S U P P L E M E N TA R Y I N F O R M AT I O N DOI: 10.1038/ncb2896 Supplementary Figure 1 Supplementary Figure 1. Sequence alignment of TERB1 homologs in vertebrates. M. musculus TERB1 was derived from our own sequencing of the cdna (AB775896). R. norvegicus (XP_226205.5), H. sapiens (NP_001129977.1), P. troglodytes (XP_001159574.2), C. lupus (XP_536822.3), G. gallus (XP_001232198.2) and X. tropicalis (XP_002933719.1) data are from the NCBI protein database. WWW.NATURE.COM/NATURECELLBIOLOGY 1
GFP DNA 5 0:00 0:30 1:00 1:30 2:00 2:30 3:00 6 7 8 9 DNA 0:00 0:30 1:00 1:30 2:00 2:30 3:00 5 6 7 8 9 Supplementary Figure 2 3D tracing of individual telomeres and heterochromatin by live imaging. Individual telomeres (arrowheads) and heterochromatin (asterisks) were traced for 3 min with 3D information (from sections 5 to 9) to evaluate the relative movements and distances shown in Fig. 2c. Bar, 5 μm. 2 WWW.NATURE.COM/NATURECELLBIOLOGY
a SUN2 -Actin b SUN2 Spermatocytes DNA SUN2 Mitotic cell Mitotic cell Spermatocytes * Haploid cells Supplementary Figure 3 SUN1 but not SUN2 associations with TERB1. a, Immunoprecipitates from mouse testis-chromatin extracts using TERB1 antibody or control IgG (same IP sample as Figure.6e) were analyzed by the indicated antibodies. SUN1, but not SUN2, associated with TERB1. b, Wild type testis suspension stained for SCP3 (red), SUN2 (green) and DNA (blue). SUN2 signals are detected at the NE in mitotic cells, but is absent in spermatocytes and haploid cells. Asterisks indicate haploid cells. Uncropped images of blots are shown in Supplementary Fig. 6. Bar, 15 μm. WWW.NATURE.COM/NATURECELLBIOLOGY 3
WT GFP- GFP- WT GFP-TREB1 GFP- Projection Projection Equator Equator Supplementary Figure 4 GFP-SUN1 and -TERB1 localizations at meiotic telomeres in wild spermatocytes. The projected and equator images of wild type spermatocytes expressing GFP-SUN1 or -TERB1 stained for SCP3 (red), GFP (green) and TRF1 (blue). Note that overexpressed GFP-SUN1 associates with the NE with telomere accumulations, while GFP-TERB1 exclusively accumulates at telomeres. Bar, 5 μm. 4 WWW.NATURE.COM/NATURECELLBIOLOGY
a MBP- HIS- MBP pull down MBP MBP- MBP- b MBP-puri cation MBP-TRFB HIS- 1 2 3 4 HIS- MBP Supplementary Figure 5 Characterization of the TERB1-TRF1 heterocomplex. a, Purifications of MBP-TERB1, HIS-TRF1 and heterocomplex from E. coli extracts. Note, MBP-TERB1 form a heterocomplex with TRF1 when they are co-expressed in the same E. coli cells. b, Purifications of MBP-TERB1-TRFB domain and HIS-TRF1 from E. coli extracts. While full length MBP-TERB1 is degraded and the stoichiometry with HIS-TRF1 is difficult to be assessed (panel a), MBP-TERB1-TRFB doesn t show any degradation band and clearly showed 1:1 stoichiometric binding with HIS-TRF1. WWW.NATURE.COM/NATURECELLBIOLOGY 5
Fig. 1c -Tubulin Fig. 6e and Fig. S3a Intensi ed SA3 SMC3 25 25 RAD21L Fig. 5b Intensi ed REC8 KASH5 SA3 SUN2 -Actin 25 -Actin Supplementary Figure 6 Entire membranes of cropped immunoblots. Blue labels on top indicate the antibody used for detection. Dashed red boxes show which region of the immunoblot were cropped for the individual figures. 6 WWW.NATURE.COM/NATURECELLBIOLOGY
Supplementary Table 1. Statistics source data for figures 5, 6 and 7 Supplementary Video Legends Supplementary Video 1. Live imaging of wild type spermatocytes. Wild type spermatocytes co-expressing GFP-TRF1 and GFP-SCP3 were cultured in medium supplemented with Hoechst 33324. Images were collected using a DeltaVision microscopy system. Exposures of 0.15 sec (for GFP) and 0.025 sec (for Hoechst 33324) were acquired every 30 sec for 5 min. Supplementary Video 2. Live imaging of wild type spermatocytes in the presence of nocodazole. Wild type spermatocytes co-expressing GFP-TRF1 and GFP- SCP3 were cultured in medium supplemented with Hoechst 33324 and nocodazole. Images were collected using a DeltaVision microscopy system. Exposures of 0.15 sec (for GFP) and 0.025 sec (for Hoechst 33324) were acquired every 30 sec for 5 min. Supplementary Video 3. Live imaging of TERB1 -/- spermatocytes. TERB1 -/- spermatocytes co-expressing GFP-TRF1 and GFP-SCP3 were cultured in medium supplemented with Hoechst 33324. Images were collected using a DeltaVision microscopy system. Exposures of 0.15 sec (for GFP) and 0.025 sec (for Hoechst 33324) were acquired every 30 sec for 5 min. Supplementary Video 4. Live imaging of SUN1 -/- spermatocytes. SUN1 -/- spermatocytes co-expressing GFP-TRF1 and GFP-SCP3 were cultured in medium supplemented with Hoechst 33324. Images were collected using a DeltaVision microscopy system. Exposures of 0.15 sec (for GFP) and 0.025 sec (for Hoechst 33324) were acquired every 30 sec for 5 min. WWW.NATURE.COM/NATURECELLBIOLOGY 7