ptk2 cells  (MatTek)

Journal: Journal of Cell Science

Article Title: Redefining colocalization analysis with a novel phasor mixing coefficient

doi: 10.1242/jcs.264388

Figure Lengend Snippet: The PMC recapitulates the expected quantification across a wide range of biomolecular association. (A–C) Examples illustrating how the PMC quantifies known high (A), moderate (B) and low (C) biomolecular associations. (A) Color mixing analysis of U2OS cells with mitochondria double-labeled by using an anti-TOMM20 antibody with both AF488-conjugated and AF555-conjugated secondary antibodies. The merged pseudo-color image (left; AF488 in cyan; AF555 in magenta) was extracted from the multispectral image. Scale bar: 10 µm. The phasor plot (middle) exhibits a single narrow cluster near the midpoint of the mixing band. The CMI (right) highlights essentially all mitochondria. The brighter regions near the nucleus reflect a slight difference in depth throughout the cell. (B) Color mixing analysis of a PtK2 cell with MHC labeled with AF488 and phosphorylated RLC labeled with AF568. The merged pseudo-color image (left; AF488 in cyan, AF568 in magenta) was extracted from the multispectral image. Scale bars: 20 µm. The phasor plot (middle) exhibits a single broad cluster centered near the midpoint of the mixing band. The cluster near the origin corresponds to the significant number of background pixels that are excluded from the mixing band. The CMI (right) highlights regions with significant mixing (solid white arrows) and minimal mixing (dashed white arrows). (C) Color mixing analysis of U2OS cells with mitochondria labeled with anti-TOMM20 antibody and an AF488-conjugated secondary antibody, and with nuclei labeled with Hoechst–JF549. The merged pseudo-color image (left; AF488 in cyan, JF549 in magenta) was extracted from the multispectral image. Scale bar: 10 µm. The phasor plot (middle) exhibits a largely bimodal cluster in which the two lobes sit near the two PCs. The mixing image (right) is largely uniformly cold, with only a region near the edge of the nucleus displaying any mixing. This small region reflects areas where the mitochondria extend over (or under) the nucleus but are still captured in the optical section. In the phasor plots, dashed gray lines indicate the bounds of the mixing band. The overlaid histograms display the S coordinate of the underling phasor cluster defined within the mixing band. (D) PCC, Manders' coefficients (M 1 and M 2 ) and the PMC (PMC 1 and PMC 2 ) for each example in A–C (from top to bottom). PCC and M 1,2 were calculated after applying an intensity threshold determined by the Otsu method (see Fig. S4 ). Importantly, PMC 1,2 capture the known association moving from high (top, A) to low (bottom, C) mixing. Individual N =3 points correspond to individual samples; line marker indicates the mean value.

Article Snippet: PtK2 cells (NBL-5; ATCC CCL-56) were grown to 80% confluency in MEM (Corning 10-010-CV) supplemented with non-essential amino acids (Sigma Aldrich, M7145), sodium pyruvate (Gibco, 11-360-070) and 10% FBS (ATCC, 30-2020), at 37°C in 5% CO 2 .

Techniques: Labeling, Marker

Journal: Journal of Cell Science

Article Title: Redefining colocalization analysis with a novel phasor mixing coefficient

doi: 10.1242/jcs.264388

Figure Lengend Snippet: The PMC exhibits less sensitivity to SNR than Pearson's or Manders’ coefficients. (A) Merged pseudo-color images (extracted from multispectral images) of PtK2 cells with MHC labeled with AF488 (cyan) and RLC labeled with AF568 (magenta) at low (i) and high (ii) SNR. Scale bars: 20 µm. (B) The mixing images corresponding to the low (left) and high (right) SNR images in A. Despite the significant increase in noise, the low-SNR mixing image largely highlights the same regions as the high-SNR image. Scale bars: 20 µm. (C) Quantifications (PCC, M 1,2 and PMC) of biomolecular association as a function of SNR. PCC and M 1,2 both predictably decrease substantially as SNR decreases. PMC 1 , in contrast, remains comparatively stable over the same range. However, PMC 2 significantly increases as SNR approaches unreasonable levels. This diverging behavior can act as an indicator of when image quality degrades. N =3 samples, error bars indicate standard error.

Article Snippet: PtK2 cells (NBL-5; ATCC CCL-56) were grown to 80% confluency in MEM (Corning 10-010-CV) supplemented with non-essential amino acids (Sigma Aldrich, M7145), sodium pyruvate (Gibco, 11-360-070) and 10% FBS (ATCC, 30-2020), at 37°C in 5% CO 2 .

Techniques: Labeling

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: Inner and outer mammalian kinetochores actively undergo large-scale shape changes in a correlated manner at metaphase. (A) Schematic of kinetochore analysis framework. (B) Representative timelapse of PtK2 cell demonstrating a representative small-scale and large-scale deforming kinetochore, visualized by eGFP-CENP-A and Hec1-Halotag with JF549 dye, with intensity linescan along the force (K-K) axis and best Gaussian fits for boxed kinetochores. Red text denotes length measurement using full-width at half maximum of Gaussian fits. Blue text denotes length measurement using AUC/peak calculation. White lines are drawn to approximate spindle shape and k-fibers. (C) Mean length of individual kinetochores over their imaging lifetime for eGFP-CENP-A and Hec1-Halotag with JF549 dye (m = 14 cells, n = 122 kinetochores; mean ± SD; Mann-Whitney test), with control 200 nm beads (in 488 and 561 nm imaging wavelengths) analyzed with the same method (n = 22). (D) Correlation between CENP-A length and Hec1 length over each timepoint. Dotted black line indicates a 1:1 comparison and red line indicates the best fit (m = 14 cells, n = 6502 timepoints; Simple linear regression). (E) Standard deviation of length of the same individual kinetochores and 200 nm beads as in (C). (F) Model cartoon depicting the sliding of parallel linkages that occur to allow for the scale of shape changes observed for CENP-A and Hec1.

Article Snippet: A PtK2 cell line stably expressing eGFP-CENP-A was generated from a wildtype PtK2 line (ATCC), infected with lentivirus for eGFP-CENP-A and selected with 0.5 μg/mL puromycin.

Techniques: Imaging, MANN-WHITNEY, Control, Comparison, Standard Deviation

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) Schematic of how kinetochore shape changes occur with merotelic attachments. (B) Representative timelapses of a PtK2 kinetochore pair (eGFP-CENP-A and Hec1-Halotag with JF549 dye) in a control cell, Hec1-9A-HaloTag overexpression cell, and 3 µM ZM447439 (ZM) treated cell, with right kinetochore displayed as stationary (grey line). (C) Mean (from left to right columns: 435 ± 60 nm, 442 ± 83 nm, 467 ± 81 nm, 461 ± 67 nm, 482 ± 92 nm, and 494 ± 96 nm) and (D) standard deviation (from left to right columns: 39 ± 17 nm, 46 ± 22 nm, 52 ± 24 nm, 44 ± 22 nm, 50 ± 28 nm, 59 ± 37 nm) of individual kinetochores imaging lifetime for eGFP-CENP-A and Hec1-Halotag with JF549 dye control (m = 14 cells, n = 122 kinetochores), Hec1-9A-Halotag (+JF549 dye) transient expression (m = 6 cells, n = 79 kinetochores), and ZM treatment (m = 16 cells, n = 86 kinetochores) (mean ± SD; Mann-Whitney test). (E) Cartoon of how kinetochore asymmetry is detected, visualized by CENP-A and Hec1 colocalized (white) with approximated k-fiber microtubules (grey lines, left) and DNA (grey area, right). Cartoon (below) depicts “tail” on the right side of the kinetochore. From the example intensity linescan, asymmetry is detected if the area under the curve (AUC) calculated for left and right side of the peak is greater than opposite side. For tail detection, the first derivative of the intensity curve is calculated. A tail is deemed present if there are >2 minima or maxima. (F) Percentage of total timepoints with Hec1 asymmetry detected for control, Hec1-9A expression, and ZM treatment. Significance from Chi-square test. (G) Percentage of total timepoints with Hec1 “tails” detected for control (371/6502 timepoints), Hec1-9A expression (280/3131 timepoints), and ZM treatment (929/4762 timepoints). Significance from Chi-square test. (H) Percentage of individual kinetochore’s lifetime with Hec1 “tails” detected for kinetochores in (C) and (D) (mean ± SEM; Mann-Whitney test). (I) Percentage of tails pointing to microtubule or DNA side for control (n = 222/371 tails towards DNA), Hec1-9A (89/280 tails towards DNA), and ZM-treated (558/929 tails towards DNA) kinetochores, for all individual timepoints with tails.

Article Snippet: A PtK2 cell line stably expressing eGFP-CENP-A was generated from a wildtype PtK2 line (ATCC), infected with lentivirus for eGFP-CENP-A and selected with 0.5 μg/mL puromycin.

Techniques: Control, Over Expression, Standard Deviation, Imaging, Expressing, MANN-WHITNEY

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: Acute force application reveals elastic mammalian kinetochores at metaphase. (A) Schematic of how kinetochore length changes occur with tension. (B) All (black line; m = 122 kinetochores; timepoints per increasing K-K bin n = 1420, 2623, 1518, 617), large-range (grey continuous line; m = 48 kinetochores; per K-K bin n = 522, 1054, 580, 246), and small-range (grey dashed line; m = 74 kinetochores; per K-K bin n = 898, 1569, 938, 371) kinetochores’ Hec1 lengths binned by K-K distance (tension), with bins with < 100 timepoints excluded. Large-range kinetochores were assigned by range of Hec1 lengths over each kinetochore’s imaging lifetime > mean range of all kinetochores. Small-range kinetochores have length ranges < mean range of all kinetochores (mean ± SEM; Mann-Whitney test done for first and last K-K distance bins). (C) Percent of K-K distance and CENP-A length increase per kinetochore during imaging lifetime; 74 ± 42% and 5 ± 10% (mean ± SD; Paired t-test). (D) Cartoon depicting assay to exert controlled force (10 µm over 18 s) on a k-fiber and its attached kinetochore with a glass microneedle, without membrane rupture ( ; ). (E) Potential outcomes of microneedle pulling before force, with high force, and after force: attached kinetochore deforms then relaxes (elastic response) or deforms and does not relax (plastic response). (F) Representative PtK2 cell timelapse of eGFP-CENP-A deforming with the microneedle pulling, and relaxing. The yellow arrowhead denotes the “front” sister kinetochore and the long yellow arrow indicates pulling. (G) K-K distance over time before, during, and after microneedle pulling for 6 kinetochores. “Initial” length is the frame before pulling begins. Time = 0 s corresponds to the time maximum K-K distance was measured during pulling. The grey box indicates the 18 s pulling period. (H) eGFP-CENP-A lengths over time before, during, and after microneedle pulling for 6 kinetochores as defined in (G). The grey box indicates the pulling period, during which kinetochore lengths were not measured due to motion blur. (I) CENP-A length before microneedle pulling (∼5 s before), at the maximum K-K distance during pulling, and 30 s after pulling. N = 6 kinetochores (paired t-test). (J) Correlation of CENP-A length and K-K distance during the relaxation period after pulling (n = 6 kinetochores). R 2 = 0.74, significantly non-zero using simple linear regression. (K) Correlation of “back” (needle distal) kinetochore CENP-A length (black dots) and K-K distance during the relaxation period after pulling overlaid on “front” (needle proximal) kinetochore lengths (grey dots) as in (I). R 2 = 0.05 for “back” kinetochores, not significant from zero using simple linear regression. (L) Length fold change (strain) of CENP-A and K-K distance (92 ± 33%) and CENP-A (63 ± 56%) from before pulling and at maximum measured K-K distance (n = 6 kinetochores; Paired t-test).

Article Snippet: A PtK2 cell line stably expressing eGFP-CENP-A was generated from a wildtype PtK2 line (ATCC), infected with lentivirus for eGFP-CENP-A and selected with 0.5 μg/mL puromycin.

Techniques: Imaging, MANN-WHITNEY, Membrane

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: Loss of condensin leads to variable metaphase kinetochore shape and loss of structural stability . (A) Cartoon depicting loss of condensin I/II expecting to lead to more compliant chromatin. (B) Relative expression of SMC2 from control (siLuciferase) and siSMC2 cells from qPCR, normalized using actin and GAPDH as housekeeping genes. (C) Mean K-K distance per kinetochore pair for PtK2 control (m = 14 cells, n = 122 kinetochores) and SMC2 RNAi cells (m = 10 cells, n = 78 kinetochores) (mean ± SD; unpaired t-test). (D) Mean (from left to right: 435 ± 60 nm, 512 ± 70 nm, 461 ± 67 nm, 554 ± 81 nm) and (E) standard deviation (from left to right: 39 ± 17 nm, 73 ± 39 nm, 44 ± 22, 84 ± 42 nm) of length per kinetochore (eGFP-CENP-A and Hec1-Halotag with JF549 dye) over the imaging lifetime for control (n = 122 kinetochores) and siSMC2 cells (n = 78 kinetochores) (mean ± SD; Mann-Whitney test). (F) Percent of K-K distance and CENP-A length increase per kinetochore during imaging lifetime for control (n = 122; 74 ± 42% and 5 ± 10%) and siSMC2 cells (n = 78; 63 ± 35% and 4 ± 18%) (mean ± SD; Mann-Whitney test). (G) Percentage of timepoints of all imaged kinetochores displaying Hec1 “tails” for control (n = 6502 timepoints) and siSMC2 (n = 4075 timepoints) cells (Chi-square test). (H) Examples of Hec1 and CENP-A distributions with 1, 2, and 3 peaks with corresponding peak heatmap (multicolor images) and detection (white cross). (I) Percentage of timepoints of all imaged CENP-A kinetochores with 1, 2, or ≥3 peaks for control (n = 6502 total timepoints) and siSMC2 (n = 4075 total timepoints) cells (bottom) (Chi-square test). (J) Percentage of kinetochores for control (n = 122 kinetochores) and siSMC2 (n = 78 kinetochores) with >1 peak (31% for control and 76% for siSMC2) and with only 1 peak (69% for control and 24% for siSMC2) during its imaging lifetime (Chi-square test). (K) Percentage of each individual kinetochore’s imaging lifetime with >1 peak in Hec1 for control (n = 122 kinetochores) and siSMC2 (n = 78 kinetochores) cells (median; Mann-Whitney test).

Article Snippet: A PtK2 cell line stably expressing eGFP-CENP-A was generated from a wildtype PtK2 line (ATCC), infected with lentivirus for eGFP-CENP-A and selected with 0.5 μg/mL puromycin.

Techniques: Expressing, Control, Standard Deviation, Imaging, MANN-WHITNEY

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: A PtK2 cell line stably expressing eGFP-CENP-A was generated from a wildtype PtK2 line (ATCC), infected with lentivirus for eGFP-CENP-A and selected with 0.5 μg/mL puromycin.

Techniques: Control