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First published online June 8, 2005
doi: 10.1242/10.1242/jcs.02402

Journal of Cell Science 118, 2755-2762 (2005)
Published by The Company of Biologists 2005
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Two novel proteins recruited by synaptonemal complex protein 1 (SYCP1) are at the centre of meiosis

Yael Costa1, Robert Speed1, Rupert Öllinger2, Manfred Alsheimer2, Colin A. Semple1, Philippe Gautier1, Klio Maratou1,*, Ivana Novak3, Christer Höög3, Ricardo Benavente2 and Howard J. Cooke1,{ddagger}

1 MRC Human Genetics Unit, Western General Hospital, Crewe Rd, Edinburgh, EH4 2XU, UK
2 Department of Cell and Developmental Biology, Biocenter of the University of Würzburg, 97074 Würzburg, Germany
3 Center for Genomics and Bioinformatics (CGB) and Department of Cell and Molecular Biology (CMB), Karolinska Institutet, Berzelius väg 35, S-171 77 Stockholm, Sweden



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  		Fig. 1. Tissue- and cell-specific expression pattern of SYCE1 and CESC1. (A) A multiple-tissue RNA blot was hybridised sequentially with Syce1, Cesc1 and ribosomal protein S26 (Vincent et al., 1993Go) probes. The signals for Syce1 and Cesc1 are at the sizes predicted for these mRNAs based on sequence databases, and are restricted to testis at this level of detection. (B-M) Frozen testis sections stained with affinity-purified anti-SYCE1 antibody (B,C), affinity-purified anti-SYCE1 antibody competed with immunizing peptide (D), anti-CESC1 IgG peak (H,I) and anti-CESC1 IgG peak competed with GST-CESC1 fusion protein (J). SYCE1 and CESC1 specifically localise to meiotic cells and in particular, to the synaptonemal complex. The same sections were stained for DNA with Hoechst 33258 (E-G, K-M). Bars are 50 µm (B,D,E,G,H,J,K,M) and 10 µm (C,F,I,L).

 


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  		Fig. 2. SYCE1 and CESC1 localise to the synapsed areas of the homologous chromosomes in mouse spermatocytes. Meiotic spread preparations from adult males were stained with anti-STAG3 (green) and anti-SYCE1 (red), anti-STAG3 (green) and anti-CESC1 (red) or anti-SYCP1 (green) and anti-SYCE1 (red). (A-C) Pachytene cell with STAG3 (A), SYCE1 (B) and merged antibody signals and DAPI staining of DNA (C). (D-F) Diplotene cell with STAG3 (D), SYCE1 (E) and merged antibody signals and DAPI staining of DNA (F). Insets show lack of staining with SYCE1 (arrowheads) of unsynapsed axes. (G-I) Diplotene cell with SYCP1 (G) SYCE1 (H) and merged antibody signals and DAPI staining of DNA (I). (J-L) Pachytene cell with STAG3 (J) CESC1 (K) and merged antibody signals and DAPI DNA staining (L). (M-O) Diplotene cell with STAG3 (M) CESC1 (N) and merged antibody signals and DAPI DNA staining (O). Insets show lack of staining with CESC1 (arrowheads) of unsynapsed axes. (P,Q) Electron microscopy images show the localisation of SYCE1 and CESC1 in relation to the central element (CE) using immuno-gold-labelled anti-SYCE1 and anti-CESC1 antibodies and electron microscopy. Bars, 7.5 µm (A-O) and 0.1 µm (P,Q).

 


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  		Fig. 3. SYCE1 and CESC1 interact with SYCP1, with each other and themselves. (A) Co-immunoprecipitation of SYCE1 with SYCP1. Testis extracts were incubated with affinity-purified anti-SYCP1, with pre-immune serum or with anti-SYCP1 and 50 ng of immunizing peptide. Complexes were recovered with protein G sepharose and analysed by western blots using anti-SYCE1 primary antibody. (B) Interaction of SYCE1 and CESC1 with SYCP1. 35S-labelled protein corresponding to the N-terminal 200 aa of SYCP1 was incubated with glutathione sepharose beads with bound glutathione-S-transferase (GST), CESC1-GST or SYCE1-GST as indicated. In contrast to GST alone, the GST fusion proteins were able to bind the N-terminal region of SYCP1. (C) SYCE1 and CESC1 homotypic and heterotypic interactions. 35S-labelled SYCE1 or CESC1 were allowed to interact with sepharose beads bound to GST, SYCE1-GST or CESC1-GST fusion proteins. CESC1 interacts with SYCE1 and with itself; SYCE1 is also able to interact with itself.

 


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  		Fig. 4. SYCE1 and CESC1 are recruited by SYCP1 in a heterologous system. (A) SYCE1 expression is both punctate and diffuse in the absence of SYCP1. (B-D) SYCE1 (B) colocalises with SYCP1 (C) to fibres and foci when coexpressed. (D) Merged antibody signals and DAPI staining (blue). (E) CESC1 has a diffuse localisation when overexpressed in COS-7 cells (F-H) Coexpressed with SYCP1 (G), CESC1 (F) localises to cytoplasmic fibres. (H) Merged antibody signals and DAPI staining (blue). Bars, 10 µm.

 


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  		Fig. 5. SYCE1 and CESC1 reproduce SYCP1 localisation in Sycp3–/– spermatocytes. (A-C) SYCE1 (B) distribution in mutant spermatocytes show the same altered distribution as SYCP1 (A), displaying gaps (arrowheads) along the chromosome axes. (C) Merged images of SYCE1 and SYCP1 signals. (D-F) CESC1 (E) also exhibits gaps (arrowheads) along chromosome axis, colocalising fully with SYCP1 (D). (F) Merged images of CESC1 and SYCP1 signals. Insets show gaps resulting from the absence of SYCP1, SYCE1 and CESC1.

 


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  		Fig. 6. Model for the distribution of SYCE1 and CESC1 within the synaptonemal complex. Dimers or multimers of CESC1 and SYCE1 associate with the N-terminal region of SYCP1. This interaction could confer additional robustness and flexibility to the SC, important for the development of recombination events. C, carboxyl-terminus; CE, central element; LE, lateral element; N, amino-terminus.

 

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© The Company of Biologists Ltd 2005