Figure 5.
MRCK promotes spindle rotation in anaphase II. (A) Time-lapse images of anaphase II in a live SrCl2-activated MII oocyte, relative to the onset of chromatid segregation (t = 0). Before activation, MII oocytes were injected with cRNAs encoding EGFP-MAP4 to label microtubules and H2B-mCherry to label chromosomes. The oocyte was treated with DMSO as a control. Images in the top row are compressed z-stacks of the microtubule and chromosome channels, spanning the entire spindle apparatus, combined with a single-plane brightfield image. The bottom row shows an expanded view of the spindle. Images are from Video 2. (B) Time-lapse images of anaphase II in a live SrCl2-activated MII oocyte treated with BDP9066. The experiment illustrates the spindle distortion phenotype. Images are from Video 3. (C) Time-lapse images of anaphase II in a live SrCl2-activated MII oocyte treated with BDP9066. The experiment illustrates the spindle breakage phenotype. Images are from Video 4. (D) Bar graph depicting the fraction of activated oocytes (%), treated with DMSO or BDP9066, showing normal spindle rotation, the spindle distortion phenotype, or the spindle breakage phenotype. (E) Maternal PN configuration in SrCl2-activated oocytes treated with DMSO (left) or BDP9066 (middle, right). Before activation, MII oocytes were injected with cRNAs encoding EGFP-MAP4 to label microtubules and H2B-mCherry to label chromosomes. Activated oocytes were imaged live 6 h after activation. In the middle image, the BDP9066-treated oocyte shows two maternal pronuclei and a distorted anaphase II spindle remnant, suggestive of spindle distortion during anaphase II. In the right image, the BDP9066-treated oocyte shows a second polar body and a maternal PN enclosed in a polar body–like protrusion (arrow), suggestive of spindle breakage during anaphase II. (F) Bar graph depicting the fraction (%) of activated oocytes containing a single maternal PN (gray), or two maternal pronuclei (yellow), when scored 6 h after activation. Oocytes were activated in the presence of DMSO or BDP9066 (1 µM) as shown in E. P value was calculated using Fisher’s exact test. (G) Spindle breakage phenotype in an SrCl2-activated MII oocyte expressing MRCKβ-K105A. The oocyte was fixed 1.5 h after activation, then stained for tubulin (green), chromatin (TO-PRO-3; magenta), and F-actin (Alexa Fluor 568–phalloidin; red). The image is a compressed z-stack of 18 consecutive confocal frames taken from Video 5 spanning the entire spindle apparatus. For clarity, a single image of the F-actin channel was overlaid. (H) Bar graph depicting the fraction (%) of SrCl2-activated oocytes injected with water (control) or with MRCKβ-K105A cRNA, showing normal spindle rotation, spindle distortion, or spindle breakage, when scored 1.5 h after activation. (I) Pronuclei staining in a control zygote, obtained by in vitro fertilization in the presence of DMSO. The zygote was fixed 6.5 h after insemination and stained for H3K4me3 (green), F-actin (Alexa Fluor 568–phalloidin; red), and chromatin (TO-PRO-3; magenta). (J) Pronuclei staining in zygotes obtained by in vitro fertilization in the presence of BDP9066. Zygotes were fixed and stained as in I. The left image illustrates the presence of two maternal pronuclei, each enclosed in a polar body–like protrusion. The right image illustrates the presence of two maternal pronuclei located deeper in the cytoplasm. (K) Bar graph depicting the fraction (%) of fertilized oocytes containing a single maternal PN (gray) or two maternal pronuclei (yellow) when fixed 6.5 h after insemination. Oocytes were fertilized in the presence of DMSO or BDP9066 (2 µM) as shown in I and J. P value was calculated using Fisher’s exact test. The number of oocytes scored is indicated in parentheses above each bar. Scale bars are 10 µm.

MRCK promotes spindle rotation in anaphase II. (A) Time-lapse images of anaphase II in a live SrCl2-activated MII oocyte, relative to the onset of chromatid segregation (t = 0). Before activation, MII oocytes were injected with cRNAs encoding EGFP-MAP4 to label microtubules and H2B-mCherry to label chromosomes. The oocyte was treated with DMSO as a control. Images in the top row are compressed z-stacks of the microtubule and chromosome channels, spanning the entire spindle apparatus, combined with a single-plane brightfield image. The bottom row shows an expanded view of the spindle. Images are from Video 2. (B) Time-lapse images of anaphase II in a live SrCl2-activated MII oocyte treated with BDP9066. The experiment illustrates the spindle distortion phenotype. Images are from Video 3. (C) Time-lapse images of anaphase II in a live SrCl2-activated MII oocyte treated with BDP9066. The experiment illustrates the spindle breakage phenotype. Images are from Video 4. (D) Bar graph depicting the fraction of activated oocytes (%), treated with DMSO or BDP9066, showing normal spindle rotation, the spindle distortion phenotype, or the spindle breakage phenotype. (E) Maternal PN configuration in SrCl2-activated oocytes treated with DMSO (left) or BDP9066 (middle, right). Before activation, MII oocytes were injected with cRNAs encoding EGFP-MAP4 to label microtubules and H2B-mCherry to label chromosomes. Activated oocytes were imaged live 6 h after activation. In the middle image, the BDP9066-treated oocyte shows two maternal pronuclei and a distorted anaphase II spindle remnant, suggestive of spindle distortion during anaphase II. In the right image, the BDP9066-treated oocyte shows a second polar body and a maternal PN enclosed in a polar body–like protrusion (arrow), suggestive of spindle breakage during anaphase II. (F) Bar graph depicting the fraction (%) of activated oocytes containing a single maternal PN (gray), or two maternal pronuclei (yellow), when scored 6 h after activation. Oocytes were activated in the presence of DMSO or BDP9066 (1 µM) as shown in E. P value was calculated using Fisher’s exact test. (G) Spindle breakage phenotype in an SrCl2-activated MII oocyte expressing MRCKβ-K105A. The oocyte was fixed 1.5 h after activation, then stained for tubulin (green), chromatin (TO-PRO-3; magenta), and F-actin (Alexa Fluor 568–phalloidin; red). The image is a compressed z-stack of 18 consecutive confocal frames taken from Video 5 spanning the entire spindle apparatus. For clarity, a single image of the F-actin channel was overlaid. (H) Bar graph depicting the fraction (%) of SrCl2-activated oocytes injected with water (control) or with MRCKβ-K105A cRNA, showing normal spindle rotation, spindle distortion, or spindle breakage, when scored 1.5 h after activation. (I) Pronuclei staining in a control zygote, obtained by in vitro fertilization in the presence of DMSO. The zygote was fixed 6.5 h after insemination and stained for H3K4me3 (green), F-actin (Alexa Fluor 568–phalloidin; red), and chromatin (TO-PRO-3; magenta). (J) Pronuclei staining in zygotes obtained by in vitro fertilization in the presence of BDP9066. Zygotes were fixed and stained as in I. The left image illustrates the presence of two maternal pronuclei, each enclosed in a polar body–like protrusion. The right image illustrates the presence of two maternal pronuclei located deeper in the cytoplasm. (K) Bar graph depicting the fraction (%) of fertilized oocytes containing a single maternal PN (gray) or two maternal pronuclei (yellow) when fixed 6.5 h after insemination. Oocytes were fertilized in the presence of DMSO or BDP9066 (2 µM) as shown in I and J. P value was calculated using Fisher’s exact test. The number of oocytes scored is indicated in parentheses above each bar. Scale bars are 10 µm.

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