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First published online 21 September 2005
doi: 10.1242/jcs.02583

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AtREC8 and AtSCC3 are essential to the monopolar orientation of the kinetochores during meiosis

Liudmila Chelysheva, Stéphanie Diallo^*, Daniel Vezon, Ghislaine Gendrot, Nathalie Vrielynck, Katia Belcram, Nathalie Rocques, Angustias Márquez-Lema, Anuj M. Bhatt, Christine Horlow, Raphaël Mercier, Christine Mézard and Mathilde Grelon^¶

Institut Jean-Pierre Bourgin, Station de Génétique et d'Amélioration des Plantes, INRA de Versailles, Route de Saint-Cyr, 78026 Versailles CEDEX, France

View larger version (76K): [in a new window]   Fig. 1. Atscc3 mutant phenotypes. (A,B) Siliques from wild-type (A) or heterozygous Atscc3-2+/– plants after clearing. (C-E) Comparison of wild-type (Wt) and homozygous mutant Atscc3-1–/– plants after 21 days (C) or 40 days (D,E) in the greenhouse. Bar, 1 cm.

View larger version (103K): [in a new window]   Fig. 2. Male sporogenesis and meiosis of wild-type (A,B,E-I) and the Atscc3-1 mutant (C,D,J-S). (A,D) DIC microscopy of male meiocytes (A,C) and of the products of male meiosis (tetrads of microspores, B and D). (E-S) DAPI staining of wild-type and mutant pollen mother cells during meiosis. (E) Pachytene, (F) diakinesis, (G) metaphase I, (H) anaphase I, and (I) end of anaphase II in wild-type meiocytes. The same stages are shown in Atscc3-1 mutant pollen mother cells: (J,O) pachytene, (K) diakinesis, (P,L,Q) metaphase I, (M,R) anaphase I and (N,S) anaphase II. Asterisks indicate pericentromeric heterochromatin during pachytene stages (E,J,O). Arrows indicate some of the abnormalities observed in the Atscc3-1 mutant: synapsis defects at pachytene (J,O), chromosome bridges (M) and chromosome fragmentation (R). Bar, 10 µm.

View larger version (48K): [in a new window]   Fig. 3. Immunolocalisation of ASY1 and AtSCC3 in wild-type and Atscc3-1 mutant cells. (A-AD) Male meiotic cells. (AE-AG) A wild-type vegetative cell, easily distinguishable from meiocytes based on its size and small nucleolus. (A,B,C) Early interphase meiocyte, probably in G1 as ASY1 labelling is very faint. (D,E,F) Interphase meiocyte, probably in G2 according to ASY1 staining, which is strong. (G,H,I) Leptotene; (J,K,L) zygotene; (M,N,O) pachytene; (P,Q,R) diakinesis; (S-X) metaphase I; (Y-AA) zygotene; (AB-AD) pachytene. Arrows in S and V indicate centromeres, and arrows in W and X indicate the AtSCC3 staining corresponding to chromosome arms. For each cell, several stains are shown: anti-ASY1 (in red), anti-SCC3 (in green), and in blue, the DAPI staining of chromatin (DAPI). For diakinesis and metaphase I cells, no ASY1 staining is shown, but an overlay of DAPI and AtSCC3 staining is shown (merge). Bar, 10 µm.

View larger version (83K): [in a new window]   Fig. 4. Immunolocalisation of ASY1 and RAD51 in wild-type and Atscc3-1 pollen mother cells. (A-H) Wild-type cells (Wt). (I-P) Atscc3-1 mutant cells. An overlay of the two signals is shown (merge), together with DAPI staining of the corresponding cell (DAPI). Bar, 10 µm.

View larger version (55K): [in a new window]   Fig. 5. DAPI staining of male meiocytes at metaphase-anaphase transition and during anaphase I. Several genotypes are shown: wild-type (Wt) (A,B), Atspo11-1-1 (C,D), Atrec8 (E,F), Atscc3-1 (G,H), double mutants Atspo11-1-1Atrec8 (I-J) and Atspo11-1-1Atscc3-1 (K,L) at metaphase-anaphase transition (left-hand column) and during anaphase I (right-hand column). Bar, 10 µm.

View larger version (83K): [in a new window]   Fig. 6. Immunolocalisation of ASY1, AtSCC3 or AtREC8 in meiocytes from wild-type (Wt), Atscc3-1 and Atrec8 plants. ASY1 (anti-ASY1) in red, and in green either AtREC8 (anti-REC8) or AtSCC3 (anti-SCC3). DAPI staining of chromatin (DAPI) is blue. Bar, 10 µm.

View larger version (62K): [in a new window]   Fig. 7. Immunolocalisation of ASY1, AtSCC3 or AtREC8 in meiocytes from double mutants Atspo11-1-1Atrec8, Atspo11-1-1Atscc3-1 or single Atspo11-1-1 mutant plants. In red, immunolocalisation of ASY1 (anti-ASY1); in green, immunolocalisation of AtREC8 (anti-REC8) or AtSCC3 (anti-SCC3); and in blue, the DAPI staining of chromatin (DAPI). Bar, 10 µm.