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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

CENP-C is required for de novo CENP-A assembly induced by the class I kinetochore components. (A) Representative images of HeLa-Int-03 cells transfected with tetR-EYFP–NSL1 or tetR-EYFP–SPC24 (green). Cells were stained with DAPI and anti-CENP-A (red) at 48 h after transfection. Arrowheads indicate the ectopic site. Scale bars: 5 μm. (B) Frequency of de novo CENP-A assembly on the ectopic site at 48 h after transfection. CENP-A signals on tetR-EYFP spots as a percentage of the total tetR-EYFP spots in each sample (n=100 cells). The red line shows KMN network components. Data shown in Fig. 2F are included for comparison. Results are mean±s.e.m. (n=3 experiments). (C) Schematic for the experiments shown in D,E and J. HeLa-Int-03 cells were first transfected with siRNA. After 24 h incubation, tetR-EYFP fusion expression vectors were transfected. Cells were stained with DAPI and each antibody. (D) Representative images of HeLa-Int-03 cells transfected with the indicated tetR-EYFP fusion proteins (green) after transfection with siRNA against CENP-A (siCENP-A). Cells were stained with DAPI and anti-CENP-C antibody (red) at 48 h after plasmid transfection. Arrowheads indicate the ectopic site. Scale bar: 5 μm. (E) Frequency of CENP-C assembly on the ectopic site determined as described in C. CENP-C signals on tetR-EYFP spots as a percentage of the total tetR-EYFP spots in each sample (n=100 cells) fixed at 24 or 48 h after plasmid transfection. Results are mean±s.e.m. (n=3 experiments). (F) Correlation between de novo CENP-A (from B) and CENP-C (from E; 48h) assembly. tetR-EYFP fusion proteins that correspond to the numerals in the graph are shown on the right. The correlation coefficient (r) and P value were calculated (except for tetR-EYFP–HJURP) and are shown in the graph. (G) CENP-C and MAD2 expression were analyzed by immunoblotting using antibodies against CENP-C, GAPDH (loading control) and MAD2. Cells were harvested at 72 h after control (siControl), CENP-C (siCENP-C) or MAD2 (siMAD2) siRNA transfection. (H) Representative images of HeLa-Int-03 cells transfected with indicated siRNAs. Cells were stained with DAPI and anti-CENP-C at 72 h after transfection. Scale bar: 5 μm. (I) 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 siRNA transfection. (J) Frequency of de novo CENP-A assembly on the ectopic site under the indicated siRNAs transfection. 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. **P<0.01; ***P<0.001 for significant differences between indicated tetR-EYFP fusion proteins or siRNA sets (Fisher's exact test). Results are mean±s.e.m. (n=3 experiments).
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JCS180786F5: CENP-C is required for de novo CENP-A assembly induced by the class I kinetochore components. (A) Representative images of HeLa-Int-03 cells transfected with tetR-EYFP–NSL1 or tetR-EYFP–SPC24 (green). Cells were stained with DAPI and anti-CENP-A (red) at 48 h after transfection. Arrowheads indicate the ectopic site. Scale bars: 5 μm. (B) Frequency of de novo CENP-A assembly on the ectopic site at 48 h after transfection. CENP-A signals on tetR-EYFP spots as a percentage of the total tetR-EYFP spots in each sample (n=100 cells). The red line shows KMN network components. Data shown in Fig. 2F are included for comparison. Results are mean±s.e.m. (n=3 experiments). (C) Schematic for the experiments shown in D,E and J. HeLa-Int-03 cells were first transfected with siRNA. After 24 h incubation, tetR-EYFP fusion expression vectors were transfected. Cells were stained with DAPI and each antibody. (D) Representative images of HeLa-Int-03 cells transfected with the indicated tetR-EYFP fusion proteins (green) after transfection with siRNA against CENP-A (siCENP-A). Cells were stained with DAPI and anti-CENP-C antibody (red) at 48 h after plasmid transfection. Arrowheads indicate the ectopic site. Scale bar: 5 μm. (E) Frequency of CENP-C assembly on the ectopic site determined as described in C. CENP-C signals on tetR-EYFP spots as a percentage of the total tetR-EYFP spots in each sample (n=100 cells) fixed at 24 or 48 h after plasmid transfection. Results are mean±s.e.m. (n=3 experiments). (F) Correlation between de novo CENP-A (from B) and CENP-C (from E; 48h) assembly. tetR-EYFP fusion proteins that correspond to the numerals in the graph are shown on the right. The correlation coefficient (r) and P value were calculated (except for tetR-EYFP–HJURP) and are shown in the graph. (G) CENP-C and MAD2 expression were analyzed by immunoblotting using antibodies against CENP-C, GAPDH (loading control) and MAD2. Cells were harvested at 72 h after control (siControl), CENP-C (siCENP-C) or MAD2 (siMAD2) siRNA transfection. (H) Representative images of HeLa-Int-03 cells transfected with indicated siRNAs. Cells were stained with DAPI and anti-CENP-C at 72 h after transfection. Scale bar: 5 μm. (I) 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 siRNA transfection. (J) Frequency of de novo CENP-A assembly on the ectopic site under the indicated siRNAs transfection. 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. **P<0.01; ***P<0.001 for significant differences between indicated tetR-EYFP fusion proteins or siRNA sets (Fisher's exact test). Results are mean±s.e.m. (n=3 experiments).

Mentions: To further explore this link, we explored the ability of several other kinetochore and CCAN components to induce de novo CENP-A assembly on the ectopic alphoidtetO array. Interestingly, components of KMN network all efficiently induced de novo CENP-A assembly in this assay (Fig. 5A,B). In contrast to these structural components of the kinetochore, ZWINT, BubR1, PP1γ and SKA1 (Fig. 5B), which are thought to be involved in regulating kinetochore activity through the spindle assembly checkpoint, did not induce de novo CENP-A assembly.Fig. 5.


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 is required for de novo CENP-A assembly induced by the class I kinetochore components. (A) Representative images of HeLa-Int-03 cells transfected with tetR-EYFP–NSL1 or tetR-EYFP–SPC24 (green). Cells were stained with DAPI and anti-CENP-A (red) at 48 h after transfection. Arrowheads indicate the ectopic site. Scale bars: 5 μm. (B) Frequency of de novo CENP-A assembly on the ectopic site at 48 h after transfection. CENP-A signals on tetR-EYFP spots as a percentage of the total tetR-EYFP spots in each sample (n=100 cells). The red line shows KMN network components. Data shown in Fig. 2F are included for comparison. Results are mean±s.e.m. (n=3 experiments). (C) Schematic for the experiments shown in D,E and J. HeLa-Int-03 cells were first transfected with siRNA. After 24 h incubation, tetR-EYFP fusion expression vectors were transfected. Cells were stained with DAPI and each antibody. (D) Representative images of HeLa-Int-03 cells transfected with the indicated tetR-EYFP fusion proteins (green) after transfection with siRNA against CENP-A (siCENP-A). Cells were stained with DAPI and anti-CENP-C antibody (red) at 48 h after plasmid transfection. Arrowheads indicate the ectopic site. Scale bar: 5 μm. (E) Frequency of CENP-C assembly on the ectopic site determined as described in C. CENP-C signals on tetR-EYFP spots as a percentage of the total tetR-EYFP spots in each sample (n=100 cells) fixed at 24 or 48 h after plasmid transfection. Results are mean±s.e.m. (n=3 experiments). (F) Correlation between de novo CENP-A (from B) and CENP-C (from E; 48h) assembly. tetR-EYFP fusion proteins that correspond to the numerals in the graph are shown on the right. The correlation coefficient (r) and P value were calculated (except for tetR-EYFP–HJURP) and are shown in the graph. (G) CENP-C and MAD2 expression were analyzed by immunoblotting using antibodies against CENP-C, GAPDH (loading control) and MAD2. Cells were harvested at 72 h after control (siControl), CENP-C (siCENP-C) or MAD2 (siMAD2) siRNA transfection. (H) Representative images of HeLa-Int-03 cells transfected with indicated siRNAs. Cells were stained with DAPI and anti-CENP-C at 72 h after transfection. Scale bar: 5 μm. (I) 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 siRNA transfection. (J) Frequency of de novo CENP-A assembly on the ectopic site under the indicated siRNAs transfection. 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. **P<0.01; ***P<0.001 for significant differences between indicated tetR-EYFP fusion proteins or siRNA sets (Fisher's exact test). Results are mean±s.e.m. (n=3 experiments).
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JCS180786F5: CENP-C is required for de novo CENP-A assembly induced by the class I kinetochore components. (A) Representative images of HeLa-Int-03 cells transfected with tetR-EYFP–NSL1 or tetR-EYFP–SPC24 (green). Cells were stained with DAPI and anti-CENP-A (red) at 48 h after transfection. Arrowheads indicate the ectopic site. Scale bars: 5 μm. (B) Frequency of de novo CENP-A assembly on the ectopic site at 48 h after transfection. CENP-A signals on tetR-EYFP spots as a percentage of the total tetR-EYFP spots in each sample (n=100 cells). The red line shows KMN network components. Data shown in Fig. 2F are included for comparison. Results are mean±s.e.m. (n=3 experiments). (C) Schematic for the experiments shown in D,E and J. HeLa-Int-03 cells were first transfected with siRNA. After 24 h incubation, tetR-EYFP fusion expression vectors were transfected. Cells were stained with DAPI and each antibody. (D) Representative images of HeLa-Int-03 cells transfected with the indicated tetR-EYFP fusion proteins (green) after transfection with siRNA against CENP-A (siCENP-A). Cells were stained with DAPI and anti-CENP-C antibody (red) at 48 h after plasmid transfection. Arrowheads indicate the ectopic site. Scale bar: 5 μm. (E) Frequency of CENP-C assembly on the ectopic site determined as described in C. CENP-C signals on tetR-EYFP spots as a percentage of the total tetR-EYFP spots in each sample (n=100 cells) fixed at 24 or 48 h after plasmid transfection. Results are mean±s.e.m. (n=3 experiments). (F) Correlation between de novo CENP-A (from B) and CENP-C (from E; 48h) assembly. tetR-EYFP fusion proteins that correspond to the numerals in the graph are shown on the right. The correlation coefficient (r) and P value were calculated (except for tetR-EYFP–HJURP) and are shown in the graph. (G) CENP-C and MAD2 expression were analyzed by immunoblotting using antibodies against CENP-C, GAPDH (loading control) and MAD2. Cells were harvested at 72 h after control (siControl), CENP-C (siCENP-C) or MAD2 (siMAD2) siRNA transfection. (H) Representative images of HeLa-Int-03 cells transfected with indicated siRNAs. Cells were stained with DAPI and anti-CENP-C at 72 h after transfection. Scale bar: 5 μm. (I) 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 siRNA transfection. (J) Frequency of de novo CENP-A assembly on the ectopic site under the indicated siRNAs transfection. 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. **P<0.01; ***P<0.001 for significant differences between indicated tetR-EYFP fusion proteins or siRNA sets (Fisher's exact test). Results are mean±s.e.m. (n=3 experiments).
Mentions: To further explore this link, we explored the ability of several other kinetochore and CCAN components to induce de novo CENP-A assembly on the ectopic alphoidtetO array. Interestingly, components of KMN network all efficiently induced de novo CENP-A assembly in this assay (Fig. 5A,B). In contrast to these structural components of the kinetochore, ZWINT, BubR1, PP1γ and SKA1 (Fig. 5B), which are thought to be involved in regulating kinetochore activity through the spindle assembly checkpoint, did not induce de novo CENP-A assembly.Fig. 5.

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