.

Fig. 1

NLRP4E is an oocyte-predominant protein. A The AA sequence alignment between NLRP4 family members, including the 75–323 AA region in NLRP4E for construction of the polyclonal antibody used in this study. B Verification of the specificity of NLRP4E polyclonal antibody. We used NLRP4E antibody to perform immunoprecipitation followed by SDS-PAGE and silver staining. The gel band (left) corresponding to the NLRP4E blot band (right) was cut and sent for MALDI. The results show that the identified protein with maximum intensity was NLRP4E. C, D RT-PCR shows that NLRP4E mRNA was the richest among NLRP4 family mRNAs. E Western blot showing that NLRP4E was predominantly expressed in the brain, liver, testis, and ovary. F Western blot showing that the NLRP4E level gradually increased in mouse ovaries from post-natal day (PND) 1 to 21 and reached a peak on PND 21. G, H Western blots (G) and immunofluorescence (H) show that NLRP4E was more predominant in oocytes than in GCs (granular cells). I Western blots show that the NLRP4E level remained constant during oocyte meiosis. J Immunofluorescence shows that NLRP4E accumulates at the cell membrane during oocyte meiosis. DAPI is shown in blue, NLRP4E in green, and actin filaments in red.  GV, germinal vesicle; GVBD, germinal vesicle breakdown; MI, metaphase I; MII, metaphase II. Scale bar, 50 μm in H, 20 μm in J. * indicates p  < 0.05

Fig. 2

NLRP4E knockdown disrupts oocyte meiosis. A, B RT-PCR and quantification showing that the NLRP4E mRNA level was significantly reduced by siRNA knockdown. C, D Western blotting shows that the NLRP4E level was significantly reduced by siRNA knockdown. E–G Quantification shows that NLRP4E knockdown significantly reduced the percentages of GVBD (E) and 1pb (the first polar body, F and G). H–J Immunofluorescence analysis shows that NLRP4E knockdown caused significant disruption of chromosomes and spindle microtubules. DNA is shown in blue and microtubules are in green. K Slices from live imaging show that in some oocytes with severe NLRP4E knockdown, the chromosomes failed to segregate at the end of meiosis. EGFP-tubulin mRNA and TagRFP-histone were injected into oocytes to label microtubules and chromosomes. L, M Immunofluorescence analysis shows that in oocytes with medium NLRP4E knockdown, the chromosomes could segregate at the end of meiosis, but spindle translocation toward the cortex was significantly blocked. Scale bar, 80 μm in G and 20 μm in other panels.  * indicates p  < 0.05; ** indicates p  < 0.01; *** indicates p  < 0.001; **** indicates p  < 0.0001

Fig. 3

NLRP4E knockdown impairs oocyte quality. A–D NLRP4E knockdown caused severe mitochondrial aggregation (A–C), and the ATP concentration was significantly decreased in NLRP4E-knockdown oocytes (D). E, F NLRP4E knockdown significantly decreased the percentage of normal fertilization (2-PN, two pronuclei). Scale bar, 20 μm.  ** indicates p  < 0.01; *** indicates p  < 0.001; **** indicates p  < 0.0001

Fig. 4

NLRP4E knockdown disrupted actin cap formation. A, B Phalloidin staining and quantification show that NLRP4E knockdown caused a significant decrease in the intensity of actin filaments at the cap region. DNA is shown in blue, and phalloidin in red. C, D Immunofluorescence analysis shows that NLRP4E knockdown caused a significant decrease in the intensity of ARP3 at the cap region. DNA is shown in blue and ARP3 in green. Scale bar, 20 μm.  * indicates p  < 0.05; **** indicates p  < 0.0001

Fig. 5

NLRP4E is activated through phosphorylation at S429 and T430. A and B We used NLRP4E antibody to perform IP, followed by phosphor protein enrichment and mass spectrometry. Three phosphor sites were identified, one at Thr165 in the NACHT domain, and the other two at Ser429 and Thr430 in a nondefined domain. Sequence alignment showed that Ser429 and Thr430 were unique among all NLRP4 members; therefore, we chose GIMDSDI(PSer)(PThr)LLD, which included Ser429 and Thr430, for custom-made phospho-specific NLRP4E antibody (p-NLRP4E antibody). C and D We preblocked p-NLRP4E antibody with GIMDSDI(PSer)(PThr)LLD, then performed western blotting with p-NLRP4E antibody. The correct-size band (red dotted line rectangle) completely disappeared, indicating that the p-NLRP4E antibody was fairly specific. E Western blot showed that the p-NLRP4E level gradually increased in mouse ovaries from PND (post-natal day) 1 to 21 and reached a peak on PND 21. F Immunofluorescence indicates that compared with NLRP4E that had an even distribution on the oocyte membrane, p-NLRP4E accumulated within the cap region. DNA is shown in blue; T-NLRP4E/p-NLRP4E is in green; actin filaments (phalloidin) are shown in red, and tubulin is in magenta. G, H In vitro maturation and quantification show that NLRP4E knockdown (KD) significantly reduced the maturation rate (percentage of 1pb). Injection of NLRP4E-WT mRNA significantly elevated the maturation rate, whereas the injected nonphospho NLRP4E-S429A & T430A mutant failed to rescue the reduced maturation rate, and the rate in the mutant group was lower than in the NLRP4E-KD group. I, J Immunofluorescence analysis shows that NLRP4E knockdown (KD) significantly reduced the intensity of actin filaments at the cap region; injected NLRP4E-WT mRNA significantly elevated the intensity, whereas the injected nonphospho NLRP4E-S429A and T430A mutant failed to rescue the reduced intensity. Scale bar, 80 μm in G, 20 μm in other panels. Different lower-case letters in H, J indicate significant differences between the two groups

Fig. 6

GRIN1 and GNAO1 activate NLRP4E, which subsequently regulates SRC and CDC42 phosphorylation and actin cap assembly. A We used NLRP4E antibody to perform IP and MALDI and identified four interacting proteins, one of which was GRIN1. B Co-IP and western blotting verified that NLRP4E interacted with GRIN1. C Co-IP- and western blotting show that GANO1interacted with NLRP4E. D Immunofluorescence shows that GANO1 colocalized with NLRP4E at the oocyte membrane. NLRP4E is shown in green, GANO1 is in red, and DNA is in blue. E–H Western blots showing that both GRIN1 (E and F) and GANO1 (G and H) knockdown significantly reduced p-NLRP4E. I, J IP-blot showing that both GANO1 (I) and NLRP4E (J) interacted with SRC. K Immunofluorescence showing that NLRP4E colocalized with SRC at the oocyte membrane. NLRP4E is shown in green, SRC is in red, and DNA is in blue. L–O Western blot showing that both GANO1 (L and M) and NLRP4E (N and O) knockdown significantly reduced p-SRC. P Co-IP blot showing that SRC interacted with CDC42. Q–T Western blots showing that both NLRP4E (Q and R) and SRC (S and T) knockdown significantly increased p-CDC42. Scale bar, 20 μm.  * indicates p  < 0.05; ** indicates p  < 0.01; *** indicates p  < 0.001; **** indicates p  < 0.0001

Fig. 7

NLRP4E directly binds and phosphorylates SRC. A, B Immunofluorescence analysis showing that NLRP4E knockdown (KD) significantly reduced the intensity of actin filaments at the cap region; injected SRC-WT-EGFP mRNA significantly elevated the intensity. DNA is shown in blue, SRC-WT-EGFP is in green, and actin filaments (phalloidin) are in red. C–E In vitro phosphorylation assay and western blot showing that purified NLRP4E-flag could dose-dependently phosphorylate SRC. Scale bar, 20 μm. Different lower-case letters indicate significant differences between the two groups

Fig. 8

Working Model. Our experimental findings suggested that GRIN1 and GNAO1 phosphorylate NLRP4E at Ser429 and Thr430, and then p-NLRP4E is translocated and accumulates in the actin cap region to activate SRC by phosphorylation at Tyr418. p-SRC could itself promote cap formation or it could also downregulate p-CDC42 at S71 and thereby promote the activity of CDC42 (the GTP-bound form) and facilitate cap formation