Pierron M et al. (2020), Centriole foci persist in starfish oocytes desp...

ECB-ART-48570

Mol Biol Cell 2020 Apr 15;319:873-880. doi: 10.1091/mbc.E19-06-0346.

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Centriole foci persist in starfish oocytes despite Polo-like kinase 1 inactivation or loss of microtubule nucleation activity.

Pierron M , Kalbfuss N , Borrego-Pinto J , Lénárt P , Gönczy P .

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Centrioles must be eliminated or inactivated from the oocyte to ensure that only the two functional centrioles contributed by the sperm are present in the zygote. Such removal can occur during oogenesis, as in Drosophila, where departure of Polo kinase from centrosomes leads to loss of microtubule nucleating activity and centriole removal. In other species, oocyte-derived centrioles are removed around the time of fertilization through incompletely understood mechanisms. Here, we use confocal imaging of live starfish oocytes and zygotes expressing markers of microtubule nucleating activity and centrioles to investigate this question. We first assay the role of Polo-like kinase 1 (Plk1) in centriole elimination. We find that although Plk1 localizes around oocyte-derived centrioles, kinase impairment with BI-2536 does not protect centrioles from removal in the bat star Patiria miniata. Moreover, we uncover that all four oocyte-derived centrioles lose microtubule nucleating activity when retained experimentally in the zygote of the radiate star Asterias forbesi. Interestingly, two such centrioles nevertheless retain the centriolar markers mEGFP::PACT and pmPoc1::mEGFP. Together, these findings indicate that centrioles can persist when Plk1 activity is impaired, as well as when microtubule nucleating activity is lacking, uncovering further diversity in the mechanisms governing centriole removal.

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Genes referenced: plk1

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	FIGURE 1:. Centriole behavior and Plk1 distribution in P. miniata oocytes. (A, B) Schematic of centriole fate during meiotic divisions of P. miniata starfish oocyte, together with overview of experimental procedure utilized in this work, not to scale; B shows higher magnification views of the region with meiotic spindles. Arrested oocytes are injected with mRNA(s) coding for the protein(s) of interest; meiotic resumption is induced by 1-methyladenine (1-MA), followed by fertilization, depending on the experiment, and then by confocal time-lapse microscopy. During the first meiotic division, pairs of centrioles, each containing a mother centriole (dark green, bearing appendages) and a daughter centriole (light green), are present at the poles of the spindle (B, meiosis I, metaphase represented). First polar body (PBI) extrusion results in the removal of 2n DNA and of a pair of centrioles from the oocyte (B, meiosis II onset). The two remaining centrioles then disengage from one another and their surrounding PCM drives the formation of the meiosis II spindle (B, meiosis II, metaphase represented). The mother centriole is invariably positioned toward the plasma membrane and thus is extruded in the second polar body (PBII), together with 1n DNA (B, meiosis II exit). The remaining daughter centriole then loses MTOC activity and is eliminated (depicted as fading away in meiosis II exit panel). Fertilization results in the sperm contributing 1n DNA and a pair of centrioles (yellow) to the zygote. Sperm-derived centrioles then duplicate, leading to two centriole pairs that recruit PCM (dark gray) and govern bipolar spindle formation during the first mitosis (A, mitosis). (C) Still images from dual-color time-lapse confocal microscopy of P. miniata oocyte expressing mRNAs encoding the microtubule marker hsEB3::mCherry3 (in magenta throughout the paper) and mEGFP::pmPlk1 (green), which localizes at centrioles (insets) and kinetochores (arrows point to three of them at metaphase of meiosis I and II). Here and in other figures, images are maximum-intensity projections of selected z-planes spanning the region of interest, and insets are 1.4×-magnified single z-plane of the boxed regions; the oocyte plasma membrane is indicated with a dashed line. Moreover, unless stated otherwise, time is indicated in minutes:seconds starting from centriole disengagement at meiosis II onset and scale bars are 5 µm.
	FIGURE 2:. Plk1 inhibition does not provoke centriole elimination in P. miniata. (A–D) Still images from dual color time-lapse confocal microscopy of P. miniata oocytes expressing hsEB3::mCherry3 to mark microtubules and mEGFP::pmCentrin2 to mark centrioles, treated with either 0.1% DMSO as a control or 10 µM BI-2536 in 0.1% DMSO, both added simultaneously with 1-MA. A, B: noncentrifuged oocytes; note that gray levels were adjusted differently in A and B, which stem from independent experimental series. C, D: centrifuged oocytes. (E) Oocyte-derived centriole number (green) and MTOC activity (magenta) over time as monitored by mEGFP::pmCentrin2 foci and hsEB3::mCherry3 (magenta), respectively, in DMSO (C) or BI-2536-treated (D) centrifuged oocytes. Each line corresponds to one oocyte. Centriole number is indicated with different shades of green and different line thicknesses. Dark or light magenta lines indicate whether microtubules are focused around centrioles or diffuse, respectively. In BI-2536 oocytes, hsEB3::mCh3 is usually diffuse before disappearing completely (n = 5/10) or sometimes refocusing in a very limited area around centrioles (n = 5/10). Small gray discs: actual data points; gray circles: ambiguous data points, either because foci are out-of-focus or because foci disappear and reappear within three frames. Small vertical black marks indicate for each oocyte when the number of detected foci drops below 3, and vertical dashed lines the resulting average time of dropping below 3 for each condition (138 min for DMSO control, 120 min for BI-2536-treated). Time is indicated in hours:minutes:seconds after 1-MA addition, as meiosis II onset is difficult to discern in BI-2536 treated oocytes. In A, the parentheses around 1-MA denote the fact that timing in this particular oocyte is merely estimated from the actual timing of NEBD and the observation that NEBD typically occurs ∼30 min after 1-MA addition.
	FIGURE 3:. Mother centrioles are extruded into polar bodies in A. forbesi oocytes. Top: still images from time-lapse confocal microscopy of A. forbesi oocyte expressing the mother centriole marker pmOdf2::mEGFP. A bright focus is observed in each polar body. In some oocytes, as illustrated here, a weak focus can also be detected at the innermost centriole at the end of meiosis II. Bottom: corresponding schematic representation.
	FIGURE 4:. Oocyte-derived centrioles lose MTOC activity when retained in A. forbesi zygotes. (A) Still images from time-lapse confocal microscopy of A. forbesi zygote treated with Latrunculin B to retain all centrioles, monitoring MTOC activity using hsEB3::Cherry3 from meiosis II onset until mitosis. Here, as well as in Figure 5, oocyte-derived centrioles are bounded by squares and sperm-derived centrioles by circles. Note that hsEB3::mCherry3 is lost from the surroundings of all four oocyte-derived centrioles, whereas the two sperm-derived centrioles (S and then S1, S2) exhibit MTOC activity. Scale bar: 10 µm. (B) Single confocal z-planes corresponding to the zygote shown in A illustrating differential MTOC activity of oocyte-derived versus sperm-derived centrioles from meiosis II to mitosis. Each image is 3.73 × 3.73 µm.
	FIGURE 5:. Two retained foci of mEGFP::PACT persist until mitosis in A. forbesi. (A) Still images from dual-color time-lapse confocal microscopy of A. forbesi zygote treated with Latrunculin B to retain all centrioles, monitoring microtubules labeled with hsEB3::mCherry3 and centrioles marked with mEGFP::PACT from meiosis II onset until mitosis. Scale bar: 10 µm; see also Supplemental Movie S1. (B) Single confocal z-planes showing MTOC activity and the presence of mEGFP::PACT foci in oocyte-derived and sperm-derived centrioles from meiosis II until mitosis. Images boxed by a dashed line for centriole 1 indicate time frames when this centriole was slightly out of focus. Note that the little signal for hsEB3::mCherry3 of centriole 3 at time point 67:14 stems from sperm-derived MTOC activity. Each image is 3.98 × 3.98 µm. (C) Oocyte-derived centriole number over time as monitored by mEGFP::PACT foci (green) and MTOC activity as monitored by hsEB3::mCherry3 (magenta). Each line corresponds to one oocyte, with asterisks indicating mitosis onset. Centriole number is indicated with different shades of green and line thicknesses. Gray filled discs: data points; gray circles: ambiguous data points due to mEGFP::PACT not being yet detected at all centrioles, to foci being out-of-focus, or to the presence of multiple, probably spurious, foci. (D) Schematic representation of centriole fate in P. miniata based on Borrego-Pinto et al. (2016a) and A. forbesi zygotes treated with Latrunculin B. The same color code is used as in Figure 1. See text for details.

References [+] :

Archambault, Polo-like kinases: conservation and divergence in their functions and regulation. 2009, Pubmed