Limits...
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

CENP-C or CENP-I can independently recruit M18BP1. (A) Schematic for the experiments shown in B, C and D. HeLa-Int-03 cells were first transfected with siRNA. After 24 h incubation, Halo fusion and tetR-EYFP fusion expression vectors were co-transfected. (B) Representative images of HeLa-Int-03 cells co-transfected with tetR-EYFP–CENP-C or tetR-EYFP–CENP-I (green) and Halo-M18BP1 after transfection with siRNA against CENP-A (siCENP-A). Cells were stained with DAPI and the Halo-tag TMR Ligand (red) at 24 h after plasmid transfection. Arrowheads indicate the ectopic site. Scale bars: 5 μm. (C) Frequency of each Halo fusion assembly on the ectopic site. Halo fusion signals on tetR-EYFP spots as a percentage of the total tetR-EYFP spots in each sample (n=50 cells) fixed at 24 h after plasmid transfection. Results are mean±s.e.m. (n=3 experiments). (D) Frequency of Halo–M18BP1 assembly on the ectopic site after transfection of control siRNA (siControl) and siRNA against CENP-A (siCENP-A), MAD2 (siMAD2), CENP-I (siCENP-I) and CENP-C (siCENP-C) as indicated. tetR-EYFP fusion proteins were co-transfected with Halo–M18BP1 after siRNA transfection. Halo-fusion signals on tetR-EYFP spots as a percentage of the total tetR-EYFP spots in each sample (n=50 cells) fixed at 24 h after plasmid transfection. Results are mean±s.e.m. (n=3 experiments). (E) M18BP1 expression was analyzed by immunoblotting using antibodies against M18BP1 and GAPDH (loading control). Cells were harvested at 72 h after each transfection with control and M18BP1 (siM18BP1) siRNA. (F) The amount of chromosomal (P) and soluble (S) CENP-A were analyzed by immunoblotting using antibodies against Lamin B1 (loading control for P), GAPDH (loading control for S) and CENP-A. Cells were harvested and fractionated at 72 h after each siRNA transfection. (G) Frequency of de novo CENP-A assembly on the ectopic site after transfection of the indicated siRNA. CENP-A signals on tetR-EYFP spots as a percentage of the total tetR-EYFP spots in each sample (n=100 cells) fixed at 48 h after plasmid transfection (72 h after siRNA transfection). Asterisks indicate significant differences from tetR-EYFP alone. ***P<0.001 (Fisher's exact test). Results are mean±s.e.m. (n=3 experiments).
© Copyright Policy - open-access
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC4696500&req=5

JCS180786F6: CENP-C or CENP-I can independently recruit M18BP1. (A) Schematic for the experiments shown in B, C and D. HeLa-Int-03 cells were first transfected with siRNA. After 24 h incubation, Halo fusion and tetR-EYFP fusion expression vectors were co-transfected. (B) Representative images of HeLa-Int-03 cells co-transfected with tetR-EYFP–CENP-C or tetR-EYFP–CENP-I (green) and Halo-M18BP1 after transfection with siRNA against CENP-A (siCENP-A). Cells were stained with DAPI and the Halo-tag TMR Ligand (red) at 24 h after plasmid transfection. Arrowheads indicate the ectopic site. Scale bars: 5 μm. (C) Frequency of each Halo fusion assembly on the ectopic site. Halo fusion signals on tetR-EYFP spots as a percentage of the total tetR-EYFP spots in each sample (n=50 cells) fixed at 24 h after plasmid transfection. Results are mean±s.e.m. (n=3 experiments). (D) Frequency of Halo–M18BP1 assembly on the ectopic site after transfection of control siRNA (siControl) and siRNA against CENP-A (siCENP-A), MAD2 (siMAD2), CENP-I (siCENP-I) and CENP-C (siCENP-C) as indicated. tetR-EYFP fusion proteins were co-transfected with Halo–M18BP1 after siRNA transfection. Halo-fusion signals on tetR-EYFP spots as a percentage of the total tetR-EYFP spots in each sample (n=50 cells) fixed at 24 h after plasmid transfection. Results are mean±s.e.m. (n=3 experiments). (E) M18BP1 expression was analyzed by immunoblotting using antibodies against M18BP1 and GAPDH (loading control). Cells were harvested at 72 h after each transfection with control and M18BP1 (siM18BP1) siRNA. (F) The amount of chromosomal (P) and soluble (S) CENP-A were analyzed by immunoblotting using antibodies against Lamin B1 (loading control for P), GAPDH (loading control for S) and CENP-A. Cells were harvested and fractionated at 72 h after each siRNA transfection. (G) Frequency of de novo CENP-A assembly on the ectopic site after transfection of the indicated siRNA. CENP-A signals on tetR-EYFP spots as a percentage of the total tetR-EYFP spots in each sample (n=100 cells) fixed at 48 h after plasmid transfection (72 h after siRNA transfection). Asterisks indicate significant differences from tetR-EYFP alone. ***P<0.001 (Fisher's exact test). Results are mean±s.e.m. (n=3 experiments).

Mentions: Using various deletion mutants of tetR-EYFP–NSL1 or tetR-EYFP–Mis12, both of which are Mis12 complex components and, hence, expected to interact with the KMN network and CENP-C (Petrovic et al., 2010; Screpanti et al., 2011), we found that NSL1 and Mis12 deletion mutants that had lost the ability to interact with CENP-C also lost the ability to induce de novo CENP-A assembly (Fig. S2A,B). We confirmed that NSL1 and Mis12 interact with CENP-C even following CENP-A depletion by small interfering RNA (siRNA)-mediated knockdown. This control minimizes the likelihood of possible secondary CENP-C recruitment through pre-assembled CENP-A (Fig. S2C–F; CENP-A depletions were also performed similarly in Figs 5, 6 and 7, and Fig. S3). We concluded that CENP-C recruited by tetR-EYFP–NSL1 or tetR-EYFP–Mis12 induces de novo CENP-A assembly on the ectopic site.


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)

CENP-C or CENP-I can independently recruit M18BP1. (A) Schematic for the experiments shown in B, C and D. HeLa-Int-03 cells were first transfected with siRNA. After 24 h incubation, Halo fusion and tetR-EYFP fusion expression vectors were co-transfected. (B) Representative images of HeLa-Int-03 cells co-transfected with tetR-EYFP–CENP-C or tetR-EYFP–CENP-I (green) and Halo-M18BP1 after transfection with siRNA against CENP-A (siCENP-A). Cells were stained with DAPI and the Halo-tag TMR Ligand (red) at 24 h after plasmid transfection. Arrowheads indicate the ectopic site. Scale bars: 5 μm. (C) Frequency of each Halo fusion assembly on the ectopic site. Halo fusion signals on tetR-EYFP spots as a percentage of the total tetR-EYFP spots in each sample (n=50 cells) fixed at 24 h after plasmid transfection. Results are mean±s.e.m. (n=3 experiments). (D) Frequency of Halo–M18BP1 assembly on the ectopic site after transfection of control siRNA (siControl) and siRNA against CENP-A (siCENP-A), MAD2 (siMAD2), CENP-I (siCENP-I) and CENP-C (siCENP-C) as indicated. tetR-EYFP fusion proteins were co-transfected with Halo–M18BP1 after siRNA transfection. Halo-fusion signals on tetR-EYFP spots as a percentage of the total tetR-EYFP spots in each sample (n=50 cells) fixed at 24 h after plasmid transfection. Results are mean±s.e.m. (n=3 experiments). (E) M18BP1 expression was analyzed by immunoblotting using antibodies against M18BP1 and GAPDH (loading control). Cells were harvested at 72 h after each transfection with control and M18BP1 (siM18BP1) siRNA. (F) The amount of chromosomal (P) and soluble (S) CENP-A were analyzed by immunoblotting using antibodies against Lamin B1 (loading control for P), GAPDH (loading control for S) and CENP-A. Cells were harvested and fractionated at 72 h after each siRNA transfection. (G) Frequency of de novo CENP-A assembly on the ectopic site after transfection of the indicated siRNA. CENP-A signals on tetR-EYFP spots as a percentage of the total tetR-EYFP spots in each sample (n=100 cells) fixed at 48 h after plasmid transfection (72 h after siRNA transfection). Asterisks indicate significant differences from tetR-EYFP alone. ***P<0.001 (Fisher's exact test). Results are mean±s.e.m. (n=3 experiments).
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4696500&req=5

JCS180786F6: CENP-C or CENP-I can independently recruit M18BP1. (A) Schematic for the experiments shown in B, C and D. HeLa-Int-03 cells were first transfected with siRNA. After 24 h incubation, Halo fusion and tetR-EYFP fusion expression vectors were co-transfected. (B) Representative images of HeLa-Int-03 cells co-transfected with tetR-EYFP–CENP-C or tetR-EYFP–CENP-I (green) and Halo-M18BP1 after transfection with siRNA against CENP-A (siCENP-A). Cells were stained with DAPI and the Halo-tag TMR Ligand (red) at 24 h after plasmid transfection. Arrowheads indicate the ectopic site. Scale bars: 5 μm. (C) Frequency of each Halo fusion assembly on the ectopic site. Halo fusion signals on tetR-EYFP spots as a percentage of the total tetR-EYFP spots in each sample (n=50 cells) fixed at 24 h after plasmid transfection. Results are mean±s.e.m. (n=3 experiments). (D) Frequency of Halo–M18BP1 assembly on the ectopic site after transfection of control siRNA (siControl) and siRNA against CENP-A (siCENP-A), MAD2 (siMAD2), CENP-I (siCENP-I) and CENP-C (siCENP-C) as indicated. tetR-EYFP fusion proteins were co-transfected with Halo–M18BP1 after siRNA transfection. Halo-fusion signals on tetR-EYFP spots as a percentage of the total tetR-EYFP spots in each sample (n=50 cells) fixed at 24 h after plasmid transfection. Results are mean±s.e.m. (n=3 experiments). (E) M18BP1 expression was analyzed by immunoblotting using antibodies against M18BP1 and GAPDH (loading control). Cells were harvested at 72 h after each transfection with control and M18BP1 (siM18BP1) siRNA. (F) The amount of chromosomal (P) and soluble (S) CENP-A were analyzed by immunoblotting using antibodies against Lamin B1 (loading control for P), GAPDH (loading control for S) and CENP-A. Cells were harvested and fractionated at 72 h after each siRNA transfection. (G) Frequency of de novo CENP-A assembly on the ectopic site after transfection of the indicated siRNA. CENP-A signals on tetR-EYFP spots as a percentage of the total tetR-EYFP spots in each sample (n=100 cells) fixed at 48 h after plasmid transfection (72 h after siRNA transfection). Asterisks indicate significant differences from tetR-EYFP alone. ***P<0.001 (Fisher's exact test). Results are mean±s.e.m. (n=3 experiments).
Mentions: Using various deletion mutants of tetR-EYFP–NSL1 or tetR-EYFP–Mis12, both of which are Mis12 complex components and, hence, expected to interact with the KMN network and CENP-C (Petrovic et al., 2010; Screpanti et al., 2011), we found that NSL1 and Mis12 deletion mutants that had lost the ability to interact with CENP-C also lost the ability to induce de novo CENP-A assembly (Fig. S2A,B). We confirmed that NSL1 and Mis12 interact with CENP-C even following CENP-A depletion by small interfering RNA (siRNA)-mediated knockdown. This control minimizes the likelihood of possible secondary CENP-C recruitment through pre-assembled CENP-A (Fig. S2C–F; CENP-A depletions were also performed similarly in Figs 5, 6 and 7, and Fig. S3). We concluded that CENP-C recruited by tetR-EYFP–NSL1 or tetR-EYFP–Mis12 induces de novo CENP-A assembly on the ectopic site.

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