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. 2020 Jan 24;23(1):100749.
doi: 10.1016/j.isci.2019.100749. Epub 2019 Nov 28.

The Transmembrane Protein Semi1 Positions Gamete Nuclei for Reciprocal Fertilization in Tetrahymena

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Free PMC article

The Transmembrane Protein Semi1 Positions Gamete Nuclei for Reciprocal Fertilization in Tetrahymena

Takahiko Akematsu et al. iScience. .
Free PMC article

Abstract

During sexual reproduction in the ciliate, Tetrahymena thermophila, cells of complementary mating type pair ("conjugate") undergo simultaneous meiosis and fertilize each other. In both mating partners only one of the four meiotic products is "selected" to escape autophagy, and this nucleus divides mitotically to produce two pronuclei. The migrating pronucleus of one cell translocates to the mating partner and fuses with its stationary pronucleus and vice versa. Selection of the designated gametic nucleus was thought to depend on its position within the cell because it always attaches to the junction with the partner cell. Here we show that a transmembrane protein, Semi1, is crucial for attachment. Loss of Semi1 causes failure to attach and consequent infertility. However, a nucleus is selected and gives rise to pronuclei regardless of Semi1 expression, indicating that attachment of a nucleus to the junction is not a precondition for selection but follows the selection process.

Keywords: Developmental Genetics; Genetics; Molecular Biology.

Conflict of interest statement

Declaration of Interests The authors declare no competing interests.

Figures

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Figure 1
Figure 1
Semi1 Mediates hMIC Attachment to the Conjugation Junction (A) Timeline of conjugation in WT (left) and semi1Δ (right) cells (see also Figures S1C and S1D) stained with DAPI, shown as fluorescence microscopy images and schematic diagrams. h: hMICs; arrows: selected hMICs undergoing gametogenic mitosis; †: degenerating unselected hMICs; arrowhead: gametic pronucleus; $: progeny MACs; #: progeny MIC. Dotted line: conjugation junction. (B) Image (left) and diagram (right) of vegetative division of a semi1Δ exconjugant, in which unexchanged gametic pronuclei are maintained (see also Figure S1E). Scale bar: 10 μm. (C) Rescue of the semi1Δ phenotype by semi1-mCherry expression. The pmCherry-SEMI1-PAC plasmid, containing a puromycin resistance marker (PAC), cadmium-inducible MTT1 promoter, and mCherry-Semi1-expression cassette, was integrated into the MAC BTU1 locus by homologous recombination. The Western blot shows that mCherry-Semi1 expression is induced by the addition of cadmium. Tubulin ɑ was the loading control. (D) Percentage of cells with normal hMIC attachment at 6 h after the initiation of conjugation (see also Figure S2) and development of progeny nuclei at 10 h. Columns and error bars represent the means and standard deviations of three independent experiments. Asterisk (*) shows a significant difference between means (p < 0.01 as calculated by Tukey's honestly significant difference [HSD] test on RStudio).
Figure 2
Figure 2
Live Cell Imaging of mCherry-Semi1 Localization (A) mCherry-Semi1 is localized to the hMIC selected for gametogenic mitosis. The bright spheres in the cytoplasm are digestive vacuoles probably incorporating overexpressed or unfolded mCherry-Semi1. (B) mCherry-Semi1 and GFP-Nup3 co-localize at the periphery of the selected hMIC in live cells. h: hMIC; arrow: selected hMIC undergoing gametogenic mitosis; †: degenerating unselected hMIC; yellow arrowheads: gametic pronucleus; magenta arrowhead: fertilized nucleus; dotted line: conjugation junction. Scale bars: 10 μm.
Figure 3
Figure 3
DNA Repair Markers Indicate that hMIC Selection Occurs without hMIC Attachment in semi1Δ Cells (A) γH2AX foci are formed in all four hMICs of both WT and semi1Δ cells but are lost in only one hMIC (arrow), concomitant with histone H3 acetylation at lysine 56 (H3K56ac). (B) Localization of EGFP-DNAPKcs in a single hMIC (arrow) in both WT and semi1Δ cells at 6 h after the initiation of conjugation. (C) Localization of Rad51 in an hMIC (arrow). (D) The position of the selected hMIC (arrow) and unselected hMICs (†) in semi1Δ cells expressing EGFP-DNAPKcs was determined using a 9 × 6 grid. (E) Heatmap showing the cytoplasmic distribution of selected hMICs. (F) Heatmap showing the cytoplasmic distribution of unselected hMIC. The heatmaps were based on data from 17 cells with a clearly defined selected MIC and 17 cells with four unselected MICs. h: hMIC; dotted line: conjugation junction; arrow: selected hMICs undergoing gametogenic mitosis; †: degenerating unselected hMIC. Scale bars: 10 μm.
Figure 4
Figure 4
Semi1 Acts on the MAC in the Absence of hMIC Selection (A) Localization of Semi1 in a spo11Δ strain, which is defective in hMIC selection. †: degenerating unselected hMIC; arrowhead: MAC bearing mCherry-Semi1; arrows: MAC elongating toward the conjugation junction. (B) In spo11Δ cells, MAC attachment to the conjugation junction occurs between 6 h and 12 h after the initiation of conjugation. (C) Left, percentage of cells showing MAC attachment at 12 h after the initiation of conjugation. Columns and error bars represent the means and standard deviations of three independent experiments. Asterisk (*) shows a significant difference (p < 0.01, as calculated by Tukey's HSD test on RStudio). Right, examples of conjugating spo11Δ semi1i (uninduced) and spo11Δ semi1i (induced) cells (see also Figure S3). Arrow: selected hMIC undergoing gametogenic mitosis; †: degenerating unselected hMIC; arrowhead: gametic pronucleus; dotted line: conjugation junction. Scale bars: 10 μm.
Figure 5
Figure 5
Zfr3-mediated Gametic Pronuclear Exchange Is Dependent on SEMI1 (A) Conjugating zfr3i cells (see also Figure S5) stained with DAPI. Top row, uninduced cells; bottom row, zfr3 RNAi induced by CdCl2. (B) Percentage of cells with normal development of progeny nuclei at 10 h after the initiation of conjugation. Columns and error bars represent the means and standard deviations of three independent experiments. Asterisk (*) shows a significant difference (p < 0.01, as calculated by Tukey's HSD test on RStudio). (C) zfr3i does not affect hMIC attachment to the conjugation junction. The rim of the selected hMIC was visualized by mCherry-Nup93. (D) EdU (red) was incorporated into the MIC (left cell) for monitoring MIC exchange. hMICs remain in the labeled cell at 5 h after the induction of meiosis. EdU labeling is seen in both cells at 10 h in the WT and is restricted to the labeled cell in the semi1Δ and zfr3i genotypes. (E and F) (E) Pronuclear arrest phenotype of zfr3i cells. Different colors in the nuclei denote different genetic compositions as shown in Figure 1A. (F) Localization of Zfr3 in WT cells. (G) Localization of Zfr3 in spo11Δ cells. White arrow: MAC elongating toward the conjugation junction. (H) Zfr3 does not localize to the selected hMIC in semi1Δ cells. (I) Zfr3 does not localize to the MAC in spo11Δ semi1i cells. (J) Semi1 localizes to the selected hMIC in zfr3i cells. (K) Semi1 localizes to the MAC in spo11Δ zfr3i cells. (F–K) Bottom: merged image. h: hMIC; yellow arrow: selected hMIC; †: degenerating unselected hMIC; arrowhead: gametic pronucleus; $: progeny MAC; #: progeny MIC; dotted line: conjugation junction. Scale bar: 10 μm.
Figure 6
Figure 6
Model of Semi1 Recruitment of the Selected hMIC to the Conjugation Junction Once an hMIC is selected at random (see also Figure S6), Semi1 binds to its rim. Semi1 recruits the selected hMIC to the conjugation junction to enable hMIC attachment to occur in a microtubule-independent manner. Semi1 also recruits Zfr3 to the surface of the selected hMIC. Zfr3 may interact with microtubules at the conjugation junction, and the Semi1–Zfr3 complex is responsible for pronuclear exchange.

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