Journal: Cell

Article Title: Kinetochore microtubule dynamics and attachment stability are regulated by Hec1.

doi: 10.1016/j.cell.2006.09.047

Figure Lengend Snippet: Figure 1. Epitope Mapping and Cellular Localization of Hec1 Monoclonal Antibody 9G3 (A) Peptides covering the sequence of human Hec1 were adsorbed onto nitrocellulose and immunoprobed with 9G3. As a control, HeLa extract was adsorbed onto the nitrocellulose at region H-12. Both the control spot and spot C-2 (amino acids 200–215) were positively identified. (B) Representation of the Ndc80 complex as predicted from previous publications (Wei et al., 2005; Ciferri et al., 2005). The asterisk marks the site on Hec1 where 9G3 binds. (C and D) Localization of 9G3 (green) and an antibody to Spc24 (red) in PtK1 cells (C) and HeLa cells (D). Linescans were carried out on sister kinet- ochore pairs from both HeLa cells (n = 40 pairs/3 cells) and PtK1 cells (n = 34 pairs/4 cells), and in all cases Hec1 localized exteriorly to Spc24 at kinetochores. (E) Western blot of whole-cell PtK1 extract with 9G3 as a probe. (F) Immunofluorescent image of a PtK1 cell injected with 9G3. To the right of each cell panel in (C), (D), and (F), a single kinetochore pair has been enlarged. The graphs represent the linescan data from the single kinetochore pair. Scale bars in (C), (D), and (F) = 5 mm.

Article Snippet: Hec1 Antibody Epitope Mapping A peptide array (containing peptides of 15 amino acids in length with a 7 amino acid overlap) covering the entire human Hec1 sequence was generated (New England Peptide, Gardner, MA).

Techniques: Sequencing, Control, Western Blot, Injection

Journal: Cell

Article Title: Kinetochore microtubule dynamics and attachment stability are regulated by Hec1.

doi: 10.1016/j.cell.2006.09.047

Figure Lengend Snippet: Figure 4. Loss of Kinetochore Oscillations and Plus-End MT Polymerization in Hec1 9G3-Injected Cells (A and B) Kinetochore behavior was analyzed by live-cell fluorescence timelapse imaging. Cells were injected with rhodamine-labeled tubulin and Alexa 488-conjugated CENP-F antibodies (A), or additionally with 9G3 (B). Images were acquired every 15 s. Selected planes are shown from the timelapse sequences (A and B, top). Selected kinetochore pairs are boxed and a time series of 12 images for each pair is shown below (A and B, bottom). Kinetochores from the control cell exhibited oscillatory behavior and periods of stretching and relaxation (example in A, bottom), whereas kinetochores from the 9G3-injected cell did not oscillate (example in B, bottom). (C and D) EB1-GFP-expressing PtK1 cells were injected with Texas Red dextran alone (C) or in combination with 9G3 (D). Images were acquired every 10 s. A region containing a kinetochore pair and the spindle poles was extracted from the timelapse sequence and shown to the right. The bright spots in extracted images are spindle poles. (E) A buffer-injected monopolar cell exhibits chromosome oscillations toward and away from the pole both prior to and after injection (top panel). In cells injected with 9G3, chromosomes stopped oscillating and moved poleward after injection (middle and bottom panels). In all panels, scale bars = 5 mm.

Article Snippet: Hec1 Antibody Epitope Mapping A peptide array (containing peptides of 15 amino acids in length with a 7 amino acid overlap) covering the entire human Hec1 sequence was generated (New England Peptide, Gardner, MA).

Techniques: Injection, Imaging, Labeling, Control, Expressing, Sequencing

Journal: Cell

Article Title: Kinetochore microtubule dynamics and attachment stability are regulated by Hec1.

doi: 10.1016/j.cell.2006.09.047

Figure Lengend Snippet: Figure 6. Aurora B Kinase Phosphoryla- tion and Regulation of Hec1 (A) Left: Aurora B/INCENP790–856 in vitro kinase assay with Histone H3 as a control substrate (lanes 1 and 2) and Hec11–230 (lanes 3 and 4). Antibody 9G3 was added to the reaction mixtures in lanes 2 and 4. Right: Normalized quantification of radioactive phosphate for lanes 1–4. Molecular weight standards are indi- cated in kilodaltons. (B) PtK1 cells were transfected with WT-GFP- Hec1 (upper row) or mutant 6A-GFP-Hec1 (bot- tom three rows) for 40 hr prior to fixation for im- munofluoresence. Scale bar = 5 mm. (C) Cells transfected with WT-GFP-Hec1 and 6A-GFP-Hec1 were scored for chromosome alignment and assigned to one of three cate- goryies: chromosomes all aligned, chromo- somes mostly aligned (1–2 chromosomes off the metaphase plate), or chromosomes mostly unaligned (fewer than 3 aligned chromosomes) (n = 52 WT-GFP-Hec1 cells; n = 40 6A-GFP- Hec1 cells). (D) Cells were scored for merotelic kineto- chores. For WT-GFP-Hec1-expressing cells: n = 19 prometaphase cells, n = 31 metaphase/ near metaphase cells, and n = 11 anaphase cells. For 6A-GFP-Hec1-expressing cells: n = 47 prometaphase cells, n = 13 metaphase/ near metaphase cells, and n = 18 anaphase cells.

Article Snippet: Hec1 Antibody Epitope Mapping A peptide array (containing peptides of 15 amino acids in length with a 7 amino acid overlap) covering the entire human Hec1 sequence was generated (New England Peptide, Gardner, MA).

Techniques: In Vitro, Kinase Assay, Control, Molecular Weight, Transfection, Mutagenesis, Expressing

Journal: Cell

Article Title: Kinetochore microtubule dynamics and attachment stability are regulated by Hec1.

doi: 10.1016/j.cell.2006.09.047

Figure Lengend Snippet: Figure 7. Model for Hec1 Regulation of kMT Dynamics and Attachment (A) Mitotic spindle arrangement in a control cell (top) in which normal Aurora B phophorylation and dephosphorylation occur. kMT plus ends exhibit dynamic instability and undergo periods of attachment and detachment. Net polymerization at plus ends of kMTs is balanced by net depolymerization at minus ends. After addition of 9G3 (bottom), the N terminus of Hec1 can no longer be phosphorylated by Aurora B, and kMT detachment and dy- namic instability are suppressed. Minus-end depolymerization is not inhibited, and active depolymerases shorten kinetochore fibers. Hyper-stretch of centromeres arises from pulling forces exerted by the centrosome-associated minus-end organizing complexes as they maintain connection with the depolymerizing minus ends of the kinetochore fibers. (B) Regulation of kMT plus-end dynamic instability and attachment strength at three possible interfaces. Interface 1 is between the N terminus of Hec1 and the MT lattice; interface 2 is between the N terminus of Hec1 and a kinetochore-binding MAP, and interface 3 is between the MAP and the MT lattice (see text for details).

Article Snippet: Hec1 Antibody Epitope Mapping A peptide array (containing peptides of 15 amino acids in length with a 7 amino acid overlap) covering the entire human Hec1 sequence was generated (New England Peptide, Gardner, MA).

Techniques: Control, De-Phosphorylation Assay, Binding Assay

Journal: Oncotarget

Article Title: ASPP1/2-PP1 complexes are required for chromosome segregation and kinetochore-microtubule attachments

doi:

Figure Lengend Snippet: a . Tandem affinity purification of ASPP1/2-containing protein complexes were conducted using MOCK HeLa cells or cells stably expressing FLAG-HA (FH)-ASPP1 or ASPP2. Associated proteins were separated by SDS-PAGE and visualized by Coomassie Blue(CB)staining. The proteins and the number of peptides identified by mass spectrometry are shown in the . b . ASPP1/2-associated protein networks. The ASPP1/2-associated proteins are grouped by functional category (node color/label). c . Endogenous ASPP1/2 interact with multiple kinetochore components. Immunoprecipitation with anti-ASPP1 or ASPP2 antibodies were performed using cell lysates prepared from HeLa cells. The presence of kinetochore components in the immunoprecipitates was detected by WB analyses with their indicated antibodies. d . Similar to (c), the presence of three PP1 catalytic subunits in the immunoprecipitates was detected by WB analyses with the indicated antibodies.

Article Snippet: Commercially available antibodies for WB were as follows: ASPP1 (ab137537; Abcam), ASPP2 (611354; BD Biosciences), Hec1 (ab3613; Abcam), Hec1 (3622-1; epitomics), α-Tubulin (1878-s; epitomics), β-Tubulin (05-661; Millipore), KNL-1 (NB100-2586; Novus), ZW10 (ab21582; Abcam), CENP-E (ab5093; Abcam), CENP-F (ab5093; Abcam), Mad1 (sc-67338; Santa Cruz), Mad2 (7938-1; epitomics), MPS1 (05-682; EMD Millipore), PP1α (1950-1; epitomics), PP1β (2029-1; epitomics), PP1γ (6646-1; epitomics), Cyclin B1 (1495-1; epitomics), CREST (15-235-0001; antibodies incorporated), p-H3(Ser10) (Sc-8656-R; Santa Cruz), Secruin (2603-1; epitomics), Myc (9E10; Sigma), Flag (M2; Sigma), HA (MM5-101R; Millipore) and Actin (AC-74; Sigma).

Techniques: Affinity Purification, Stable Transfection, Expressing, SDS Page, Staining, Mass Spectrometry, Functional Assay, Immunoprecipitation

Journal: Oncotarget

Article Title: ASPP1/2-PP1 complexes are required for chromosome segregation and kinetochore-microtubule attachments

doi:

Figure Lengend Snippet: a . Tandem affinity purification of the Hec1-containing protein complex was conducted using HeLa cells stably expressing FLAG-HA (FH)-Hec1. Associated proteins were separated by SDS-PAGE and visualized by CB staining. The proteins and the number of peptides identified by mass spectrometry analysis are shown in the . b . Endogenous Hec1 interaction with ASPP1/2 and PP1α. Immunoprecipitation with anti-Hec1 antibody was performed using cell lysates prepared from HeLa cells. The presence of proteins in the immunoprecipitates was detected by WB analyses using the indicated antibodies. c . iASPP cannot interact with Hec1. 293T cells were co-transfected with Myc-Hec1 and FH-ASPP (ASPP1, ASPP2 or iASPP) constructs. After 24 hr, cell lysates were prepared for immunoprecipitation with the anti-Flag antibody and detected by WB analyses using the indicated antibodies. d . Schematic representation of ASPP2 deletion mutants. Binding capacity of ASPP2 WT or mutants to Hec1 is indicated with the symbols. e . Identification of Hec1-binding domain in ASPP2. 293T cells were co-transfected with Myc-Hec1 and FH-ASPP2-WT or deletion mutants. After 24 hr, cell lysates were prepared for immunoprecipitation with anti-FLAG antibody and detected by WB analyses. f . ASPP1/2 facilitate the interaction between Hec1 and PP1α in a PP1-binding dependent manner. 293T cells were co-transfected with indicated constructs. After 24 hr, cell lysates were prepared for immunoprecipitation with the anti-Flag antibody and detected by WB analyses using indicated antibodies. g . ASPP1/2 co-depletion reduces the endogenous interaction between Hec1 and PP1α. HeLa cells were transfected with the control or ASPP1/2 siRNAs. After 48 hr, cell lysates were prepared for immunoprecipitation with anti-Hec1 antibody and detected by WB analyses using the indicated antibodies.

Article Snippet: Commercially available antibodies for WB were as follows: ASPP1 (ab137537; Abcam), ASPP2 (611354; BD Biosciences), Hec1 (ab3613; Abcam), Hec1 (3622-1; epitomics), α-Tubulin (1878-s; epitomics), β-Tubulin (05-661; Millipore), KNL-1 (NB100-2586; Novus), ZW10 (ab21582; Abcam), CENP-E (ab5093; Abcam), CENP-F (ab5093; Abcam), Mad1 (sc-67338; Santa Cruz), Mad2 (7938-1; epitomics), MPS1 (05-682; EMD Millipore), PP1α (1950-1; epitomics), PP1β (2029-1; epitomics), PP1γ (6646-1; epitomics), Cyclin B1 (1495-1; epitomics), CREST (15-235-0001; antibodies incorporated), p-H3(Ser10) (Sc-8656-R; Santa Cruz), Secruin (2603-1; epitomics), Myc (9E10; Sigma), Flag (M2; Sigma), HA (MM5-101R; Millipore) and Actin (AC-74; Sigma).

Techniques: Affinity Purification, Stable Transfection, Expressing, SDS Page, Staining, Mass Spectrometry, Immunoprecipitation, Transfection, Construct, Binding Assay

Journal: Oncotarget

Article Title: ASPP1/2-PP1 complexes are required for chromosome segregation and kinetochore-microtubule attachments.

doi: 10.18632/oncotarget.6355

Figure Lengend Snippet: Figure 4: ASPP1/2 co-depletion causes SAC hyperactivation. a. Localization of Mad1, Mad2 and Mps1 in ASPP1/2 co-depleted HeLa cells. HeLa cells were transfected with control or ASPP1/2 siRNAs for 48 hr, and treated with nocodazole for 12 hr and then released into fresh media for 1-2 hr before fixation. Cells were stained with antibodies against the indicated SAC proteins (red), together with kinetochores (CREST, green) and DNA (blue). The figures show confocal images of cells at prometaphase and metaphase. Insets are magnified images of the boxed areas. Scale bar = 10 µm. b. Quantification of the fluorescence intensity of the SAC proteins normalized to the fluorescence intensity of CREST staining are shown. For quantifications, ~30 mitotic cells were measured for each experiment and condition. Error bars, SEM *p<0.01 from triplicates. c. WB analyses of cell lysates prepared from control and ASPP1/2 co-depleted HeLa cells using the indicated antibodies.

Article Snippet: Commercially available antibodies for WB were as follows: ASPP1 (ab137537; Abcam), ASPP2 (611354; BD Biosciences), Hec1 (ab3613; Abcam), Hec1 (3622-1; epitomics), α-Tubulin (1878- s; epitomics), β-Tubulin (05-661; Millipore), KNL1 (NB100-2586; Novus), ZW10 (ab21582; Abcam), CENP-E (ab5093; Abcam), CENP-F (ab5093; Abcam), Mad1 (sc-67338; Santa Cruz), Mad2 (7938-1; epitomics), MPS1 (05-682; EMD Millipore), PP1α (1950-1; epitomics), PP1β (2029-1; epitomics), PP1γ (6646-1; epitomics), Cyclin B1 (1495-1; epitomics), CREST (15- 235-0001; antibodies incorporated), p-H3(Ser10) (Sc8656-R; Santa Cruz), Secruin (2603-1; epitomics), Myc (9E10; Sigma), Flag (M2; Sigma), HA (MM5-101R; Millipore) and Actin (AC-74; Sigma).

Techniques: Transfection, Control, Staining, Fluorescence

Journal: bioRxiv

Article Title: Mammalian metaphase kinetochores are elastic and require condensin for robust structure and function

doi: 10.64898/2025.12.23.696255

Figure Lengend Snippet: (A) Representative PtK2 SMC2 RNAi cell timelapse of eGFP-CENP-A deforming with the microneedle pulling, and relaxing. Yellow box shows the zoomed in pair on the right, cyan arrow shows direction of needle movement, white arrowheads highlight the “front” kinetochore, and grey arrowheads highlight the “back” kinetochore. (B) K-K distance over time before, during, and after microneedle pulling for n = 9 kinetochores. “Initial” length is the frame just before pulling begins. Time = 0 s corresponds to the time maximum K-K distance was reached and measured during pulling, highlighting relaxation dynamics of each pulled kinetochore. The grey box indicates the pulling period. (C) CENP-A lengths over time before, during, and after microneedle pulling for 9 kinetochores as defined in (B). (D) CENP-A length at the frame before microneedle pulling, at the maximum K-K distance during pulling, and 30 s after pulling in siSMC2 cells (n = 9 kinetochores; Paired t-test). (E) Percent of K-K distance and CENP-A length increase from the frame before pulling begins to maximum measured K-K distance (n = 9 kinetochores; Paired t-test). (F) eGFP-CENP-A images at maximum measured K-K distance and 30s afterwards of control deformation and two siSMC2 kinetochore deformations exhibiting dramatic “tails”. (G) Percentage of control (n = 6) and siSMC2 (n = 9) kinetochore pulls with persistent “tails” for > 30 s during the relaxation period. (H) Timelapse comparing representative K-K distance relaxation for a control pull and for a fast relaxation SMC2 RNAi pull during the needle hold. White box indicates zoomed in pair. Grey dashed lines project the kinetochores’ movements over time. (I) Examples of non-detached (15 s after maximum K-K) and detached kinetochore (10 s after maximum K-K) from siSMC2 pulling experiments with fast K-K relaxation rates. Inset is of the front kinetochore with a linescan for tubulin intensity and kinetochore intensity; corresponding plots on the right annotated with sections of intensity signal corresponding to k-fiber presence or not. (J) K-K distance change after maximum K-K distance (t = 0) for control (black, n = 6 kinetochores), and detached, siSMC2 kinetochores (red and brown line, n = 2/9 kinetochores) pulls. (K) Percentage of pulls that led to detachment events in control and siSMC2 cells based on two criteria: fast K-K distance relaxation as in (J) and loss of tubulin signal attached to the kinetochore as in (I).

Article Snippet: The following primary antibodies were used: mouse anti-Hec1 (1:1000; Novus Biologicals; NB100–338), chicken anti-GFP (1:500; Aves Lab Inc.; GFP-1010), rabbit anti-alpha tubulin (1:500; Abcam; ab18251), rat anti-alpha tubulin (1:2000; Bio-Rad; REFMCA77G).

Techniques: Control