Borrego-Pinto J et al. (2016), Distinct mechanisms eliminate mother and daught...

ECB-ART-44587

J Cell Biol 2016 Mar 28;2127:815-27. doi: 10.1083/jcb.201510083.

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Distinct mechanisms eliminate mother and daughter centrioles in meiosis of starfish oocytes.

Borrego-Pinto J , Somogyi K , Karreman MA , König J , Müller-Reichert T , Bettencourt-Dias M , Gönczy P , Schwab Y , Lénárt P .

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Centriole elimination is an essential process that occurs in female meiosis of metazoa to reset centriole number in the zygote at fertilization. How centrioles are eliminated remains poorly understood. Here we visualize the entire elimination process live in starfish oocytes. Using specific fluorescent markers, we demonstrate that the two older, mother centrioles are selectively removed from the oocyte by extrusion into polar bodies. We show that this requires specific positioning of the second meiotic spindle, achieved by dynein-driven transport, and anchorage of the mother centriole to the plasma membrane via mother-specific appendages. In contrast, the single daughter centriole remaining in the egg is eliminated before the first embryonic cleavage. We demonstrate that these distinct elimination mechanisms are necessary because if mother centrioles are artificially retained, they cannot be inactivated, resulting in multipolar zygotic spindles. Thus, our findings reveal a dual mechanism to eliminate centrioles: mothers are physically removed, whereas daughters are eliminated in the cytoplasm, preparing the egg for fertilization.

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Genes referenced: dnah3 LOC100893907 LOC115919910 LOC583082 LOC594261 odf2 tubgcp2

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	Figure 1. Identification and characterization of centriole markers in starfish. (A) Overview of identified starfish (P. miniata) homologues of centriolar and PCM proteins compared with those of select model organisms. Black plus sign indicates the presence of a clear homologue; gray plus sign indicates functional homology with divergent amino acid sequence. Mother centriole–specific markers are shown in green and daughter centriole–specific markers are shown in pink (see also schematic representation on the top left). Genes coding for proteins shown in bold were used in this study. (B–E) Fluorescent protein fusions of indicated centriolar proteins were validated as live cell markers in the ciliated epithelium of starfish embryos. HiLyte647 tubulin labels the cilium (orange arrowheads). pmPoc1-mCherry and mEGFP-pmCentrin-2 label both mother and daughter centrioles; pmOdf2-mEGFP and pmChibby-mEGFP are mother-specific markers; hsCentrobin-mEGFP is a daughter-specific marker. Maximum intensity projections of 2–5 confocal sections are shown. Bars, 1 µm.
	Figure 2. Mother centrioles are invariably extruded into the polar bodies, leaving a single daughter centriole in the mature egg. (A–C) Oocytes injected with and expressing the indicated fluorescent markers were imaged by 3D confocal microscopy throughout meiosis, starting at metaphase I. Overview images are maximum intensity projections of the entire z-stacks; insets are single confocal sections of the regions marked on the overviews. Here and in other figures, unless otherwise indicated, the dashed line indicates the oocyte contour based on the transmitted light image (not depicted). z-Stacks were acquired every 30–60 s, and time is shown in mm:ss. Bars: (overview images) 5 µm; (insets) 1 µm. (A) pmPoc1-mEGFP labels centrioles and microtubules (see Video 1). (B) pmOdf2-mEGFP specifically labels mother centrioles; Cy3-tubulin labels microtubules (see Video 2). (C) hsCentrobin-mEGFP labels daughter centrioles; pmPoc1-mCherry labels centrioles and microtubules (see Video 3). (D) Distribution of mother and daughter centrioles after PBII extrusion determined using the indicated centriolar markers. In all oocytes examined, the two mother centrioles were extruded into the polar bodies (configuration I); extrusion of the daughter centriole into PBII (configuration II) was never observed. An Odf2-mEGFP–expressing oocyte, coinjected with Cy3-Tubulin, is shown as an example on the left (maximum intensity projection of a z-stack; processed to remove autofluorescence as described in Materials and methods). Bar, 5 µm.
	Figure 3. The mother centriole is transported and anchored to the cell membrane shortly after PBI extrusion. (A) pmOdf2-mEGFP labels mother centrioles; EB3-mCherry3 labels microtubule plus ends. The left panel shows maximum intensity projections for selected time points, the right panel shows the region around the centriole for all frames. Bars: (left) 5 µm; (right) 1 µm. (B) pmPoc1-mEGFP imaged in 3D at high temporal resolution (see Video 4). 3D volume rendering of the data overlaid with an isosurface reconstruction of the cell outline (gray). Identified positions of centrioles are shown as green and pink spheres for mother and daughter centrioles, respectively. z-Stacks were acquired every 12 s. Bar, 5 µm. (C) Example of retrieved 3D coordinates of a mother centriole and the closest point on the plasma membrane. Time point selected from the dataset in B. (D) Example of a 3D mother centriole trajectory during the transport phase showing the linear fit (dashed line) to estimate the speed of motion. (E) Plot of centriole distance from the plasma membrane over time for the oocyte shown in B. (F) Dashed squares indicate the area shown in insets (see Video 5). Asterisk in the first frame shows nuclear position after centrifugation. t = 0 is shortly after NEBD. 3D rendering as in B. Bars: (main) 40; (inset) 5 µm. (G) Distance measurements of mother and daughter centrioles to the plasma membrane over time for the oocyte shown in F. M1 and M2 are mother centrioles; D1 and D2 are daughter centrioles identified based on their microtubule nucleating activity at the end of meiosis. (H) Centrioles were tracked in an oocyte expressing pmPoc1-mEGFP and hsEB3-mCherry3 starting from anaphase I onset (only pmPoc1-mEGFP is shown). 3D rendering as in B. Bar, 5 µm (see Video 6). (I) Plot of centriole distance from the plasma membrane over time for the oocyte shown in H.
	Figure 4. The mother centriole is anchored to the cell membrane via appendages. (A) The mother centriole labeled by pmOdf2-mEGFP colocalizes with the cell membrane labeled by FM4-64 in metaphase II. Bar, 5 µm. (B) Oocytes expressing pmPoc1-mEGFP were arrested in metaphase II by MG-132. Mother centriole anchoring was assessed in control cells (DMSO) and in oocytes treated with nocodazole, taxol, or cytochalasin D. 3D volume renderings are shown as in Fig. 3 B. t = 0 corresponds to the time of drug addition, time is in mm:ss. (C) Distance of mother and daughter centrioles to the plasma membrane over time for each oocyte shown in B. Orange vertical lines indicate when the drug effect becomes visible. (D) Serial semi-thin section (130 nm) electron micrographs showing the area encompassing the MII spindle and PBI. The panel shows a montage of selected serial sections containing the four centrioles. (E) 3D reconstruction of the serially sectioned volume. (F) High-magnification images of the four centrioles, showing the relevant sections for each of them. Vesicles are identified by an orange asterisk. Orange arrowheads indicate electron-dense connections to the plasma membrane, reminiscent of mother centriole appendages. (G) Top panel shows another example of a mother centriole attached to vesicles (orange asterisk) via electron dense connections reminiscent of appendages during metaphase II (arrowheads). Bottom panel shows a mother centriole still anchored to the plasma membrane after PBII extrusion; arrowheads indicate attachment points to the plasma membrane. Schematic overviews of the whole area and position of the mother centriole within (green sphere) are depicted. Bars: (D and E) 1 µm; (F and G) 0.5 µm.
	Figure 5. Mother and daughter centrioles follow different fates at the end of meiosis. (A) High-magnification view of a daughter centriole losing microtubule nucleating activity (here visualized by hsEB3-mCherry3), followed by centriole elimination as indicated by loss of the hsCentrobin-mEGFP focus. t = 0 (mm:ss) is set at PBII cytokinesis; panels show single confocal sections. Bar, 2 µm. (B) Quantification of the timing of microtubule nucleating activity loss and of centriole elimination. Top bars indicate the mean times and SDs; bottom bars show data for all individual oocytes imaged. (C) An oocyte expressing pmPoc1-mEGFP and hsEB3-mCherry3 was centrifuged to spatially separate spindle and centrioles. Centrioles were then imaged from MI to the end of meiosis (see Video 7). Maximum intensity z-projections of selected frames are shown. Time in mm:ss; scale bar: 10 µm. (D) Quantification of hsEB3-mCherry3 intensities of individual centrioles. Background-corrected mean fluorescence intensities were measured in a sphere 3 µm in diameter and plotted over time for the oocyte shown in (C). Mother centrioles were identified by their attachment to the plasma membrane. (E) Oocytes expressing hsEB3-mCherry3 and pmOdf2-mEGFP were fertilized and then treated with latB at MII (one mother centriole retained; see Video 9). Top panels show the major events of the process (maximum intensity projections). The centrioles are monitored by hsEB3-mCherry3. The green square in the first frame shows the initial position of the mother centriole identified by pmOdf2-mEGFP (green) and hsEB3-mCherry3 (magenta) at the time of latB addition. t = 0 is set at anaphase II onset. Blue arrowheads indicate sperm, green and pink arrowheads the maternal mother and daughter asters, respectively. The blue and white dashed lines delineate the zygotic nucleus after pronuclear fusion and the cell membrane, respectively. Bottom panels show a higher magnification of a single confocal section (every 2.35 min, the daughter centriole lost activity and therefore could not be followed after the fourth time point as indicated by gray squares). Time is in mm:ss. Bars: (top) 10 µm; (bottom) 5 µm. (F) The left panel shows a control oocyte treated with DMSO and fertilized (see Video 8); the middle panel shows a fertilized oocyte treated with latB at MII (one mother retained, see Video 9); the right panel shows a fertilized oocyte after latB treatment at MI (two mothers retained, see Video 10). For each case, a frame at metaphase of the first zygotic mitosis is shown (maximum intensity projections). On the corresponding schematics on the right, maternal mother and daughter centrioles are shown in green and in pink, respectively. Maternal daughter centrioles after elimination are shown with dashed pink lines. Sperm centrioles are shown in blue and the new generation of zygotic daughter centrioles in violet. The same color code is used for the arrowheads marking centrioles on the images. Bar, 10 µm.

References [+] :

Albertson, Segregation of holocentric chromosomes at meiosis in the nematode, Caenorhabditis elegans. 1993, Pubmed