OSD1 promotes meiotic progression via APC/C inhibition and forms a regulatory network with TDM and CYCA1;2/TAM

Abstract

Cell cycle control is modified at meiosis compared to mitosis, because two divisions follow a single DNA replication event. Cyclin-dependent kinases (CDKs) promote progression through both meiosis and mitosis, and a central regulator of their activity is the APC/C (Anaphase Promoting Complex/Cyclosome) that is especially required for exit from mitosis. We have shown previously that OSD1 is involved in entry into both meiosis I and meiosis II in Arabidopsis thaliana; however, the molecular mechanism by which OSD1 controls these transitions has remained unclear. Here we show that OSD1 promotes meiotic progression through APC/C inhibition. Next, we explored the functional relationships between OSD1 and the genes known to control meiotic cell cycle transitions in Arabidopsis. Like osd1, cyca1;2/tam mutation leads to a premature exit from meiosis after the first division, while tdm mutants perform an aberrant third meiotic division after normal meiosis I and II. Remarkably, while tdm is epistatic to tam, osd1 is epistatic to tdm. We further show that the expression of a non-destructible CYCA1;2/TAM provokes, like tdm, the entry into a third meiotic division. Finally, we show that CYCA1;2/TAM forms an active complex with CDKA;1 that can phosphorylate OSD1 in vitro. We thus propose that a functional network composed of OSD1, CYCA1;2/TAM, and TDM controls three key steps of meiotic progression, in which OSD1 is a meiotic APC/C inhibitor.

Figure 1. Structural comparison of OSD1 and Mes1 proteins.

OSD1 and Mes1 share co-aligned putative APC/C interacting domains.

Figure 2. OSD1 interacts with CCS52A1 through its D-BOX and MR-tail.

(A) Yeast 2-hybrid experiments showed that among the 14 APC/C subunits tested, OSD1 interacts only with CCS52A1. (B) CDC20s and CCS52s TAP elutions probed with anti-OSD1 antibody. OSD1 is detected in all elutions but CDC20.3. The anti-CBP recognizes the Calmodulin Binding Protein stretch in the TAP tag and served as loading control. (C) OSD1 with mutation of a putative non-conserved D-BOX (OSD1ΔD') or mutation of its GxEN-box (OSD1ΔGxEN) still interacts with CCS52A1. In contrast, this interaction is abolished by the mutation of the conserved D-box (OSD1ΔD) and reduced by the deletion of the MR-tail (OSD1ΔMR).

Figure 3. Complementation test of osd1-3 by wild-type and mutated versions of OSD1.

Male meiotic products stained by toluidine blue: (A) a dyad of spores from the osd1-3 mutant. (B) A tetrad of spores from osd1 complemented by the wild type OSD1 gene. Note that one of the spores is out of focus because they are organized in a tetrahedron. (C to I) Male meiotic products from osd1-3 transformed by versions of the OSD1 gene with a GxEN-box mutation (OSD1ΔGxEN), a D-box mutation (OSD1ΔD), a MR-tail mutation (OSD1ΔMR) or combination of these mutations. Some versions induced complementation, with a majority of tetrads (E, H), while others did not restore tetrad formation (C, D, F, G, I). Scale bar = 10 µM.

Figure 4. Expression of OSD1 in mouse oocytes provokes a metaphase I arrest.

Germinal Vesicle (GV) stage mouse oocytes were injected with mRNA encoding the indicated OSD1 constructs. (A) Immunofluorescence on fixed oocytes in prometaphase I showing equal expression of the different OSD1 constructs with anti-OSD1 antibody. (B) Histone H2B-RFP encoding mRNA was injected together with the indicated OSD1 mRNA to follow chromosome movements. Oocytes were induced to enter the first meiotic division in a synchronized manner, and followed by live imaging. Shown are images from the DIC channel and collapsed images of 8 z-sections of 2 µm to visualize H2B-RFP labelled chromosomes at selected time points. (C) Chromosome spreads at the end of the movie. Chromosomes were stained with propidium iodide (red), and kinetochores with CREST serum (green). Only in OSD1 injected oocytes chromosomes have not been separated in meiosis I.

Figure 5. Meiotic chromosome spreads of wild type, osd1-3, tam-2, and tdm-3 single mutants.

(A to F) wild type. (A) pachytene, (B) diakinesis, (C) metaphase I, (D) telophase I, (E) metaphase II, (F) telophase II. (G to I) osd1-3. (G) Pachytene (H) metaphase I, (I) telophase I. (J to L) tam-2. (J) Pachytene, (K) Metaphase I, (L) telophase I. (M to R) tdm-3. (M) Pachytene (N) Metaphase I (O) metaphase II (P) telophase I (Q) aberrant third division (R) resulting telophase III. Scale bar = 10 µM.

Figure 6. Epistasis analysis between OSD1, TAM, and TDM.

Meiotic spreads of (A to C) osd1-3/tam-2 double mutant, (D to F) osd1-3/tdm-3 double mutant, (G to L) tam-2/tdm-3 double mutant and (M to O) osd1-3/tam-2/tdm-3 triple mutant. Scale bar = 10 µM.

Figure 7. CDKA;1 is activated by CYCA1;2/TAM and phosphorylates OSD1 in vitro.

HisMBP-CYCA1;2/TAM was co-expressed with StrepIII-CDKA;1 and GST-Cak1 in E. coli. HisMBP-CYCA1;2/TAM proteins were purified by means of a Ni-NTA column. HisMBP-OSD1 (Top) or Histone H1 (middle) were used as substrates in the kinase reaction. Coomassie blue staining of the gel shows equal loading of the respective substrate. StrepIII-CDKs co-purified with HisMBP-CYCA1;2/TAM were detected with strep-tactin HRP, showing identical amount of the kinases in each reaction (bottom).

Figure 8. TAMΔD provokes the entry into a third meiotic division.

(A to F) Meiotic spreads of wild type plants transformed by TAMΔD. (A) Pachytene. (B) Metaphase I. (C) Metaphase II. (D) late anaphase II (E) Aberrant third division (F) Resulting telophase III with seven nuclei. (G) Meiotic product stained by toluidine blue. Scale bar = 10 µM. (H) Alexander staining of an anther, showing the complete absence of pollen grains (Compare to Figure S5A). Scale bar = 100 µM.

Figure 9. Four spindles form at meiosis III in TAMΔD plants.

Immunolocalization of tubulin during meiosis. DNA appears in red and tubulin in green. (A and B) Wild type. One spindle is visible at metaphase I (C) and two at metaphase II (B). (A to C) tdm. After regular meiosis I (C) and meiosis II (D), tdm meiocytes enter a third division of meiosis with the formation of four spindles (E) . (F to H) Wild type transformed by TAMΔD. Like in tdm, TAMΔD meiocytes perform a third meiotic division with the formation of four spindles (H).

Figure 10. TAMΔD in osd1.

(A and B) Meiotic spreads of osd1-3 transformed by TAMΔD. (A) Metaphase I. (B) Telophase I. (C) Meiotic product stained by toluidine blue. Scale bar = 10 µM. (D) Alexander staining of an anther, showing the complete absence of pollen grains. Scale bar = 100 µM.

Figure 11. OSD1 and UVI4 are synthetically essential for female gametogenesis and somatic growth.

(A and B) Cleared female gametophyte. Nuclei and nucleoli have been artificially highlighted in blue and red, respectively. (A) Wild type at the 8 nuclei stage. A nucleolus is visible in the center of each nucleus (arrows) (B) A female gametophyte in the uvi4+/− osd1-2+/− plant at a comparable stage, showing a single giant nucleus with a massive nucleolus. (C to D) Double staining of female gametophytes with DAPI and propidium iodide. The DNA is stained in blue and the nucleoli appear in red. (C) Wild type at the 8 nuclei stage (arrows) (D) One female gametophyte in uvi4+/− osd1-2+/− containing a single large nucleus with a great amount of DNA, and a large nucleolus. Scale bar = 10 µM. (E) A 5 weeks old osd1/uvi4 double mutant. Scale bar = 5 mm.