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First published online 29 August 2006
doi: 10.1242/jcs.03155

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The Arabidopsis SKP1 homolog ASK1 controls meiotic chromosome remodeling and release of chromatin from the nuclear membrane and nucleolus

¹ Department of Chemistry and Biochemistry, Miami University, Oxford, OH 45056, USA
² Department of Biology and the Huck Institutes of the Life Sciences, The Pennsylvania State University, University Park, PA 16802, USA
³ Department of Gene Technology, Tallinn University of Technology, Akadeemia tee 15, 19086 Tallinn, Estonia

View larger version (82K): [in a new window]   Fig. 1. Alterations in nucleolar structure of ask1-1 plants are observed in intact male meiocytes. (a) Prophase I spreads of meiotic chromosomes in wild-type (A-D) and ask1-1 (E-H) cells prepared using Carnoy fixation with enzymatic digestion and acid clearing. (A,E) interphase; (B,F) leptotene; (C,G) zygotene; (D,H) pachytene. Alterations in the association of chromatin with the nucleolus and the nuclear membrane are not observed. (b) Meiotic chromosome analysis in wild-type (A-D and I-L) and ask1-1 (E-H and M-P) cells fixed in parafomaldehyde and lightly squashed without additional treatment. (A,E) interphase; (B,C,F,G) leptotene; (D,H,I,M) zygotene; (J,N) pachytene; (K,L,O,P) diakinesis. Large arrows indicate the position of the nucleolus and nucleolus-associated chromatin in ask1-1. Small arrow points to the attachment of chromatin to the nuclear envelope in ask1-1. Meiotic stage assignment for ask1-1 meiocytes is based on chromatin structure and the stage of the surrounding tapetal cells (Wang et al., 2004). Bars, 10 µm.

View larger version (107K): [in a new window]   Fig. 2. Electron micrographs of Arabidopsis meiocytes showing chromosomes at different stages of prophase I. (A-D) Wild type. (A) Interphase; (B) laptotene/zygotene, note most chromosomes are resolved away from and to one side of the nucleolus; (C) pachytene; (D) diplotene/diakinesis. (E-H) ask1-1. (E) Interphase; (F) zygotene, showing the close association of unsynapsed chromosome ends or rDNA with the nucleolus; (G) pachytene; (H) diplotene. Note the multiple attachment sites of chromatin to the nuclear envelope (small arrows) and the nucleolus (large arrows). Nu, nucleolus. Bar, 1 µm.

View larger version (147K): [in a new window]   Fig. 3. Alterations in rDNA structure in meiocytes of ask1-1 plants. FISH using an rDNA probe on meiotic chromosomes in wild-type (A-D,I-L) and ask1-1 (E-H,M-P) plants. Merged photos of biotin-labeled rDNA signals (green) on chromosomes counterstained with DAPI (red) are shown. (A,E) Early leptotene; (B,F) zygotene; (C,G) pachytene; (D,H) diakinesis; (I,M) premetaphase; (J,N) metaphase I/early anaphase I; (K,O) telophase I; (L,P) metaphase II. Arrow shows the single rDNA region. Eleven chromosomes are observed suggesting the breakage of rDNA from chromosomes II and IV. Assignment of meiotic stages in ask1-1 is based on chromosome morphology and the stage of the surrounding tapetal cells (Wang et al., 2004). Bar, 10 µm.

View larger version (109K): [in a new window]   Fig. 4. Telomeres exhibit a prolonged attachment to the nucleolus in the meiocytes of ask1-1 plants. (a) Telomere FISH on meiotic chromosomes in wild-type (A-D) and ask1-1 (E-H) cells prepared using standard fixation techniques and chromosome spreading. (A,E) Interphase; (B,F) leptotene; (C,G) zygotene; (D,H) pachytene. No significant differences are observed in the distribution of telomere signals. (b) Telomere FISH on meiotic chromosomes in paraformaldehyde-fixed wild-type (A-D) and ask1-1 (E-H) cells. Arrows indicate the position of the nucleolus. (A,E) Interphase; (B,F) leptotene; (C,G) zygotene; (D,H) pachytene. Note the persistent association of telomere signals with the nucleolus in ask1-1 meiocytes but not in wild-type meiocytes. Assignment of meiotic stages in ask1-1 is based on chromosome morphology and the stage of the surrounding tapetal cells. Bars, 10 µm.

View larger version (57K): [in a new window]   Fig. 5. Immunolocalization of modified histones during meiosis. Merged photos of anti-histone antibody (green) and meiotic chromosomes (red) are shown. Assignment of meiotic stages in ask1-1 meiocytes is based on chromosome morphology and the stage of the surrounding tapetal cells. (a) Immunolocalization of dimethyl-histone H3 lysine 4 in wild-type meiocytes. (A) interphase; (B) leptotene/zygotene transition; (C) pachytene. (D) metaphase I. Arrow indicates weak signal at pachytene. ask1-1 meiocytes exhibited similar labeling patterns (data not shown). (b) Immunolocalization of acetylated histone 3 (positions 9 and 14) in wild-type (A-C,G-I) and ask1-1 (D-F,J-L) meiocytes. (A,D) Interphase; (B,E) leptotene; (C,F) zygotene; (G,J) pachytene; (H,K) metaphase I; (I,L) telophase I. (c) Immunolocalization of dimethyl-histone 3 lysine 9 in wild-type (A-C) and ask1-1 (D-F) meiocytes. (A,D) Interphase/early leptotene; (B,E) zygotene; (C,F) pachytene. Note the nucleolus in ask1-1 is also stained with DAPI (red) at later stages, suggesting the persistent association of chromatin with nucleolus in the mutant. Nu, nucleolus. Bars, 10 µm.

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