Regulation of mouse oocyte microtubule and organelle dynamics by PADI6 and the cytoplasmic lattices

Abstract

Organelle positioning and movement in oocytes is largely mediated by microtubules (MTs) and their associated motor proteins. While yet to be studied in germ cells, cargo trafficking in somatic cells is also facilitated by specific recognition of acetylated MTs by motor proteins. We have previously shown that oocyte-restricted PADI6 is essential for formation of a novel oocyte-restricted fibrous structure, the cytoplasmic lattices (CPLs). Here, we show that α-tubulin appears to be associated with the PADI6/CPL complex. Next, we demonstrate that organelle positioning and redistribution is defective in PADI6-null oocytes and that alteration of MT polymerization or MT motor activity does not induce organelle redistribution in these oocytes. Finally, we report that levels of acetylated microtubules are dramatically suppressed in the cytoplasm of PADI6-null oocytes, suggesting that the observed organelle redistribution failure is due to defects in stable cytoplasmic MTs. These results demonstrate that the PADI6/CPL superstructure plays a key role in regulating MT-mediated organelle positioning and movement.

Figure 1. Co-localization and interaction of α-tubulin and PADI6 in murine GV oocytes, mature oocytes, and early embryos

(A-B) Co-localization of α-tubulin and PADI6 by confocal microscopy before (A) and after (B) treatment with 0.1% of Triton X-100 to release soluble proteins. PADI6 and α-tubulin co-localization is highlighted in merged images. Spindle MTs are indicated by white arrows in panel A. Cortical region is indicated by arrow in panel B. The co-localization of DDX6 and PADI6 serves as a control. (C) Immunoprecipitation of α-tubulin from oocyte extracts using anti-PADI6 antibodies. PI = PADI6 pre-immune sera. (D) Ultra-structure of cytoplasmic lattices (CPLs) and co-localization of α-tubulin and PADI6 at the CPLs by immuno-EM. CPL structures are indicated by white arrows in upper (low magnification) and lower (high magnification) left TEM images and are presented here for reference. Immuno-EM image (right panel) with α-tubulin and PADI6-coated gold particles indicated by black arrows and arrow head, respectively. Inset shows PADI6 pre-immune sera. (E) IIF-confocal analysis of tubulin localization and levels in wild-type and Padi6-null oocytes prior to and following extraction with Triton X-100. (F) Western blot analysis of α-tubulin and PADI6 levels in the insoluble pellet of wild type and Padi6-null oocytes following extraction with Triton. (G) Staining intensity of α-tubulin bands from wild-type and Padi6-null oocytes in Figure F. See also Figure S1, S2 and Table S1.

Figure 2. Abnormal meiotic spindle configuration in mature Padi6-null oocytes

(A) Spindle MT structure in live oocytes. MTs and DNA were stained with Oregon green 488 Taxol and Hoechst 33342 (magenta), respectively. (B) PFA-fixed oocytes were stained with antibodies to α-tubulin (green) and PADI6 (red). (C) Localization of Aurora kinase A (green) on spindles from Padi6 wild-type and mutant oocytes. (D) Same as (C) except oocytes were stained with anti-phospho-histone H3 Ser10 antibody (red). (E) Measurement of spindle apparatus from Padi6 wild-type and mutant oocytes. Chro., chromosome; *, P < 0.05; **, P < 0.001; ***, P < 0.0001; n.s., not significant. Numbers are average ± SEM.

Figure 3. Endoplasmic reticulum (ER) and mitochondrial positioning and redistribution during maturation is altered in Padi6-null oocytes

(A) ER and mitochondria localization in GV-stage PFA-fixed oocytes following staining with antibodies to calreticulin (red) and Cytochrome C (green). DIC, differential interference contrast. (B) Time course of organelle redistribution during oocyte maturation. DNA was stained using Hoechst 33342 (blue) and MT was stained with Oregon green 488 Taxol. ER and mitochondria were stained with ER tracker and Mito Tracker, respectively. Oocytes were collected at 3-hour intervals during in vitro culture, fixed, prepared for IIF and imaged by confocal microscopy. (C) Organelle localization in ovulated oocytes recovered from oviducts at 13 hours post hCG and prepared as in (A). Arrow indicates cortical ER aggregates. (D) TEM images of the metaphase II-arrested wild-type and Padi6-null oocyte cortex. The lower panels in (D) are high magnification images. ER and mitochondria are indicated by white arrows and asterisks, respectively. CPLs are indicated by black arrows. (E) Morphometric analyses of CPLs and organelles in the cortical and middle region of wild-type and Padi6-null oocytes. *, P < 0.05; ***, P < 0.0001; n.s., not significant. Numbers are mean ± S.D. of percentage per oocyte. See also Movie S1 and S2.

Figure 3. Endoplasmic reticulum (ER) and mitochondrial positioning and redistribution during maturation is altered in Padi6-null oocytes

(A) ER and mitochondria localization in GV-stage PFA-fixed oocytes following staining with antibodies to calreticulin (red) and Cytochrome C (green). DIC, differential interference contrast. (B) Time course of organelle redistribution during oocyte maturation. DNA was stained using Hoechst 33342 (blue) and MT was stained with Oregon green 488 Taxol. ER and mitochondria were stained with ER tracker and Mito Tracker, respectively. Oocytes were collected at 3-hour intervals during in vitro culture, fixed, prepared for IIF and imaged by confocal microscopy. (C) Organelle localization in ovulated oocytes recovered from oviducts at 13 hours post hCG and prepared as in (A). Arrow indicates cortical ER aggregates. (D) TEM images of the metaphase II-arrested wild-type and Padi6-null oocyte cortex. The lower panels in (D) are high magnification images. ER and mitochondria are indicated by white arrows and asterisks, respectively. CPLs are indicated by black arrows. (E) Morphometric analyses of CPLs and organelles in the cortical and middle region of wild-type and Padi6-null oocytes. *, P < 0.05; ***, P < 0.0001; n.s., not significant. Numbers are mean ± S.D. of percentage per oocyte. See also Movie S1 and S2.

Figure 4. Alteration of MT polymerization does not induce organelle redistribution in Padi6-null oocytes

MTs were stained using an anti-α-tubulin antibody in PFA-fixed oocytes while the ER and mitochondria were stained using ER Tracker (red), Mito Tracker (magenta). (A) Control experiment documenting the localization of MTs, ER, and mitochondria in wild type MII-arrested oocytes following DMSO treatment. (B) Localization of MTs, ER and mitochondria in wild type and PADI6-null oocytes following taxol treatment. Arrow indicates taxol-induced organelle clustering around the enlarged wild-type oocyte spindle apparatus. (C) Distribution of mitochondria in wild type and PADI6-null oocytes following nocodazole treatment. (D) Same as (C) except oocytes were pretreated with vinblastine. Mitochondria in (C) and (D) were visualized using Mito Tracker. All oocytes were stained with Hoechst 33342 (blue) to visualize DNA. See also Figure S4.

Figure 5. Padi6-null oocytes display reduced levels of cytoplasmic acetylated α-tubulin and treatment of these oocytes with MT motor inhibitors does not affect organelle distribution

(A-F) Confocal IIF and western blot analysis of acetylated α-tubulin localization and levels in wild-type and Padi6-null GV-stage (A-B), MII-arrested oocytes (C-D), and TSA-treated in vitro matured oocytes (E-F). Cytoplasmic acetylated tubulin was indicated by arrows. (G-H) Effect of MT motor inhibitors on organelle distribution in oocytes. Padi6 wild-type (G) and mutant (H) GV-stage oocytes were cultured with the MT motor inhibitors, vanadate or AMP-PNP, for 3 hours, stained with Tubulin Tracker (green), ER Tracker (red), Mito Tracker (magenta), and Hoechst 33342 (blue), and imaged by confocal microscopy. See also Figure S5.