cenp-t Search Results


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  • 99
    Thermo Fisher silencer select pre designed sirna
    A portion of the N-terminal tail of <t>CENP-A</t> recruits CENP-C and CENP-T to the LacO array. (A) Diagram of the chimeric histones targeted to the LacO array in U2OS-LacO cells and assessed for recruitment of CENP-C and CENP-T. (B and C) Quantitation of CENP-C (B) and CENP-T (C) intensity at the LacO array normalized to the mean intensity at endogenous centromeres. (D and E) Representative images of CENP-C (D) and CENP-T (E) recruitment to the LacO by H3 1–29+CATD+135–140 fused to LacI and an HA tag. (F, H, and J) Quantitation of CENP-C and CENP-T intensity at the LacO array for HA-LacI–fused H3 1–29+CATD+135–140 in cells treated with CENP-T (F), CENP-C (H), and CENP-N (J) <t>siRNA</t> normalized to the mean intensity at the LacO array in cells treated with GAPDH siRNA. (G, I, and K) Representative images of CENP-C and CENP-T recruitment to the LacO array by HA-LacI–fused H3 1–29+CATD+135–140 in cells treated with CENP-T (G), CENP-C (I), and CENP-N (K) siRNA. (L and N) Quantitation of CENP-C (L) and CENP-T (N) intensity at the LacO array. Error bars show SEM. *, P
    Silencer Select Pre Designed Sirna, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 3493 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    91
    Millipore rabbit anti cenp t
    <t>CENP-A</t> α-amino methylation is required for CCAN formation. ( a ) Schematic of the experiment. ( b ) Representative images of the cells immunostained for <t>CENP-T</t> and CENP-C. Centromeric CENP-T was decreased upon CENP-A knockout and is rescued by replacement of the wild type CENP-A but not with methylation mutant. ( c , d ) Quantitation of CENP-T and CENP-C at the centromere after removal of endogenous CENP-A by cre-recombinase. n =2. A minimum 30 cells in two biological replicates were used for the quantitation. ( e ) Representative images of cells immunostained for CENP-I and CENP-B. CENP-I level decreased when endogenous CENP-A was removed by Cre expression and is rescued by replacement with the wild type CENP-A but not with methylation mutant. ( f , g ) Quantitation of CENP-I and CENP-B at the centromere after removing endogenous CENP-A by cre-recombinase. Box-and-whisker plots. Central lines, medians; whiskers, range 5–95 percentile; outliers not shown. ( h ) Schematic of the experiment in I-K. eGFP tagged CENP-A wild type and mutant integrated RPE-CENP-A -/F cells were infected with cre-recombinase virus to remove endogenous CENP-A. Individual colonies were picked after 14 days and expanded. The cells were treated with 0.1 μM Nocodazole for 6 h and then released for one hour, fixed and DAPI stained ( i ) Scale bar, 10 μm. Cells expressing the methylation mutant showed significantly high percentage of ( j ) lagging chromosomes and ( k ) micronuclei formation in the absence of Nocodazole treatment. The experiments were done three times. Mean and s.d. shown. ( l ) Representative images of the RPE cells immunostained for NDC80. Kinetochore localization of NDC80 was decreased in methylation mutant CENP-A. ( m ) Quantitation of NDC80 at the kinetochore after removal of endogenous CENP-A by cre-recombinase and replacement of either wild type or mutant CENP-A. A minimum of 8 prometaphse cells were used for the quantitation. Indicated P values were determined by t -test. Scale bars, 10 μm.
    Rabbit Anti Cenp T, supplied by Millipore, used in various techniques. Bioz Stars score: 91/100, based on 6 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    92
    Cocalico anti cenp t
    Labeling of proteins transiently associated with <t>CENP-A</t> and H3.1 nucleosomes. (A) Schematic representation of the experimental approach. (B) (C) Representative images of cells stably expressing indicated proteins fused to the BirA* ligase and HA tag, and incubated with medium supplemented with or without biotin for 5 hours. DNA is visualized by DAPI staining, immunofluorescence for <t>CENP-T</t> is shown in red, biotinylated proteins (B) or BirA*-HA fusion proteins (C) are shown in green. Scale bar is 5μm. (D) Streptavidin purification of biotinylated proteins from indicated cells lines analyzed by immunoblot. Cell media was supplemented with or without biotin for 24 hours. (E) Graph showing SILAC comparison between CENP-A-BirA*-HA biotinylation profile (heavy) versus biotinylation profile of H3.1-BirA-HA* (light) in cells undergoing S phase. Corresponding H/L scores for selected proteins are displayed. The graph represents an average of two independently performed experiments +/− SEM.
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    90
    Abcam 1 200 rabbit polyclonal anti cenp t
    Labeling of proteins transiently associated with <t>CENP-A</t> and H3.1 nucleosomes. (A) Schematic representation of the experimental approach. (B) (C) Representative images of cells stably expressing indicated proteins fused to the BirA* ligase and HA tag, and incubated with medium supplemented with or without biotin for 5 hours. DNA is visualized by DAPI staining, immunofluorescence for <t>CENP-T</t> is shown in red, biotinylated proteins (B) or BirA*-HA fusion proteins (C) are shown in green. Scale bar is 5μm. (D) Streptavidin purification of biotinylated proteins from indicated cells lines analyzed by immunoblot. Cell media was supplemented with or without biotin for 24 hours. (E) Graph showing SILAC comparison between CENP-A-BirA*-HA biotinylation profile (heavy) versus biotinylation profile of H3.1-BirA-HA* (light) in cells undergoing S phase. Corresponding H/L scores for selected proteins are displayed. The graph represents an average of two independently performed experiments +/− SEM.
    1 200 Rabbit Polyclonal Anti Cenp T, supplied by Abcam, used in various techniques. Bioz Stars score: 90/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    85
    Aprogen anti cenp t antiserum production
    Labeling of proteins transiently associated with <t>CENP-A</t> and H3.1 nucleosomes. (A) Schematic representation of the experimental approach. (B) (C) Representative images of cells stably expressing indicated proteins fused to the BirA* ligase and HA tag, and incubated with medium supplemented with or without biotin for 5 hours. DNA is visualized by DAPI staining, immunofluorescence for <t>CENP-T</t> is shown in red, biotinylated proteins (B) or BirA*-HA fusion proteins (C) are shown in green. Scale bar is 5μm. (D) Streptavidin purification of biotinylated proteins from indicated cells lines analyzed by immunoblot. Cell media was supplemented with or without biotin for 24 hours. (E) Graph showing SILAC comparison between CENP-A-BirA*-HA biotinylation profile (heavy) versus biotinylation profile of H3.1-BirA-HA* (light) in cells undergoing S phase. Corresponding H/L scores for selected proteins are displayed. The graph represents an average of two independently performed experiments +/− SEM.
    Anti Cenp T Antiserum Production, supplied by Aprogen, used in various techniques. Bioz Stars score: 85/100, based on 12 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    86
    Bethyl anti cenp t
    The <t>CENP-T/W</t> complex is required in each mitosis and does not exhibit persistent binding to centromeres. (A) CENPs -T and -W were depleted by RNAi in HeLa cells, resulting in defects in spindle assembly assayed by immunofluorescence for tubulin (green) and centromeres (red) with DNA stained with DAPI (blue). CENP-W depleted cells exhibit a high frequency of multipolar spindles. Depletion of CENP-T was less severe resulting in characteristic fusiform spindle structures. (B) CENP-W is required for robust mitosis in each cell cycle. Mitotic kinetics were assayed using histone H2B-GFP HeLa cells treated with CENP-W siRNA for 48 (blue circles) or 24 h (red triangles), scoring time between NEB (defined as onset of deformations in nuclear chromatin) and anaphase onset and are plotted by rank order of individual cells. Mean time for scrambled siRNA control (dark X) and untreated cells (light X) is shown as a green line, with +1 standard deviation (SD) marked with a red line. Inset graph reports the percentage of cells with NEB-anaphase times in excess of +1 SD and the average delay in anaphase onset. (C) Experimental schematic describes the pulse chase approach to assay the heritability of CLIP tagged CENPs -T and -W. (D) CENPs -T and -W do not persist at centromeres. CLIP signal intensity coincident with centromeres was quantified at the time of pulse and 24 h later. While CENP-A-SNAP signal was depleted by approximately 50%, as expected, CENP-T-CLIP and CENP-W-CLIP signal was reduced to background levels after 24 h.
    Anti Cenp T, supplied by Bethyl, used in various techniques. Bioz Stars score: 86/100, based on 4 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    94
    GeneTex antibodies mouse polyclonal anti cenp t antibody
    The <t>CENP-T/W</t> complex is required in each mitosis and does not exhibit persistent binding to centromeres. (A) CENPs -T and -W were depleted by RNAi in HeLa cells, resulting in defects in spindle assembly assayed by immunofluorescence for tubulin (green) and centromeres (red) with DNA stained with DAPI (blue). CENP-W depleted cells exhibit a high frequency of multipolar spindles. Depletion of CENP-T was less severe resulting in characteristic fusiform spindle structures. (B) CENP-W is required for robust mitosis in each cell cycle. Mitotic kinetics were assayed using histone H2B-GFP HeLa cells treated with CENP-W siRNA for 48 (blue circles) or 24 h (red triangles), scoring time between NEB (defined as onset of deformations in nuclear chromatin) and anaphase onset and are plotted by rank order of individual cells. Mean time for scrambled siRNA control (dark X) and untreated cells (light X) is shown as a green line, with +1 standard deviation (SD) marked with a red line. Inset graph reports the percentage of cells with NEB-anaphase times in excess of +1 SD and the average delay in anaphase onset. (C) Experimental schematic describes the pulse chase approach to assay the heritability of CLIP tagged CENPs -T and -W. (D) CENPs -T and -W do not persist at centromeres. CLIP signal intensity coincident with centromeres was quantified at the time of pulse and 24 h later. While CENP-A-SNAP signal was depleted by approximately 50%, as expected, CENP-T-CLIP and CENP-W-CLIP signal was reduced to background levels after 24 h.
    Antibodies Mouse Polyclonal Anti Cenp T Antibody, supplied by GeneTex, used in various techniques. Bioz Stars score: 94/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    90
    Santa Cruz Biotechnology cenp t sirna
    The <t>CENP-T/W</t> complex is required in each mitosis and does not exhibit persistent binding to centromeres. (A) CENPs -T and -W were depleted by RNAi in HeLa cells, resulting in defects in spindle assembly assayed by immunofluorescence for tubulin (green) and centromeres (red) with DNA stained with DAPI (blue). CENP-W depleted cells exhibit a high frequency of multipolar spindles. Depletion of CENP-T was less severe resulting in characteristic fusiform spindle structures. (B) CENP-W is required for robust mitosis in each cell cycle. Mitotic kinetics were assayed using histone H2B-GFP HeLa cells treated with CENP-W siRNA for 48 (blue circles) or 24 h (red triangles), scoring time between NEB (defined as onset of deformations in nuclear chromatin) and anaphase onset and are plotted by rank order of individual cells. Mean time for scrambled siRNA control (dark X) and untreated cells (light X) is shown as a green line, with +1 standard deviation (SD) marked with a red line. Inset graph reports the percentage of cells with NEB-anaphase times in excess of +1 SD and the average delay in anaphase onset. (C) Experimental schematic describes the pulse chase approach to assay the heritability of CLIP tagged CENPs -T and -W. (D) CENPs -T and -W do not persist at centromeres. CLIP signal intensity coincident with centromeres was quantified at the time of pulse and 24 h later. While CENP-A-SNAP signal was depleted by approximately 50%, as expected, CENP-T-CLIP and CENP-W-CLIP signal was reduced to background levels after 24 h.
    Cenp T Sirna, supplied by Santa Cruz Biotechnology, used in various techniques. Bioz Stars score: 90/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    95
    Addgene inc cenp t
    The <t>CENP-T/W</t> complex is required in each mitosis and does not exhibit persistent binding to centromeres. (A) CENPs -T and -W were depleted by RNAi in HeLa cells, resulting in defects in spindle assembly assayed by immunofluorescence for tubulin (green) and centromeres (red) with DNA stained with DAPI (blue). CENP-W depleted cells exhibit a high frequency of multipolar spindles. Depletion of CENP-T was less severe resulting in characteristic fusiform spindle structures. (B) CENP-W is required for robust mitosis in each cell cycle. Mitotic kinetics were assayed using histone H2B-GFP HeLa cells treated with CENP-W siRNA for 48 (blue circles) or 24 h (red triangles), scoring time between NEB (defined as onset of deformations in nuclear chromatin) and anaphase onset and are plotted by rank order of individual cells. Mean time for scrambled siRNA control (dark X) and untreated cells (light X) is shown as a green line, with +1 standard deviation (SD) marked with a red line. Inset graph reports the percentage of cells with NEB-anaphase times in excess of +1 SD and the average delay in anaphase onset. (C) Experimental schematic describes the pulse chase approach to assay the heritability of CLIP tagged CENPs -T and -W. (D) CENPs -T and -W do not persist at centromeres. CLIP signal intensity coincident with centromeres was quantified at the time of pulse and 24 h later. While CENP-A-SNAP signal was depleted by approximately 50%, as expected, CENP-T-CLIP and CENP-W-CLIP signal was reduced to background levels after 24 h.
    Cenp T, supplied by Addgene inc, used in various techniques. Bioz Stars score: 95/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    90
    Covance cenp t
    Model of centromere function mediated by centromeric chromatin and DNA sequences At exit of mitosis, centromeric chromatin replication and identity is mediated by <t>CENP-A</t> (in red) deposition via interaction with HJURP. CENP-A then mediates the assembly of CENP-C (in green) in mid-G1 followed by CENP-N/L (in orange) during S-phase. These steps might be interconnected. At this point, CENP-A becomes dispensable for mitotic centromere function as long as CENP-B (in light blue) is stably bound to centromeric sequences to support CENP-C binding. Assembly of the other subunits of the CCAN, such as <t>CENP-T/W</t> (in yellow) and HIKM (in brown), allows the full recruitment of the kinetochore complex (in grey) required to mediate centromere function. In summary, we propose that the kinetochore is tethered to the centromere through a dual linkage of CENP-A chromatin and CENP-B-bound DNA sequences, as the two major links from the DNA to the kinetochore to mediate successful chromosome segregation.
    Cenp T, supplied by Covance, used in various techniques. Bioz Stars score: 90/100, based on 7 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    90
    Gallus BioPharmaceuticals gallus gallus cenp t
    The B. mori <t>CENP-T</t> protein is essential in vivo . A) CRISPR/Cas9-introduced mutation in the Cenp-T coding sequence. Top: Alignment between wild-type (WT) and mutant (+7) sequences showing a seven base-pair insertion of the Cenp-T gene. Bottom: Schematic of WT and truncated protein products in the CRISPR mutant. PAM site (red), premature stop codon (blue) and Cas9 cleavage site (red arrow) are indicated. B) Hatching rate of the WT and Cenp-T mutant strains. The percentages of hatched (grey), unhatched (green) and eggs that died prior to hatching (black) are indicated for the different crossing patterns between WT (+/+) and heterozygous Cenp-T mutants (+/KO or KO/+). The number of independent crosses n is shown above. The raw data are shown in Table S3. C) Percentages of genotypes from larvae derived from the cross between two heterozygous Cenp-T mutants. The number n of analyzed larvae is indicated. The raw data are shown in Table S4.
    Gallus Gallus Cenp T, supplied by Gallus BioPharmaceuticals, used in various techniques. Bioz Stars score: 90/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    90
    Abcam rabbit anti cenp t antibodies
    Summary of the permitted and restricted assembly steps in human cell extracts. (A) Experimental approaches to reconstitute kinetochore assembly and function in vitro using mitotic HeLa cell extracts and the main conclusions derived from this study. (B) Simplified scheme indicating the permitted assembly steps (green check-mark), moderately/weakly efficient reactions (blue check-mark), and restricted interactions (red crosses). Symbols “D” indicate phospho-mimetic substitutions in <t>CENP-T.</t>
    Rabbit Anti Cenp T Antibodies, supplied by Abcam, used in various techniques. Bioz Stars score: 90/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Abcam rabbit anti cenp t antibody affinity purified
    Summary of the permitted and restricted assembly steps in human cell extracts. (A) Experimental approaches to reconstitute kinetochore assembly and function in vitro using mitotic HeLa cell extracts and the main conclusions derived from this study. (B) Simplified scheme indicating the permitted assembly steps (green check-mark), moderately/weakly efficient reactions (blue check-mark), and restricted interactions (red crosses). Symbols “D” indicate phospho-mimetic substitutions in <t>CENP-T.</t>
    Rabbit Anti Cenp T Antibody Affinity Purified, supplied by Abcam, used in various techniques. Bioz Stars score: 90/100, based on 10 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Abcam rabbit polyclonal anti cenp t antibody
    Summary of the permitted and restricted assembly steps in human cell extracts. (A) Experimental approaches to reconstitute kinetochore assembly and function in vitro using mitotic HeLa cell extracts and the main conclusions derived from this study. (B) Simplified scheme indicating the permitted assembly steps (green check-mark), moderately/weakly efficient reactions (blue check-mark), and restricted interactions (red crosses). Symbols “D” indicate phospho-mimetic substitutions in <t>CENP-T.</t>
    Rabbit Polyclonal Anti Cenp T Antibody, supplied by Abcam, used in various techniques. Bioz Stars score: 99/100, based on 6 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Millipore rabbit polyclonal anti cenp t
    Summary of the permitted and restricted assembly steps in human cell extracts. (A) Experimental approaches to reconstitute kinetochore assembly and function in vitro using mitotic HeLa cell extracts and the main conclusions derived from this study. (B) Simplified scheme indicating the permitted assembly steps (green check-mark), moderately/weakly efficient reactions (blue check-mark), and restricted interactions (red crosses). Symbols “D” indicate phospho-mimetic substitutions in <t>CENP-T.</t>
    Rabbit Polyclonal Anti Cenp T, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 8 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    95
    Thermo Fisher tokyo n a cenp t mutant b mori
    Summary of the permitted and restricted assembly steps in human cell extracts. (A) Experimental approaches to reconstitute kinetochore assembly and function in vitro using mitotic HeLa cell extracts and the main conclusions derived from this study. (B) Simplified scheme indicating the permitted assembly steps (green check-mark), moderately/weakly efficient reactions (blue check-mark), and restricted interactions (red crosses). Symbols “D” indicate phospho-mimetic substitutions in <t>CENP-T.</t>
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    Image Search Results


    A portion of the N-terminal tail of CENP-A recruits CENP-C and CENP-T to the LacO array. (A) Diagram of the chimeric histones targeted to the LacO array in U2OS-LacO cells and assessed for recruitment of CENP-C and CENP-T. (B and C) Quantitation of CENP-C (B) and CENP-T (C) intensity at the LacO array normalized to the mean intensity at endogenous centromeres. (D and E) Representative images of CENP-C (D) and CENP-T (E) recruitment to the LacO by H3 1–29+CATD+135–140 fused to LacI and an HA tag. (F, H, and J) Quantitation of CENP-C and CENP-T intensity at the LacO array for HA-LacI–fused H3 1–29+CATD+135–140 in cells treated with CENP-T (F), CENP-C (H), and CENP-N (J) siRNA normalized to the mean intensity at the LacO array in cells treated with GAPDH siRNA. (G, I, and K) Representative images of CENP-C and CENP-T recruitment to the LacO array by HA-LacI–fused H3 1–29+CATD+135–140 in cells treated with CENP-T (G), CENP-C (I), and CENP-N (K) siRNA. (L and N) Quantitation of CENP-C (L) and CENP-T (N) intensity at the LacO array. Error bars show SEM. *, P

    Journal: The Journal of Cell Biology

    Article Title: Both tails and the centromere targeting domain of CENP-A are required for centromere establishment

    doi: 10.1083/jcb.201412011

    Figure Lengend Snippet: A portion of the N-terminal tail of CENP-A recruits CENP-C and CENP-T to the LacO array. (A) Diagram of the chimeric histones targeted to the LacO array in U2OS-LacO cells and assessed for recruitment of CENP-C and CENP-T. (B and C) Quantitation of CENP-C (B) and CENP-T (C) intensity at the LacO array normalized to the mean intensity at endogenous centromeres. (D and E) Representative images of CENP-C (D) and CENP-T (E) recruitment to the LacO by H3 1–29+CATD+135–140 fused to LacI and an HA tag. (F, H, and J) Quantitation of CENP-C and CENP-T intensity at the LacO array for HA-LacI–fused H3 1–29+CATD+135–140 in cells treated with CENP-T (F), CENP-C (H), and CENP-N (J) siRNA normalized to the mean intensity at the LacO array in cells treated with GAPDH siRNA. (G, I, and K) Representative images of CENP-C and CENP-T recruitment to the LacO array by HA-LacI–fused H3 1–29+CATD+135–140 in cells treated with CENP-T (G), CENP-C (I), and CENP-N (K) siRNA. (L and N) Quantitation of CENP-C (L) and CENP-T (N) intensity at the LacO array. Error bars show SEM. *, P

    Article Snippet: For siRNA experiments, cells were treated with 20 µM CENP-T siRNAs (Silencer Select; catalog no. 4392420, siRNA ID s36976; target sequence 5′-AGAAGUGCCUAGAUAAAUA-3′; Life Technologies), CENP-C siRNAs (ON-TARGETplus SMARTpool; catalog no. L-003251; target sequences 5′-GCGAAUAGAUUAUCAAGGA-3′, 5′-CCAUAAACCUCACCCAGUA-3′, 5′-UUUGAUAGGUGAAACAGUA-3′, and 5′-CGAAGUUGAUAGAGGAUGA-3′; GE Healthcare), CENP-N siRNAs (siGENOME SMARTpool; catalog no. M-015872-02-005; target sequences 5′-CUACCUACGUGGUGUACUA-3′, 5′-AUACACCGCUUCUGGGUCA-3′, 5′-GAUCCAUCUGUGUGAGGAA-3′, and 5′-GUAAAUUUCCGACAGAGAA-3′; GE Healthcare), or GAPDH siRNAs (ON-TARGETplus SMARTpool; catalog no. D-001830; target sequence 5′-GUCAACGGAUUUGGUCGUA-3′; GE Healthcare).

    Techniques: Quantitation Assay

    CENP-A α-amino methylation is required for CCAN formation. ( a ) Schematic of the experiment. ( b ) Representative images of the cells immunostained for CENP-T and CENP-C. Centromeric CENP-T was decreased upon CENP-A knockout and is rescued by replacement of the wild type CENP-A but not with methylation mutant. ( c , d ) Quantitation of CENP-T and CENP-C at the centromere after removal of endogenous CENP-A by cre-recombinase. n =2. A minimum 30 cells in two biological replicates were used for the quantitation. ( e ) Representative images of cells immunostained for CENP-I and CENP-B. CENP-I level decreased when endogenous CENP-A was removed by Cre expression and is rescued by replacement with the wild type CENP-A but not with methylation mutant. ( f , g ) Quantitation of CENP-I and CENP-B at the centromere after removing endogenous CENP-A by cre-recombinase. Box-and-whisker plots. Central lines, medians; whiskers, range 5–95 percentile; outliers not shown. ( h ) Schematic of the experiment in I-K. eGFP tagged CENP-A wild type and mutant integrated RPE-CENP-A -/F cells were infected with cre-recombinase virus to remove endogenous CENP-A. Individual colonies were picked after 14 days and expanded. The cells were treated with 0.1 μM Nocodazole for 6 h and then released for one hour, fixed and DAPI stained ( i ) Scale bar, 10 μm. Cells expressing the methylation mutant showed significantly high percentage of ( j ) lagging chromosomes and ( k ) micronuclei formation in the absence of Nocodazole treatment. The experiments were done three times. Mean and s.d. shown. ( l ) Representative images of the RPE cells immunostained for NDC80. Kinetochore localization of NDC80 was decreased in methylation mutant CENP-A. ( m ) Quantitation of NDC80 at the kinetochore after removal of endogenous CENP-A by cre-recombinase and replacement of either wild type or mutant CENP-A. A minimum of 8 prometaphse cells were used for the quantitation. Indicated P values were determined by t -test. Scale bars, 10 μm.

    Journal: Nature Communications

    Article Title: α-amino trimethylation of CENP-A by NRMT is required for full recruitment of the centromere

    doi: 10.1038/ncomms14678

    Figure Lengend Snippet: CENP-A α-amino methylation is required for CCAN formation. ( a ) Schematic of the experiment. ( b ) Representative images of the cells immunostained for CENP-T and CENP-C. Centromeric CENP-T was decreased upon CENP-A knockout and is rescued by replacement of the wild type CENP-A but not with methylation mutant. ( c , d ) Quantitation of CENP-T and CENP-C at the centromere after removal of endogenous CENP-A by cre-recombinase. n =2. A minimum 30 cells in two biological replicates were used for the quantitation. ( e ) Representative images of cells immunostained for CENP-I and CENP-B. CENP-I level decreased when endogenous CENP-A was removed by Cre expression and is rescued by replacement with the wild type CENP-A but not with methylation mutant. ( f , g ) Quantitation of CENP-I and CENP-B at the centromere after removing endogenous CENP-A by cre-recombinase. Box-and-whisker plots. Central lines, medians; whiskers, range 5–95 percentile; outliers not shown. ( h ) Schematic of the experiment in I-K. eGFP tagged CENP-A wild type and mutant integrated RPE-CENP-A -/F cells were infected with cre-recombinase virus to remove endogenous CENP-A. Individual colonies were picked after 14 days and expanded. The cells were treated with 0.1 μM Nocodazole for 6 h and then released for one hour, fixed and DAPI stained ( i ) Scale bar, 10 μm. Cells expressing the methylation mutant showed significantly high percentage of ( j ) lagging chromosomes and ( k ) micronuclei formation in the absence of Nocodazole treatment. The experiments were done three times. Mean and s.d. shown. ( l ) Representative images of the RPE cells immunostained for NDC80. Kinetochore localization of NDC80 was decreased in methylation mutant CENP-A. ( m ) Quantitation of NDC80 at the kinetochore after removal of endogenous CENP-A by cre-recombinase and replacement of either wild type or mutant CENP-A. A minimum of 8 prometaphse cells were used for the quantitation. Indicated P values were determined by t -test. Scale bars, 10 μm.

    Article Snippet: The antibodies used for immunofluorescence were: mouse anti-CENP-A (1:1,000, Cat#13939 Abcam), rabbit anti-me3-CENP-A (1:200), mouse anti-CENP-B (1:250), rabbit anti-CENP-T (1:2,000), rabbit anti-CENP-I (1:1,000), mouse anti-CENP-C (1:1,000), rabbit anti-γ-tubulin (1:1,000; Cat# T5192 Sigma), mouse anti-centrin (1:1,000; Cat# 04-1624 Millipore), mouse anti-α-tubulin (1:1,000).

    Techniques: Methylation, Knock-Out, Mutagenesis, Quantitation Assay, Expressing, Whisker Assay, Infection, Staining

    Loss of CENP-T causes multipolar spindle formation in a p53 dependent fashion. ( a ) Schematic of the experiment in B-D. eGFP tagged CENP-A wild type and mutant integrated RPE-CENP-A -/F cells were infected with cre-recombinase virus to remove endogenous CENP-A. Individual colonies were picked after 14 days and cells were infected with p53 shRNA. The p53 shRNA integrated clones were selected with puromycin. The cells were then synchronized and fixed. ( b ) Western blot showing the efficiency of p53 knockdown. ( c ) Cells showing multipolar spindles in MT1 CENP-A replaced cells but not in wild-type. ( d ) Percentage of cells showing multipolar cells in wild type and mutant cells, based on three independent experiments. ( e ) Western blots showing siRNA mediated knockdown of CENP-T and CENP-I in HeLa T-REx cells. ( f ) Loss of CENP-T and CENP-I leads to mitotic arrest, based on three independent experiments. ( g , h ) CENP-T knockdown but not CENP-I cause multipolar spindle in HeLa T-Rex cells, based on three independent experiments. ( i ) CENP-T knockdown causes multipolar spindles only in p53 null HCT116 cells but not in p53 wild-type HCT116 cells, based on three independent experiments. Error bars indicate s.d. Indicated P value was determined by χ 2 test. Scale bars, 10 μm.

    Journal: Nature Communications

    Article Title: α-amino trimethylation of CENP-A by NRMT is required for full recruitment of the centromere

    doi: 10.1038/ncomms14678

    Figure Lengend Snippet: Loss of CENP-T causes multipolar spindle formation in a p53 dependent fashion. ( a ) Schematic of the experiment in B-D. eGFP tagged CENP-A wild type and mutant integrated RPE-CENP-A -/F cells were infected with cre-recombinase virus to remove endogenous CENP-A. Individual colonies were picked after 14 days and cells were infected with p53 shRNA. The p53 shRNA integrated clones were selected with puromycin. The cells were then synchronized and fixed. ( b ) Western blot showing the efficiency of p53 knockdown. ( c ) Cells showing multipolar spindles in MT1 CENP-A replaced cells but not in wild-type. ( d ) Percentage of cells showing multipolar cells in wild type and mutant cells, based on three independent experiments. ( e ) Western blots showing siRNA mediated knockdown of CENP-T and CENP-I in HeLa T-REx cells. ( f ) Loss of CENP-T and CENP-I leads to mitotic arrest, based on three independent experiments. ( g , h ) CENP-T knockdown but not CENP-I cause multipolar spindle in HeLa T-Rex cells, based on three independent experiments. ( i ) CENP-T knockdown causes multipolar spindles only in p53 null HCT116 cells but not in p53 wild-type HCT116 cells, based on three independent experiments. Error bars indicate s.d. Indicated P value was determined by χ 2 test. Scale bars, 10 μm.

    Article Snippet: The antibodies used for immunofluorescence were: mouse anti-CENP-A (1:1,000, Cat#13939 Abcam), rabbit anti-me3-CENP-A (1:200), mouse anti-CENP-B (1:250), rabbit anti-CENP-T (1:2,000), rabbit anti-CENP-I (1:1,000), mouse anti-CENP-C (1:1,000), rabbit anti-γ-tubulin (1:1,000; Cat# T5192 Sigma), mouse anti-centrin (1:1,000; Cat# 04-1624 Millipore), mouse anti-α-tubulin (1:1,000).

    Techniques: Mutagenesis, Infection, shRNA, Clone Assay, Western Blot

    Labeling of proteins transiently associated with CENP-A and H3.1 nucleosomes. (A) Schematic representation of the experimental approach. (B) (C) Representative images of cells stably expressing indicated proteins fused to the BirA* ligase and HA tag, and incubated with medium supplemented with or without biotin for 5 hours. DNA is visualized by DAPI staining, immunofluorescence for CENP-T is shown in red, biotinylated proteins (B) or BirA*-HA fusion proteins (C) are shown in green. Scale bar is 5μm. (D) Streptavidin purification of biotinylated proteins from indicated cells lines analyzed by immunoblot. Cell media was supplemented with or without biotin for 24 hours. (E) Graph showing SILAC comparison between CENP-A-BirA*-HA biotinylation profile (heavy) versus biotinylation profile of H3.1-BirA-HA* (light) in cells undergoing S phase. Corresponding H/L scores for selected proteins are displayed. The graph represents an average of two independently performed experiments +/− SEM.

    Journal: Developmental cell

    Article Title: Inheritance of CENP-A nucleosomes during DNA replication requires HJURP

    doi: 10.1016/j.devcel.2018.09.003

    Figure Lengend Snippet: Labeling of proteins transiently associated with CENP-A and H3.1 nucleosomes. (A) Schematic representation of the experimental approach. (B) (C) Representative images of cells stably expressing indicated proteins fused to the BirA* ligase and HA tag, and incubated with medium supplemented with or without biotin for 5 hours. DNA is visualized by DAPI staining, immunofluorescence for CENP-T is shown in red, biotinylated proteins (B) or BirA*-HA fusion proteins (C) are shown in green. Scale bar is 5μm. (D) Streptavidin purification of biotinylated proteins from indicated cells lines analyzed by immunoblot. Cell media was supplemented with or without biotin for 24 hours. (E) Graph showing SILAC comparison between CENP-A-BirA*-HA biotinylation profile (heavy) versus biotinylation profile of H3.1-BirA-HA* (light) in cells undergoing S phase. Corresponding H/L scores for selected proteins are displayed. The graph represents an average of two independently performed experiments +/− SEM.

    Article Snippet: Fixed cells were incubated in blocking solution (0.1% Triton-X in PBS, 0.2% BSA, 2% FBS) for 1.5 h at RT, and incubated with indicated primary antibodies for 1.5 h. anti-CENP-T (custom polyclonal antibody, Cocalico), anti-CENP-A (MA1–20832, ThermoFisher) and anti-HA antibodies (mAb HA.11) were used at 1:6000, 1:1000, and 1:1000 dilution, respectively and detected using fluorophore conjugated secondary antibodies (Cy3, Cy5 or FITC, Jackson Immuno Inc.).

    Techniques: Labeling, Stable Transfection, Expressing, Incubation, Staining, Immunofluorescence, Purification

    HJURP is required for CENPA retention across S phase (A) Schematic representation DLD1-Tir1 cell line where HJURP was endogenously tagged with AID-YFP at both alleles. (B) Immunoblot analysis showing the efficiency of IAA dependent HJURP degradation. Ponceau was used as a loading control. The blots in B were separated into two sections due to removal of extraneous samples present on the same blot. (C) Clonogenic assay using the parental DLD1-Tir1 and HJURP-AID-YFP cells plus or minus IAA treatment for 10 days. (D) Representative images of DLD1-Tir1 cells in G1/S arrest and G2 phase. Insets are showing single centromeres and sister centromeres in G1/S and G2 phase cells, respectively. DNA was visualized by DAPI, immunofluorescence for CENP-T is shown in green and CENP-A is shown in red. Scale bar is 2μm. (E) Schematic representation of the experiment in F and H. (F)(H) Representative images of cells in G2 phase and mitosis, respectively, and treated as shown in E. DNA was visualized by DAPI, immunofluorescence for CENP-T is shown in green and CENP-A is shown in red. Scale bar is indicated. (G)(I) Quantification of F and H, respectively. The data normalized to G1/S phase condition (G) and untreated condition (I) within cell lines. Normalized data from four (G) or three (I) independent experiments was plotted using box-and-whisker plot: 5–95 percentile, n > 2946 (G) and 8414 centromeres (I). The statistical significance was calculated using unpaired t-test and the p values are indicated. For reference, red line indicates level of CENP-A retention in (G) untreated parental control in G2 phase or (I) untreated parental control in mitosis. The percent loss of CENP-A was calculated to be 31.53% and 36.68% in G and I, respectively.

    Journal: Developmental cell

    Article Title: Inheritance of CENP-A nucleosomes during DNA replication requires HJURP

    doi: 10.1016/j.devcel.2018.09.003

    Figure Lengend Snippet: HJURP is required for CENPA retention across S phase (A) Schematic representation DLD1-Tir1 cell line where HJURP was endogenously tagged with AID-YFP at both alleles. (B) Immunoblot analysis showing the efficiency of IAA dependent HJURP degradation. Ponceau was used as a loading control. The blots in B were separated into two sections due to removal of extraneous samples present on the same blot. (C) Clonogenic assay using the parental DLD1-Tir1 and HJURP-AID-YFP cells plus or minus IAA treatment for 10 days. (D) Representative images of DLD1-Tir1 cells in G1/S arrest and G2 phase. Insets are showing single centromeres and sister centromeres in G1/S and G2 phase cells, respectively. DNA was visualized by DAPI, immunofluorescence for CENP-T is shown in green and CENP-A is shown in red. Scale bar is 2μm. (E) Schematic representation of the experiment in F and H. (F)(H) Representative images of cells in G2 phase and mitosis, respectively, and treated as shown in E. DNA was visualized by DAPI, immunofluorescence for CENP-T is shown in green and CENP-A is shown in red. Scale bar is indicated. (G)(I) Quantification of F and H, respectively. The data normalized to G1/S phase condition (G) and untreated condition (I) within cell lines. Normalized data from four (G) or three (I) independent experiments was plotted using box-and-whisker plot: 5–95 percentile, n > 2946 (G) and 8414 centromeres (I). The statistical significance was calculated using unpaired t-test and the p values are indicated. For reference, red line indicates level of CENP-A retention in (G) untreated parental control in G2 phase or (I) untreated parental control in mitosis. The percent loss of CENP-A was calculated to be 31.53% and 36.68% in G and I, respectively.

    Article Snippet: Fixed cells were incubated in blocking solution (0.1% Triton-X in PBS, 0.2% BSA, 2% FBS) for 1.5 h at RT, and incubated with indicated primary antibodies for 1.5 h. anti-CENP-T (custom polyclonal antibody, Cocalico), anti-CENP-A (MA1–20832, ThermoFisher) and anti-HA antibodies (mAb HA.11) were used at 1:6000, 1:1000, and 1:1000 dilution, respectively and detected using fluorophore conjugated secondary antibodies (Cy3, Cy5 or FITC, Jackson Immuno Inc.).

    Techniques: Clonogenic Assay, Immunofluorescence, Whisker Assay

    HJURP is required for CENPA inheritance of existing CENP-A nucleosomes. (A) Schematic representation of the DLD1-Tir1 cell line where HJURP was endogenously tagged with AID-YFP and CENP-A was endogenously tagged with the SNAP tag. (B) The immunofluorescence images of the localization profile of HJURP-AID-YFP in cell line shown in A. (C) Immunoblot analysis of the efficiency of IAA dependent HJURP degradation during mitosis demonstrated by staining with HJURP antibody. Ponceau staining was used as a loading control. The blots were separated in two sections due to removal of empty lanes from the original blot. (D) (F) Schematic representation of the experiment (top). Representative images of cells at indicated time points and treated as shown in the top panel. DNA was visualized by DAPI, immunofluorescence for CENP-T is shown in green and CENP-A is shown in red (bottom). Scale bar is 2μm. (E)(G) Quantification of D and F, respectively. The data was normalized to initial G1/S condition (E) or initial signal (G) within each individual experiment. Normalized data from two (E) or three (G) independent experiments was plotted using box-and-whisker plot: 5–95 percentile, n at least 4674 (E) and 2295 (G). The statistical significance was calculated using unpaired t-test and the p values are indicated. For reference, red line indicates the median level of CENP-A retention in untreated control. The percent loss of CENP-A was calculated to be 38.41% and 27.91% in E and G, respectively.

    Journal: Developmental cell

    Article Title: Inheritance of CENP-A nucleosomes during DNA replication requires HJURP

    doi: 10.1016/j.devcel.2018.09.003

    Figure Lengend Snippet: HJURP is required for CENPA inheritance of existing CENP-A nucleosomes. (A) Schematic representation of the DLD1-Tir1 cell line where HJURP was endogenously tagged with AID-YFP and CENP-A was endogenously tagged with the SNAP tag. (B) The immunofluorescence images of the localization profile of HJURP-AID-YFP in cell line shown in A. (C) Immunoblot analysis of the efficiency of IAA dependent HJURP degradation during mitosis demonstrated by staining with HJURP antibody. Ponceau staining was used as a loading control. The blots were separated in two sections due to removal of empty lanes from the original blot. (D) (F) Schematic representation of the experiment (top). Representative images of cells at indicated time points and treated as shown in the top panel. DNA was visualized by DAPI, immunofluorescence for CENP-T is shown in green and CENP-A is shown in red (bottom). Scale bar is 2μm. (E)(G) Quantification of D and F, respectively. The data was normalized to initial G1/S condition (E) or initial signal (G) within each individual experiment. Normalized data from two (E) or three (G) independent experiments was plotted using box-and-whisker plot: 5–95 percentile, n at least 4674 (E) and 2295 (G). The statistical significance was calculated using unpaired t-test and the p values are indicated. For reference, red line indicates the median level of CENP-A retention in untreated control. The percent loss of CENP-A was calculated to be 38.41% and 27.91% in E and G, respectively.

    Article Snippet: Fixed cells were incubated in blocking solution (0.1% Triton-X in PBS, 0.2% BSA, 2% FBS) for 1.5 h at RT, and incubated with indicated primary antibodies for 1.5 h. anti-CENP-T (custom polyclonal antibody, Cocalico), anti-CENP-A (MA1–20832, ThermoFisher) and anti-HA antibodies (mAb HA.11) were used at 1:6000, 1:1000, and 1:1000 dilution, respectively and detected using fluorophore conjugated secondary antibodies (Cy3, Cy5 or FITC, Jackson Immuno Inc.).

    Techniques: Immunofluorescence, Staining, Whisker Assay

    MCM2 binds CENP-A and is involved in its maintenance during DNA replication (A) Schematic representation of constructs used in B and C. The CENP-A and H3.1 domain structure is shown. The alignment of an 8 amino acid stretch corresponding to both histones demonstrates the conservation of Arginine 63 and Lysine 64 between the variants. (B) MBP-MCM2-HBD in vitro pull down demonstrating the interaction with indicated histone variants in the wild type and mutant form. (C) Table indicating Kd values measured SPR to assess the strength of interaction between MBP-MCM2-HBD and indicated histone variants in the wild type and mutant form. (D) Representative images of HeLa cells expressing either CENP-A WT -GFP or CENP-A RK- > AA -GFP mutant at indicated cell cycle stages. DNA was visualized by DAPI, immunofluorescence for CENP-T is shown in red, expressed CENP-A was detected by GFP signal. Scale bar is 2μm. (E) Quantification of D. The data vas plotted using box-and-whisker plot: 5–95 percentile, n > 5660. The percent change of the levels of centromeric CENP-A WT -GFP and CENP-A RK- > AA -GFP forms between experimental time points is indicated, (***) indicates p value

    Journal: Developmental cell

    Article Title: Inheritance of CENP-A nucleosomes during DNA replication requires HJURP

    doi: 10.1016/j.devcel.2018.09.003

    Figure Lengend Snippet: MCM2 binds CENP-A and is involved in its maintenance during DNA replication (A) Schematic representation of constructs used in B and C. The CENP-A and H3.1 domain structure is shown. The alignment of an 8 amino acid stretch corresponding to both histones demonstrates the conservation of Arginine 63 and Lysine 64 between the variants. (B) MBP-MCM2-HBD in vitro pull down demonstrating the interaction with indicated histone variants in the wild type and mutant form. (C) Table indicating Kd values measured SPR to assess the strength of interaction between MBP-MCM2-HBD and indicated histone variants in the wild type and mutant form. (D) Representative images of HeLa cells expressing either CENP-A WT -GFP or CENP-A RK- > AA -GFP mutant at indicated cell cycle stages. DNA was visualized by DAPI, immunofluorescence for CENP-T is shown in red, expressed CENP-A was detected by GFP signal. Scale bar is 2μm. (E) Quantification of D. The data vas plotted using box-and-whisker plot: 5–95 percentile, n > 5660. The percent change of the levels of centromeric CENP-A WT -GFP and CENP-A RK- > AA -GFP forms between experimental time points is indicated, (***) indicates p value

    Article Snippet: Fixed cells were incubated in blocking solution (0.1% Triton-X in PBS, 0.2% BSA, 2% FBS) for 1.5 h at RT, and incubated with indicated primary antibodies for 1.5 h. anti-CENP-T (custom polyclonal antibody, Cocalico), anti-CENP-A (MA1–20832, ThermoFisher) and anti-HA antibodies (mAb HA.11) were used at 1:6000, 1:1000, and 1:1000 dilution, respectively and detected using fluorophore conjugated secondary antibodies (Cy3, Cy5 or FITC, Jackson Immuno Inc.).

    Techniques: Construct, In Vitro, Mutagenesis, SPR Assay, Expressing, Immunofluorescence, Whisker Assay

    The CENP-T/W complex is required in each mitosis and does not exhibit persistent binding to centromeres. (A) CENPs -T and -W were depleted by RNAi in HeLa cells, resulting in defects in spindle assembly assayed by immunofluorescence for tubulin (green) and centromeres (red) with DNA stained with DAPI (blue). CENP-W depleted cells exhibit a high frequency of multipolar spindles. Depletion of CENP-T was less severe resulting in characteristic fusiform spindle structures. (B) CENP-W is required for robust mitosis in each cell cycle. Mitotic kinetics were assayed using histone H2B-GFP HeLa cells treated with CENP-W siRNA for 48 (blue circles) or 24 h (red triangles), scoring time between NEB (defined as onset of deformations in nuclear chromatin) and anaphase onset and are plotted by rank order of individual cells. Mean time for scrambled siRNA control (dark X) and untreated cells (light X) is shown as a green line, with +1 standard deviation (SD) marked with a red line. Inset graph reports the percentage of cells with NEB-anaphase times in excess of +1 SD and the average delay in anaphase onset. (C) Experimental schematic describes the pulse chase approach to assay the heritability of CLIP tagged CENPs -T and -W. (D) CENPs -T and -W do not persist at centromeres. CLIP signal intensity coincident with centromeres was quantified at the time of pulse and 24 h later. While CENP-A-SNAP signal was depleted by approximately 50%, as expected, CENP-T-CLIP and CENP-W-CLIP signal was reduced to background levels after 24 h.

    Journal: PLoS Biology

    Article Title: Premitotic Assembly of Human CENPs -T and -W Switches Centromeric Chromatin to a Mitotic State

    doi: 10.1371/journal.pbio.1001082

    Figure Lengend Snippet: The CENP-T/W complex is required in each mitosis and does not exhibit persistent binding to centromeres. (A) CENPs -T and -W were depleted by RNAi in HeLa cells, resulting in defects in spindle assembly assayed by immunofluorescence for tubulin (green) and centromeres (red) with DNA stained with DAPI (blue). CENP-W depleted cells exhibit a high frequency of multipolar spindles. Depletion of CENP-T was less severe resulting in characteristic fusiform spindle structures. (B) CENP-W is required for robust mitosis in each cell cycle. Mitotic kinetics were assayed using histone H2B-GFP HeLa cells treated with CENP-W siRNA for 48 (blue circles) or 24 h (red triangles), scoring time between NEB (defined as onset of deformations in nuclear chromatin) and anaphase onset and are plotted by rank order of individual cells. Mean time for scrambled siRNA control (dark X) and untreated cells (light X) is shown as a green line, with +1 standard deviation (SD) marked with a red line. Inset graph reports the percentage of cells with NEB-anaphase times in excess of +1 SD and the average delay in anaphase onset. (C) Experimental schematic describes the pulse chase approach to assay the heritability of CLIP tagged CENPs -T and -W. (D) CENPs -T and -W do not persist at centromeres. CLIP signal intensity coincident with centromeres was quantified at the time of pulse and 24 h later. While CENP-A-SNAP signal was depleted by approximately 50%, as expected, CENP-T-CLIP and CENP-W-CLIP signal was reduced to background levels after 24 h.

    Article Snippet: Antibodies: anti-CENP-T (Bethyl), anti-CENP-W (C6ORF173 antibody Abcam, ab75827), anti-tubulin monoclonal (DN1A Sigma), anti-H3P Ser10 (Millipore), cyclin-B (Upstate).

    Techniques: Binding Assay, Immunofluorescence, Staining, Standard Deviation, Pulse Chase, Cross-linking Immunoprecipitation

    CENPs -T and -W assemble predominantly in late S-phase and G2. (A) Schematic description of the CLIP quench-chase-pulse-chase experiment used to assay the timing of assembly of CLIP tagged CENPs -T and -W at centromeres using synchronized HeLa cells. (B) CLIP-tagged CENPs -T and -W are localized at centromeres prior to the onset of anaphase, indicating that newly synthesized CENP-T and CENP-W assemble at the centromere in the proximal cell cycle. (C) Progressive assembly of pulsed CENP-T and CENP-W in S-phase and G2. Cells were labelled with PCNA and phospho-histone H3 antibodies to document position in the cell cycle. Cells judged to be in earlier stages of S-phase have no detectable CLIP signal at centromeres, while cells later in S-phase and G2 have robust centromere-associated CLIP signal. (D) Centromere-associated CENP-T-CLIP and CENP-W-CLIP fluorescence were quantified relative to progression through the cell cycle, showing an increased signal intensity at centromeres coinciding with progression through S-phase and G2.

    Journal: PLoS Biology

    Article Title: Premitotic Assembly of Human CENPs -T and -W Switches Centromeric Chromatin to a Mitotic State

    doi: 10.1371/journal.pbio.1001082

    Figure Lengend Snippet: CENPs -T and -W assemble predominantly in late S-phase and G2. (A) Schematic description of the CLIP quench-chase-pulse-chase experiment used to assay the timing of assembly of CLIP tagged CENPs -T and -W at centromeres using synchronized HeLa cells. (B) CLIP-tagged CENPs -T and -W are localized at centromeres prior to the onset of anaphase, indicating that newly synthesized CENP-T and CENP-W assemble at the centromere in the proximal cell cycle. (C) Progressive assembly of pulsed CENP-T and CENP-W in S-phase and G2. Cells were labelled with PCNA and phospho-histone H3 antibodies to document position in the cell cycle. Cells judged to be in earlier stages of S-phase have no detectable CLIP signal at centromeres, while cells later in S-phase and G2 have robust centromere-associated CLIP signal. (D) Centromere-associated CENP-T-CLIP and CENP-W-CLIP fluorescence were quantified relative to progression through the cell cycle, showing an increased signal intensity at centromeres coinciding with progression through S-phase and G2.

    Article Snippet: Antibodies: anti-CENP-T (Bethyl), anti-CENP-W (C6ORF173 antibody Abcam, ab75827), anti-tubulin monoclonal (DN1A Sigma), anti-H3P Ser10 (Millipore), cyclin-B (Upstate).

    Techniques: Cross-linking Immunoprecipitation, Pulse Chase, Synthesized, Fluorescence

    CENP-T and -W assembly occur through a dynamic exchange mechanism in S-phase and G2. (A) Asynchronous HeLa cells expressing CENP-T and CENP-W CLIP were used to assay timing of assembly in an unperturbed cell population. Cells were cell cycle staged by counterstaining with PCNA and phospho-histone H3. (B) Cells were classified on the basis of CLIP signal at centromeres and cell cycle stage. (C) FRAP of GFP derivatives of CENP-T only occurs during late S-phase and G2 indicating loading takes place during this period. (D) Centromere-associated GFP-CENP-T was photobleached and allowed to recover to approximately 40%. Following a second bleach event, recovery continued reaching approximately 40%, indicating an exchange-based dynamic loading process during this time period. (E) Double FRAP of GFP-CENP-W, as for CENP-T. (F) Fluorescence loss after photoactivation. Photoactivatable GFP-CENP-W was activated in G2 cells. Following a chase period of 3 h, the majority of the fluorescent signal had dissociated from the centromere.

    Journal: PLoS Biology

    Article Title: Premitotic Assembly of Human CENPs -T and -W Switches Centromeric Chromatin to a Mitotic State

    doi: 10.1371/journal.pbio.1001082

    Figure Lengend Snippet: CENP-T and -W assembly occur through a dynamic exchange mechanism in S-phase and G2. (A) Asynchronous HeLa cells expressing CENP-T and CENP-W CLIP were used to assay timing of assembly in an unperturbed cell population. Cells were cell cycle staged by counterstaining with PCNA and phospho-histone H3. (B) Cells were classified on the basis of CLIP signal at centromeres and cell cycle stage. (C) FRAP of GFP derivatives of CENP-T only occurs during late S-phase and G2 indicating loading takes place during this period. (D) Centromere-associated GFP-CENP-T was photobleached and allowed to recover to approximately 40%. Following a second bleach event, recovery continued reaching approximately 40%, indicating an exchange-based dynamic loading process during this time period. (E) Double FRAP of GFP-CENP-W, as for CENP-T. (F) Fluorescence loss after photoactivation. Photoactivatable GFP-CENP-W was activated in G2 cells. Following a chase period of 3 h, the majority of the fluorescent signal had dissociated from the centromere.

    Article Snippet: Antibodies: anti-CENP-T (Bethyl), anti-CENP-W (C6ORF173 antibody Abcam, ab75827), anti-tubulin monoclonal (DN1A Sigma), anti-H3P Ser10 (Millipore), cyclin-B (Upstate).

    Techniques: Expressing, Cross-linking Immunoprecipitation, Fluorescence

    Analysis of CENP-T and -W in the cell cycle. (A) HeLa cells were fractionated across the cell cycle using a double thymidine protocol. The relative abundance of CENP-T (red) and -W (blue) transcripts were measured by qPCR. CENP-A (dashed grey) is shown as a reference. No significant periodic RNA accumulation was observed. (B) Protein obtained from cell cycle fractions was examined by Western blot for CENP-T and CENP-W relative to tubulin (loading control), cyclin B, and phospho-histone H3 (Ser10). (C) The relative abundance of centromere-associated CENP-W was estimated using a cell line constitutively expressing a CLIP-tagged fusion protein. CENP-W-CLIP was labelled with CLIP-505 at steady state and fluorescence intensity quantified. Cells were counterstained for CENP-A to define centromeres and for PCNA and phospho-histone H3 to resolve the cell cycle stage of individual cells (see Figure S2 ). Cells were scored as S-phase (PCNA-positive), G2 and M (phospho-histone H3-positive), or G1 (negative for either PCNA or H3P). Early and late S-phase designations were made on the basis of PNCA distribution. (D) An example of cell staining showing CLIP-505-CENP-W assembly in a pair of cells in S-phase and undetected in a pair of G2 cells.

    Journal: PLoS Biology

    Article Title: Premitotic Assembly of Human CENPs -T and -W Switches Centromeric Chromatin to a Mitotic State

    doi: 10.1371/journal.pbio.1001082

    Figure Lengend Snippet: Analysis of CENP-T and -W in the cell cycle. (A) HeLa cells were fractionated across the cell cycle using a double thymidine protocol. The relative abundance of CENP-T (red) and -W (blue) transcripts were measured by qPCR. CENP-A (dashed grey) is shown as a reference. No significant periodic RNA accumulation was observed. (B) Protein obtained from cell cycle fractions was examined by Western blot for CENP-T and CENP-W relative to tubulin (loading control), cyclin B, and phospho-histone H3 (Ser10). (C) The relative abundance of centromere-associated CENP-W was estimated using a cell line constitutively expressing a CLIP-tagged fusion protein. CENP-W-CLIP was labelled with CLIP-505 at steady state and fluorescence intensity quantified. Cells were counterstained for CENP-A to define centromeres and for PCNA and phospho-histone H3 to resolve the cell cycle stage of individual cells (see Figure S2 ). Cells were scored as S-phase (PCNA-positive), G2 and M (phospho-histone H3-positive), or G1 (negative for either PCNA or H3P). Early and late S-phase designations were made on the basis of PNCA distribution. (D) An example of cell staining showing CLIP-505-CENP-W assembly in a pair of cells in S-phase and undetected in a pair of G2 cells.

    Article Snippet: Antibodies: anti-CENP-T (Bethyl), anti-CENP-W (C6ORF173 antibody Abcam, ab75827), anti-tubulin monoclonal (DN1A Sigma), anti-H3P Ser10 (Millipore), cyclin-B (Upstate).

    Techniques: Real-time Polymerase Chain Reaction, Western Blot, Expressing, Cross-linking Immunoprecipitation, Fluorescence, Staining

    MHF1 and MHF2 are present in two functionally distinct complexes. (A) Mass spectrometric analysis shows that MHF1 (CENP-S) and MHF2 (CENP-X) are present in both FANCM and MHF1 immunoprecipitates isolated from the chromatin fraction of HeLa cells by either FANCM or MHF1 antibody, whereas other centromere proteins (CENP-T, CENP-K, CENP-U, CENP-H) were detected only in the MHF1 immunoprecipitate. (B , C) Immunoblotting shows that MHF1, MHF2, FANCM and other components of the FA core and Bloom complexes were present in both FANCM and MHF1 immunoprecipitates, but CENP-K and CENP-T were detected only in the MHF1 immunoprecipitate. (D) Immunofluorescence analyses of wild-type (WT) or FANCM −/− DT40 cells with different antibodies (green) as indicated. DNA was stained by DAPI (blue). The signal intensities of CENP-T, Ndc80 and CENP-S at each kinetochore detected by immunofluorescence were measured relative to an adjacent background signal ( n > 10 cells; average intensity ± SD). The scale bar represents 10 μm. (E) Immunoblotting shows the levels of FANCD2 and CENP-T in CENP-T conditional knockout (KO) DT40 cells with or without the treatment of tetracycline (tet) for 24 h. The ratio between the monoubiquitinated and non-ubiquitinated FANCD2 (L/S) is shown. Cells were treated with MMC (50 ng/ml) for 16 h. Actin was used as a loading control. (F) Histograms showing the levels of spontaneous SCEs in CENP-T conditional KO DT40 cells with or without the treatment of tet for 24 h. The mean number of SCEs per metaphase is listed. P -value was calculated using the Student's t -test. (G) .

    Journal: Cell Research

    Article Title: The histone-fold complex MHF is remodeled by FANCM to recognize branched DNA and protect genome stability

    doi: 10.1038/cr.2014.42

    Figure Lengend Snippet: MHF1 and MHF2 are present in two functionally distinct complexes. (A) Mass spectrometric analysis shows that MHF1 (CENP-S) and MHF2 (CENP-X) are present in both FANCM and MHF1 immunoprecipitates isolated from the chromatin fraction of HeLa cells by either FANCM or MHF1 antibody, whereas other centromere proteins (CENP-T, CENP-K, CENP-U, CENP-H) were detected only in the MHF1 immunoprecipitate. (B , C) Immunoblotting shows that MHF1, MHF2, FANCM and other components of the FA core and Bloom complexes were present in both FANCM and MHF1 immunoprecipitates, but CENP-K and CENP-T were detected only in the MHF1 immunoprecipitate. (D) Immunofluorescence analyses of wild-type (WT) or FANCM −/− DT40 cells with different antibodies (green) as indicated. DNA was stained by DAPI (blue). The signal intensities of CENP-T, Ndc80 and CENP-S at each kinetochore detected by immunofluorescence were measured relative to an adjacent background signal ( n > 10 cells; average intensity ± SD). The scale bar represents 10 μm. (E) Immunoblotting shows the levels of FANCD2 and CENP-T in CENP-T conditional knockout (KO) DT40 cells with or without the treatment of tetracycline (tet) for 24 h. The ratio between the monoubiquitinated and non-ubiquitinated FANCD2 (L/S) is shown. Cells were treated with MMC (50 ng/ml) for 16 h. Actin was used as a loading control. (F) Histograms showing the levels of spontaneous SCEs in CENP-T conditional KO DT40 cells with or without the treatment of tet for 24 h. The mean number of SCEs per metaphase is listed. P -value was calculated using the Student's t -test. (G) .

    Article Snippet: Anti-human CENP-T antibody was purchased from Bethyl Laboratories, Inc.

    Techniques: Isolation, Immunofluorescence, Staining, Knock-Out

    Model of centromere function mediated by centromeric chromatin and DNA sequences At exit of mitosis, centromeric chromatin replication and identity is mediated by CENP-A (in red) deposition via interaction with HJURP. CENP-A then mediates the assembly of CENP-C (in green) in mid-G1 followed by CENP-N/L (in orange) during S-phase. These steps might be interconnected. At this point, CENP-A becomes dispensable for mitotic centromere function as long as CENP-B (in light blue) is stably bound to centromeric sequences to support CENP-C binding. Assembly of the other subunits of the CCAN, such as CENP-T/W (in yellow) and HIKM (in brown), allows the full recruitment of the kinetochore complex (in grey) required to mediate centromere function. In summary, we propose that the kinetochore is tethered to the centromere through a dual linkage of CENP-A chromatin and CENP-B-bound DNA sequences, as the two major links from the DNA to the kinetochore to mediate successful chromosome segregation.

    Journal: Cell reports

    Article Title: CENP-A is dispensable for mitotic centromere function after initial centromere/kinetochore assembly

    doi: 10.1016/j.celrep.2016.10.084

    Figure Lengend Snippet: Model of centromere function mediated by centromeric chromatin and DNA sequences At exit of mitosis, centromeric chromatin replication and identity is mediated by CENP-A (in red) deposition via interaction with HJURP. CENP-A then mediates the assembly of CENP-C (in green) in mid-G1 followed by CENP-N/L (in orange) during S-phase. These steps might be interconnected. At this point, CENP-A becomes dispensable for mitotic centromere function as long as CENP-B (in light blue) is stably bound to centromeric sequences to support CENP-C binding. Assembly of the other subunits of the CCAN, such as CENP-T/W (in yellow) and HIKM (in brown), allows the full recruitment of the kinetochore complex (in grey) required to mediate centromere function. In summary, we propose that the kinetochore is tethered to the centromere through a dual linkage of CENP-A chromatin and CENP-B-bound DNA sequences, as the two major links from the DNA to the kinetochore to mediate successful chromosome segregation.

    Article Snippet: Cells were fixed in 4% formaldehyde at room temperature or in methanol at −20°C for 10 min. Incubations with primary antibodies were conducted in blocking buffer for 1 hr at room temperature using the following antibodies: CENP-A (Abcam, 1:1500), CENP-C (MBL, 1:1000), CENP-B (Abcam, 1:1000), ACA (Antibodies Inc, 1:500), Hec1 (Abcam, 1:1000), Dsn1 (1:1000, a gift from A. Desai, Ludwig, San Diego), CENP-I (a gift from Song-Tao Liu, University of Toledo, OH), DM1A (α-tubulin, 1:2000), CENP-T (Covance, 1:5000) and HA-11 (Covance, 1:1000).

    Techniques: Stable Transfection, Binding Assay

    CENP-A is required to regulate early steps of centromere assembly, such as for CENP-C and CENP-N, but not CENP-T (A) Schematic of the experiments shown in B and C. (B) Representative immunofluorescence images show de novo CENP-C AID-mRFP loading at centromeres. (C) Bar graphs showing centromere intensities of CENP-C AID-mRFP in the indicated cell lines. Error bars represent the SEM of three independent experiments. Individual Σn = ~30 cells, Σn = 25 centromeres for cell. Unpaired t test: *** p

    Journal: Cell reports

    Article Title: CENP-A is dispensable for mitotic centromere function after initial centromere/kinetochore assembly

    doi: 10.1016/j.celrep.2016.10.084

    Figure Lengend Snippet: CENP-A is required to regulate early steps of centromere assembly, such as for CENP-C and CENP-N, but not CENP-T (A) Schematic of the experiments shown in B and C. (B) Representative immunofluorescence images show de novo CENP-C AID-mRFP loading at centromeres. (C) Bar graphs showing centromere intensities of CENP-C AID-mRFP in the indicated cell lines. Error bars represent the SEM of three independent experiments. Individual Σn = ~30 cells, Σn = 25 centromeres for cell. Unpaired t test: *** p

    Article Snippet: Cells were fixed in 4% formaldehyde at room temperature or in methanol at −20°C for 10 min. Incubations with primary antibodies were conducted in blocking buffer for 1 hr at room temperature using the following antibodies: CENP-A (Abcam, 1:1500), CENP-C (MBL, 1:1000), CENP-B (Abcam, 1:1000), ACA (Antibodies Inc, 1:500), Hec1 (Abcam, 1:1000), Dsn1 (1:1000, a gift from A. Desai, Ludwig, San Diego), CENP-I (a gift from Song-Tao Liu, University of Toledo, OH), DM1A (α-tubulin, 1:2000), CENP-T (Covance, 1:5000) and HA-11 (Covance, 1:1000).

    Techniques: Immunofluorescence

    The B. mori CENP-T protein is essential in vivo . A) CRISPR/Cas9-introduced mutation in the Cenp-T coding sequence. Top: Alignment between wild-type (WT) and mutant (+7) sequences showing a seven base-pair insertion of the Cenp-T gene. Bottom: Schematic of WT and truncated protein products in the CRISPR mutant. PAM site (red), premature stop codon (blue) and Cas9 cleavage site (red arrow) are indicated. B) Hatching rate of the WT and Cenp-T mutant strains. The percentages of hatched (grey), unhatched (green) and eggs that died prior to hatching (black) are indicated for the different crossing patterns between WT (+/+) and heterozygous Cenp-T mutants (+/KO or KO/+). The number of independent crosses n is shown above. The raw data are shown in Table S3. C) Percentages of genotypes from larvae derived from the cross between two heterozygous Cenp-T mutants. The number n of analyzed larvae is indicated. The raw data are shown in Table S4.

    Journal: bioRxiv

    Article Title: CenH3-independent kinetochore assembly in Lepidoptera requires CENP-T

    doi: 10.1101/836262

    Figure Lengend Snippet: The B. mori CENP-T protein is essential in vivo . A) CRISPR/Cas9-introduced mutation in the Cenp-T coding sequence. Top: Alignment between wild-type (WT) and mutant (+7) sequences showing a seven base-pair insertion of the Cenp-T gene. Bottom: Schematic of WT and truncated protein products in the CRISPR mutant. PAM site (red), premature stop codon (blue) and Cas9 cleavage site (red arrow) are indicated. B) Hatching rate of the WT and Cenp-T mutant strains. The percentages of hatched (grey), unhatched (green) and eggs that died prior to hatching (black) are indicated for the different crossing patterns between WT (+/+) and heterozygous Cenp-T mutants (+/KO or KO/+). The number of independent crosses n is shown above. The raw data are shown in Table S3. C) Percentages of genotypes from larvae derived from the cross between two heterozygous Cenp-T mutants. The number n of analyzed larvae is indicated. The raw data are shown in Table S4.

    Article Snippet: Similarly, HMM profile searches within known protein structures identified the known Gallus gallus CENP-T as the best hit (Figure S2B).

    Techniques: In Vivo, CRISPR, Mutagenesis, Sequencing, Derivative Assay

    Identification of kinetochore components including CENP-T in CenH3-deficient Lepidoptera. A) Schematic kinetochore organization in vertebrates and fungi. Boxes indicate subcomplexes within inner and outer kinetochores. Kinetochore components present in Lepidoptera are highlighted in bold ([ 29 ], this study). B) The table lists the number of peptides and coverages (in parenthesis) of S. frugiperda proteins identified by mass spectrometry, which were enriched in the kinetochore IPs over the control. The corresponding homologs in B. mori and descriptions based on homology predictions are listed alongside. C) Top: Graphical representation of the B. mori CENP-T and G. gallus CENP-T sequence features showing the location of the histone fold domains (blue box), CENP-T family specific extension (green box), Arginine-rich ( E value 4.7×10 −5 and E value 6.0×10 −7 , respectively) and Proline-rich regions ( E value 3.7×10 −4 and E value 3.4×10 −8 , respectively). Bottom: Multiple alignment of the Cterminal extension of vertebrate and insect CENP-T proteins. B. mori (above) and G. gallus (below) secondary-structure predictions are derived from the Jpred4 (α-helical regions are in red; β-strands in yellow) [ 75 ]. Background colouring of the residues is based on the clustalx colouring scheme. Full-length insect CENP-T sequences and accession numbers if available are listed in Table S2.

    Journal: bioRxiv

    Article Title: CenH3-independent kinetochore assembly in Lepidoptera requires CENP-T

    doi: 10.1101/836262

    Figure Lengend Snippet: Identification of kinetochore components including CENP-T in CenH3-deficient Lepidoptera. A) Schematic kinetochore organization in vertebrates and fungi. Boxes indicate subcomplexes within inner and outer kinetochores. Kinetochore components present in Lepidoptera are highlighted in bold ([ 29 ], this study). B) The table lists the number of peptides and coverages (in parenthesis) of S. frugiperda proteins identified by mass spectrometry, which were enriched in the kinetochore IPs over the control. The corresponding homologs in B. mori and descriptions based on homology predictions are listed alongside. C) Top: Graphical representation of the B. mori CENP-T and G. gallus CENP-T sequence features showing the location of the histone fold domains (blue box), CENP-T family specific extension (green box), Arginine-rich ( E value 4.7×10 −5 and E value 6.0×10 −7 , respectively) and Proline-rich regions ( E value 3.7×10 −4 and E value 3.4×10 −8 , respectively). Bottom: Multiple alignment of the Cterminal extension of vertebrate and insect CENP-T proteins. B. mori (above) and G. gallus (below) secondary-structure predictions are derived from the Jpred4 (α-helical regions are in red; β-strands in yellow) [ 75 ]. Background colouring of the residues is based on the clustalx colouring scheme. Full-length insect CENP-T sequences and accession numbers if available are listed in Table S2.

    Article Snippet: Similarly, HMM profile searches within known protein structures identified the known Gallus gallus CENP-T as the best hit (Figure S2B).

    Techniques: Mass Spectrometry, Sequencing, Derivative Assay

    Ectopic CENP-T and CENP-I are sufficient to recruit the outer kinetochore in S. frugiperda cells. A) Representative images showing localization pattern of endogenous CENP-T, Dsn1 and Spc24/25 (in grey) in Sf9-LacO cells transiently expressing LacI-GFP (top row), CENP-T-GFP-LacI (second row) and ΔN-CENP-T-GFP-LacI (bottom row) constructs (in green). Endogenous CENP-T and Dsn1 (in grey) colocalize with CENP-T-GFP-LacI and ΔN-CENP-T-GFP-LacI, but not with LacI-GFP foci. Scale bar: 5μm. B) Quantifications of mean fluorescence intensity ratio of CENP-T, Dsn1 and Spc24/25 at the LacI foci compared to levels of CENP-T, Dsn1 and Spc24/25, respectively at endogenous sites (n= 10 cells analyzed). Statistical significance was tested using the Mann and Whitney test (p

    Journal: bioRxiv

    Article Title: CenH3-independent kinetochore assembly in Lepidoptera requires CENP-T

    doi: 10.1101/836262

    Figure Lengend Snippet: Ectopic CENP-T and CENP-I are sufficient to recruit the outer kinetochore in S. frugiperda cells. A) Representative images showing localization pattern of endogenous CENP-T, Dsn1 and Spc24/25 (in grey) in Sf9-LacO cells transiently expressing LacI-GFP (top row), CENP-T-GFP-LacI (second row) and ΔN-CENP-T-GFP-LacI (bottom row) constructs (in green). Endogenous CENP-T and Dsn1 (in grey) colocalize with CENP-T-GFP-LacI and ΔN-CENP-T-GFP-LacI, but not with LacI-GFP foci. Scale bar: 5μm. B) Quantifications of mean fluorescence intensity ratio of CENP-T, Dsn1 and Spc24/25 at the LacI foci compared to levels of CENP-T, Dsn1 and Spc24/25, respectively at endogenous sites (n= 10 cells analyzed). Statistical significance was tested using the Mann and Whitney test (p

    Article Snippet: Similarly, HMM profile searches within known protein structures identified the known Gallus gallus CENP-T as the best hit (Figure S2B).

    Techniques: Expressing, Construct, Fluorescence

    Homologs of CENP-T are present in all other CenH3-deficient insects. Holocentric insect orders and species are indicated in blue, and inferred multiple transitions to holocentric chromosomes are labeled with “H”. Using protein homology searches of genomes or assembled transcriptomes, the ability or inability to find CenH3 and CENP-T homologs are indicated by a black or white box, respectively. Note that while our previous studies did not identify any holocentric insect species that encodes for CenH3 [ 29 ], searches in additional organisms revealed the presence of putative CenH3 proteins in water striders (Hemiptera). This result provides new insights into the transition to CenH3-deficient holocentric architectures in that the change in centromeric architecture appears to precede the loss of CenH3, perhaps by providing the necessary conditions to allow the loss of this otherwise essential component.

    Journal: bioRxiv

    Article Title: CenH3-independent kinetochore assembly in Lepidoptera requires CENP-T

    doi: 10.1101/836262

    Figure Lengend Snippet: Homologs of CENP-T are present in all other CenH3-deficient insects. Holocentric insect orders and species are indicated in blue, and inferred multiple transitions to holocentric chromosomes are labeled with “H”. Using protein homology searches of genomes or assembled transcriptomes, the ability or inability to find CenH3 and CENP-T homologs are indicated by a black or white box, respectively. Note that while our previous studies did not identify any holocentric insect species that encodes for CenH3 [ 29 ], searches in additional organisms revealed the presence of putative CenH3 proteins in water striders (Hemiptera). This result provides new insights into the transition to CenH3-deficient holocentric architectures in that the change in centromeric architecture appears to precede the loss of CenH3, perhaps by providing the necessary conditions to allow the loss of this otherwise essential component.

    Article Snippet: Similarly, HMM profile searches within known protein structures identified the known Gallus gallus CENP-T as the best hit (Figure S2B).

    Techniques: Labeling

    Depletion of CCAN components affects CENP-T and outer kinetochore recruitment in B. mori cells. A) Representative images of mitotic BmN4-SID1 cells showing the levels of endogenous CENP-T, Dsn1 and Spc24/25 with and without depletion of inner (CENP-T, CENP-I, CENP-M, CENP-N) and outer (Dsn1 and Spc24) kinetochore components. Scale bar: 10μm. B) Quantification of mean fluorescence intensity for CENP-T, Dsn1 and Spc24/25 signals in the control or upon kinetochore depletions. Statistical significance was tested using the Mann and Whitney test (p

    Journal: bioRxiv

    Article Title: CenH3-independent kinetochore assembly in Lepidoptera requires CENP-T

    doi: 10.1101/836262

    Figure Lengend Snippet: Depletion of CCAN components affects CENP-T and outer kinetochore recruitment in B. mori cells. A) Representative images of mitotic BmN4-SID1 cells showing the levels of endogenous CENP-T, Dsn1 and Spc24/25 with and without depletion of inner (CENP-T, CENP-I, CENP-M, CENP-N) and outer (Dsn1 and Spc24) kinetochore components. Scale bar: 10μm. B) Quantification of mean fluorescence intensity for CENP-T, Dsn1 and Spc24/25 signals in the control or upon kinetochore depletions. Statistical significance was tested using the Mann and Whitney test (p

    Article Snippet: Similarly, HMM profile searches within known protein structures identified the known Gallus gallus CENP-T as the best hit (Figure S2B).

    Techniques: Fluorescence

    Summary of the permitted and restricted assembly steps in human cell extracts. (A) Experimental approaches to reconstitute kinetochore assembly and function in vitro using mitotic HeLa cell extracts and the main conclusions derived from this study. (B) Simplified scheme indicating the permitted assembly steps (green check-mark), moderately/weakly efficient reactions (blue check-mark), and restricted interactions (red crosses). Symbols “D” indicate phospho-mimetic substitutions in CENP-T.

    Journal: bioRxiv

    Article Title: Reconstitution of human kinetochore in mitotic cell extracts reveals permitted and restricted assembly steps

    doi: 10.1101/2020.07.15.203109

    Figure Lengend Snippet: Summary of the permitted and restricted assembly steps in human cell extracts. (A) Experimental approaches to reconstitute kinetochore assembly and function in vitro using mitotic HeLa cell extracts and the main conclusions derived from this study. (B) Simplified scheme indicating the permitted assembly steps (green check-mark), moderately/weakly efficient reactions (blue check-mark), and restricted interactions (red crosses). Symbols “D” indicate phospho-mimetic substitutions in CENP-T.

    Article Snippet: Subsequently, beads with anti-rabbit antibodies were coated with 50 µg ml−1 rabbit anti-CENP-T antibodies (Abcam) or 120 µg ml−1 rabbit anti-CENP-C antibodies (custom-made by Covance and affinity-purified in-house ( )).

    Techniques: In Vitro, Derivative Assay

    CENP-T robustly binds recombinant, but not native, Ndc80 complexes. (A) Schematic representation of recombinant CENP-T/W protein. The phospho-mimetic CENP-T/W construct contains substitutions, indicated by red dots (left). Schematic representation of recombinant Ndc80 constructs (right); see Supplementary Figure 1A for a comparison with wild-type Ndc80. (B and C) Representative images of coverslip-immobilized beads in bright field (top) and GFP (bottom) channels, showing recruitment of GFP-fused Ndc80 complexes to recombinant wild-type (B) or phospho-mimetic (C) CENP-T/W, which has no fluorescent tags. (D and E) Average GFP-brightness (mean with SD) of wild-type (D) or phospho-mimetic (E) CENP-T/W-coated beads incubated with various Ndc80 or Mis12 proteins, as in panels (B and C). Each point is derived from an independent experiment and represents the average brightness of > 30 beads. P-values were calculated by unpaired t-test: ***, p

    Journal: bioRxiv

    Article Title: Reconstitution of human kinetochore in mitotic cell extracts reveals permitted and restricted assembly steps

    doi: 10.1101/2020.07.15.203109

    Figure Lengend Snippet: CENP-T robustly binds recombinant, but not native, Ndc80 complexes. (A) Schematic representation of recombinant CENP-T/W protein. The phospho-mimetic CENP-T/W construct contains substitutions, indicated by red dots (left). Schematic representation of recombinant Ndc80 constructs (right); see Supplementary Figure 1A for a comparison with wild-type Ndc80. (B and C) Representative images of coverslip-immobilized beads in bright field (top) and GFP (bottom) channels, showing recruitment of GFP-fused Ndc80 complexes to recombinant wild-type (B) or phospho-mimetic (C) CENP-T/W, which has no fluorescent tags. (D and E) Average GFP-brightness (mean with SD) of wild-type (D) or phospho-mimetic (E) CENP-T/W-coated beads incubated with various Ndc80 or Mis12 proteins, as in panels (B and C). Each point is derived from an independent experiment and represents the average brightness of > 30 beads. P-values were calculated by unpaired t-test: ***, p

    Article Snippet: Subsequently, beads with anti-rabbit antibodies were coated with 50 µg ml−1 rabbit anti-CENP-T antibodies (Abcam) or 120 µg ml−1 rabbit anti-CENP-C antibodies (custom-made by Covance and affinity-purified in-house ( )).

    Techniques: Recombinant, Construct, Incubation, Derivative Assay

    Strategies for reconstructing kinetochores and our experimental approach. (A) Principal architecture of mitotic kinetochore and its binding to microtubules; see text for details. CCAN, constitutive centromere associated network. Letters “P” on CENP-T indicate phosphorylation-dependent activation of its binding to Ndc80 and Mis12 complexes; letters “P” on Mis12 indicate phosphorylation-dependent activation of its binding to CENP-C. (B) Previous experimental approaches to reconstitute kinetochore assembly and function in vitro ; see text for additional references. (C) Schematic of GFP-fused proteins stably expressed in HeLa cells. Experiments with Ndc80 complexes used three different cell lines: Nuf2-GFP (shown), GFP-Spc24, and GFP-Spc25. (D) Key steps of our experimental approach. Left image shows representative HeLa cell that was fixed and stained with propidium iodide to reveal DNA; green signal is from Mis12-GFP. Ruptured cells (images in the middle) were centrifuged to remove insoluble cellular components. Graph shows concentration of GFP-fused kinetochore proteins in clarified cell extracts. Each colored point represents average bead brightness from independent experiments, during which 50–100 beads were analyzed. For more detailed statistics, see Source data. Black lines show mean with standard error of the mean (SEM). The column for native Ndc80-GFP combines data from cell lines expressing GFP-fused Nuf2, Spc24, and Spc25.

    Journal: bioRxiv

    Article Title: Reconstitution of human kinetochore in mitotic cell extracts reveals permitted and restricted assembly steps

    doi: 10.1101/2020.07.15.203109

    Figure Lengend Snippet: Strategies for reconstructing kinetochores and our experimental approach. (A) Principal architecture of mitotic kinetochore and its binding to microtubules; see text for details. CCAN, constitutive centromere associated network. Letters “P” on CENP-T indicate phosphorylation-dependent activation of its binding to Ndc80 and Mis12 complexes; letters “P” on Mis12 indicate phosphorylation-dependent activation of its binding to CENP-C. (B) Previous experimental approaches to reconstitute kinetochore assembly and function in vitro ; see text for additional references. (C) Schematic of GFP-fused proteins stably expressed in HeLa cells. Experiments with Ndc80 complexes used three different cell lines: Nuf2-GFP (shown), GFP-Spc24, and GFP-Spc25. (D) Key steps of our experimental approach. Left image shows representative HeLa cell that was fixed and stained with propidium iodide to reveal DNA; green signal is from Mis12-GFP. Ruptured cells (images in the middle) were centrifuged to remove insoluble cellular components. Graph shows concentration of GFP-fused kinetochore proteins in clarified cell extracts. Each colored point represents average bead brightness from independent experiments, during which 50–100 beads were analyzed. For more detailed statistics, see Source data. Black lines show mean with standard error of the mean (SEM). The column for native Ndc80-GFP combines data from cell lines expressing GFP-fused Nuf2, Spc24, and Spc25.

    Article Snippet: Subsequently, beads with anti-rabbit antibodies were coated with 50 µg ml−1 rabbit anti-CENP-T antibodies (Abcam) or 120 µg ml−1 rabbit anti-CENP-C antibodies (custom-made by Covance and affinity-purified in-house ( )).

    Techniques: Binding Assay, Activation Assay, In Vitro, Stable Transfection, Staining, Concentration Assay, Expressing