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CENP-C and CENP-I are key connecting factors for kinetochore and CENP-A assembly.

Shono N, Ohzeki J, Otake K, Martins NM, Nagase T, Kimura H, Larionov V, Earnshaw WC, Masumoto H - J. Cell. Sci. (2015)

Bottom Line: We showed that these components work by recruiting CENP-C and subsequently recruiting M18BP1.Furthermore, we found that CENP-I can also recruit M18BP1 and, as a consequence, enhances M18BP1 assembly on centromeres in the downstream of CENP-C.Thus, we suggest that CENP-C and CENP-I are key factors connecting kinetochore to CENP-A assembly.

View Article: PubMed Central - PubMed

Affiliation: Laboratory of Cell Engineering, Department of Frontier Research, Kazusa DNA Research Institute, 2-6-7 Kazusa-Kamatari, Kisarazu, Chiba 292-0818, Japan Division of Biological Science, Graduate School of Science, Nagoya University, Furo-cho, Chikusa-ku, Nagoya, Aichi 464-8602, Japan.

No MeSH data available.


Related in: MedlinePlus

Centromere assembly of newly synthesized CENP-A requires CENP-C and is enhanced through CENP-I. (A) Representative images of HeLa-Int-03 cells transfected with control siRNA (siControl) or siRNA against CENP-C (siCENP-C), CENP-I (siCENP-i) or both, together with siRNA against MAD2 (siMAD2). Cells were stained with DAPI, anti-CENP-C (red) and anti-CENP-I (green) at 72 h after transfection. Scale bars: 5 μm. (B) Normalized CENP-C and CENP-I dot signals in each sample at 36 h and 72 h after siRNA transfection. ***P<0.001 (Mann–Whitney test). Results are mean±s.e.m. (n=39–48 cells). (C,E) Representative images of HeLa-Int-03 M18BP1–Halo cells (C) or HeLa-Int-03 SNAP–CENP-A cells (E) transfected with the indicated siRNAs. Cells were stained with DAPI, anti-α-tubulin (green) and the Halo-tag TMR Ligand (red) at 36 h after co-transfection (C). A schematic for the experiment in E is shown above the images. Pre-existing SNAP–CENP-A was quenched with block ligand until 9 h before fixation, and then newly synthesized SNAP–CENP-A was labeled with SNAP TMR Ligand before fixation at 36 h after siRNA transfection (E). Scale bars: 5 μm. (D,F) Normalized M18BP1–Halo (D) or normalized new CENP-A (F) dot signals in each sample. Asterisks indicate significant differences. *P<0.05; **P<0.01; ***P<0.001; n.s., not significant (Mann–Whitney test). Results are mean±s.e.m. (n=43–50 cells). (G) CENP-C and CENP-I coordinate centromere ‘function’ and ‘epigenetics’. Solid lines show recruitment activity observed in our present experiments and dashed lines show recruitment activity indicated previously. The CENP-C N-terminus assembles kinetochore through the Mis12 complex (function) and its C-terminus assembles newly synthesized CENP-A through M18BP1 (epigenetics). CENP-I recruited by the CENP-C domain II (amino acids 72–425) supports both pathways. Each class is also involved in maintenance of CENP-A assembly (see Discussion).
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JCS180786F8: Centromere assembly of newly synthesized CENP-A requires CENP-C and is enhanced through CENP-I. (A) Representative images of HeLa-Int-03 cells transfected with control siRNA (siControl) or siRNA against CENP-C (siCENP-C), CENP-I (siCENP-i) or both, together with siRNA against MAD2 (siMAD2). Cells were stained with DAPI, anti-CENP-C (red) and anti-CENP-I (green) at 72 h after transfection. Scale bars: 5 μm. (B) Normalized CENP-C and CENP-I dot signals in each sample at 36 h and 72 h after siRNA transfection. ***P<0.001 (Mann–Whitney test). Results are mean±s.e.m. (n=39–48 cells). (C,E) Representative images of HeLa-Int-03 M18BP1–Halo cells (C) or HeLa-Int-03 SNAP–CENP-A cells (E) transfected with the indicated siRNAs. Cells were stained with DAPI, anti-α-tubulin (green) and the Halo-tag TMR Ligand (red) at 36 h after co-transfection (C). A schematic for the experiment in E is shown above the images. Pre-existing SNAP–CENP-A was quenched with block ligand until 9 h before fixation, and then newly synthesized SNAP–CENP-A was labeled with SNAP TMR Ligand before fixation at 36 h after siRNA transfection (E). Scale bars: 5 μm. (D,F) Normalized M18BP1–Halo (D) or normalized new CENP-A (F) dot signals in each sample. Asterisks indicate significant differences. *P<0.05; **P<0.01; ***P<0.001; n.s., not significant (Mann–Whitney test). Results are mean±s.e.m. (n=43–50 cells). (G) CENP-C and CENP-I coordinate centromere ‘function’ and ‘epigenetics’. Solid lines show recruitment activity observed in our present experiments and dashed lines show recruitment activity indicated previously. The CENP-C N-terminus assembles kinetochore through the Mis12 complex (function) and its C-terminus assembles newly synthesized CENP-A through M18BP1 (epigenetics). CENP-I recruited by the CENP-C domain II (amino acids 72–425) supports both pathways. Each class is also involved in maintenance of CENP-A assembly (see Discussion).

Mentions: In order to check whether the conclusions of tethering experiments could be extended to CENP-A assembly at endogenous centromeres, we first tested the interdependency of kinetochore assembly of CENP-C and CENP-I. Interestingly, CENP-I levels at kinetochores fell dramatically after CENP-C depletion. In contrast, CENP-C levels at kinetochores were unaffected after 72 h of CENP-I depletion (Fig. 8A,B).Fig. 8.


CENP-C and CENP-I are key connecting factors for kinetochore and CENP-A assembly.

Shono N, Ohzeki J, Otake K, Martins NM, Nagase T, Kimura H, Larionov V, Earnshaw WC, Masumoto H - J. Cell. Sci. (2015)

Centromere assembly of newly synthesized CENP-A requires CENP-C and is enhanced through CENP-I. (A) Representative images of HeLa-Int-03 cells transfected with control siRNA (siControl) or siRNA against CENP-C (siCENP-C), CENP-I (siCENP-i) or both, together with siRNA against MAD2 (siMAD2). Cells were stained with DAPI, anti-CENP-C (red) and anti-CENP-I (green) at 72 h after transfection. Scale bars: 5 μm. (B) Normalized CENP-C and CENP-I dot signals in each sample at 36 h and 72 h after siRNA transfection. ***P<0.001 (Mann–Whitney test). Results are mean±s.e.m. (n=39–48 cells). (C,E) Representative images of HeLa-Int-03 M18BP1–Halo cells (C) or HeLa-Int-03 SNAP–CENP-A cells (E) transfected with the indicated siRNAs. Cells were stained with DAPI, anti-α-tubulin (green) and the Halo-tag TMR Ligand (red) at 36 h after co-transfection (C). A schematic for the experiment in E is shown above the images. Pre-existing SNAP–CENP-A was quenched with block ligand until 9 h before fixation, and then newly synthesized SNAP–CENP-A was labeled with SNAP TMR Ligand before fixation at 36 h after siRNA transfection (E). Scale bars: 5 μm. (D,F) Normalized M18BP1–Halo (D) or normalized new CENP-A (F) dot signals in each sample. Asterisks indicate significant differences. *P<0.05; **P<0.01; ***P<0.001; n.s., not significant (Mann–Whitney test). Results are mean±s.e.m. (n=43–50 cells). (G) CENP-C and CENP-I coordinate centromere ‘function’ and ‘epigenetics’. Solid lines show recruitment activity observed in our present experiments and dashed lines show recruitment activity indicated previously. The CENP-C N-terminus assembles kinetochore through the Mis12 complex (function) and its C-terminus assembles newly synthesized CENP-A through M18BP1 (epigenetics). CENP-I recruited by the CENP-C domain II (amino acids 72–425) supports both pathways. Each class is also involved in maintenance of CENP-A assembly (see Discussion).
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JCS180786F8: Centromere assembly of newly synthesized CENP-A requires CENP-C and is enhanced through CENP-I. (A) Representative images of HeLa-Int-03 cells transfected with control siRNA (siControl) or siRNA against CENP-C (siCENP-C), CENP-I (siCENP-i) or both, together with siRNA against MAD2 (siMAD2). Cells were stained with DAPI, anti-CENP-C (red) and anti-CENP-I (green) at 72 h after transfection. Scale bars: 5 μm. (B) Normalized CENP-C and CENP-I dot signals in each sample at 36 h and 72 h after siRNA transfection. ***P<0.001 (Mann–Whitney test). Results are mean±s.e.m. (n=39–48 cells). (C,E) Representative images of HeLa-Int-03 M18BP1–Halo cells (C) or HeLa-Int-03 SNAP–CENP-A cells (E) transfected with the indicated siRNAs. Cells were stained with DAPI, anti-α-tubulin (green) and the Halo-tag TMR Ligand (red) at 36 h after co-transfection (C). A schematic for the experiment in E is shown above the images. Pre-existing SNAP–CENP-A was quenched with block ligand until 9 h before fixation, and then newly synthesized SNAP–CENP-A was labeled with SNAP TMR Ligand before fixation at 36 h after siRNA transfection (E). Scale bars: 5 μm. (D,F) Normalized M18BP1–Halo (D) or normalized new CENP-A (F) dot signals in each sample. Asterisks indicate significant differences. *P<0.05; **P<0.01; ***P<0.001; n.s., not significant (Mann–Whitney test). Results are mean±s.e.m. (n=43–50 cells). (G) CENP-C and CENP-I coordinate centromere ‘function’ and ‘epigenetics’. Solid lines show recruitment activity observed in our present experiments and dashed lines show recruitment activity indicated previously. The CENP-C N-terminus assembles kinetochore through the Mis12 complex (function) and its C-terminus assembles newly synthesized CENP-A through M18BP1 (epigenetics). CENP-I recruited by the CENP-C domain II (amino acids 72–425) supports both pathways. Each class is also involved in maintenance of CENP-A assembly (see Discussion).
Mentions: In order to check whether the conclusions of tethering experiments could be extended to CENP-A assembly at endogenous centromeres, we first tested the interdependency of kinetochore assembly of CENP-C and CENP-I. Interestingly, CENP-I levels at kinetochores fell dramatically after CENP-C depletion. In contrast, CENP-C levels at kinetochores were unaffected after 72 h of CENP-I depletion (Fig. 8A,B).Fig. 8.

Bottom Line: We showed that these components work by recruiting CENP-C and subsequently recruiting M18BP1.Furthermore, we found that CENP-I can also recruit M18BP1 and, as a consequence, enhances M18BP1 assembly on centromeres in the downstream of CENP-C.Thus, we suggest that CENP-C and CENP-I are key factors connecting kinetochore to CENP-A assembly.

View Article: PubMed Central - PubMed

Affiliation: Laboratory of Cell Engineering, Department of Frontier Research, Kazusa DNA Research Institute, 2-6-7 Kazusa-Kamatari, Kisarazu, Chiba 292-0818, Japan Division of Biological Science, Graduate School of Science, Nagoya University, Furo-cho, Chikusa-ku, Nagoya, Aichi 464-8602, Japan.

No MeSH data available.


Related in: MedlinePlus