Table S3 ). (G) Scheme showing 97 Hz and 2 Hz SPT workflow. Three-state model fits to 97 Hz SPT data using Spot-On was used to derive fractions and diffusion coefficients of fast diffusing ( F fast , D fast ), slow diffusing ( F slow , D slow ), and bound PARP ( F bound , D bound ) molecules. Further, 2 Hz SPT data were fit using a two-phase exponential model to derive fractions and duration of transient (Fraction transient, τ transient ) and stable (Fraction stable, τ stable ) PARP binding events. (H and I) Pie chart illustrations summarizing the derivation of overall fractions of Halo-PARP1 (in H) and Halo-PARP2 (in I) engaging in transient and stable binding, slow diffusion, and fast diffusion from 97 to 2 Hz SPT experiments. The bound, slow, and fast-diffusing fractions (in i) were determined using Spot-On’s three-state model fitting to 97 Hz SPT data. The bound fraction (in ii) in Halo-PARP1 and Halo-PARP2 cells was analyzed by 2 Hz SPT and fit to a two-phase exponential model. Data acquired for H2B-Halo were used for photobleaching correction, and a correction factor (see ) was applied to obtain the true fraction of transiently and stably binding Halo-PARP molecules (in iii). These data were compiled together to obtain the overall fractions of endogenous Halo-PARP1 and Halo-PARP2 molecules (in iv). (J) Normalized and photobleaching corrected recovery curves from FRAP experiments performed on Halo-PARP1 (blue circles) and Halo-PARP2 (red circles). H2B-Halo (green circles) was used for photobleaching correction. A two-phase exponential model (solid line) was fit to the FRAP data. Error bars represent SD from 11 to 18 cells from ≥3 independent replicates. " width="100%" height="100%">
Journal: iScience
Article Title: Dynamics of endogenous PARP1 and PARP2 during DNA damage revealed by live-cell single-molecule imaging
doi: 10.1016/j.isci.2022.105779
Figure Lengend Snippet: Live-cell single-molecule microscopy reveals fraction of stably bound PARP1 and PARP2 in undamaged cells (A) (i) Schematic describing the covalent binding of JF646 dye to the HaloTag. (ii) Sample cropped frames from a representative 97 Hz SPT movie depicting the trajectory of a single PARP1 molecule. The 640 nm excitation laser was used continuously for imaging while the camera exposure time was 10.3 ms. (B and C) Single-particle trajectories (length of >2) over 30 s for Halo-PARP1 (in B) or Halo-PARP2 (in C) in a single representative nucleus. (D) Fraction bound ( F bound ) of Halo-PARP1 and Halo-PARP2 in undamaged cells inferred from Spot-On’s three-state model fitting to 97 Hz SPT data. Bar graphs show the mean F bound ± SEM obtained from ≥42,000 trajectories (>3 detections) from ≥52 cells from ≥5 independent replicates (represented by dots), each of which were fitted separately. Statistical difference between the two groups was determined using unpaired t-test. (E) Cumulative distribution function (CDF) of displacements for Halo-PARP1 and Halo-PARP2 (representative Δ τ = 30 ms) in undamaged cells. Individual curves depict data merged from ≥42,000 trajectories (>3 detections) from ≥52 cells from ≥5 independent replicates. (F) A log-log plot showing the uncorrected survival probability (1-CDF) of individual Halo-PARP1 and Halo-PARP2 molecules and their respective two-phase exponential model fits (solid curves) to 2 Hz SPT data in undamaged cells. Each curve represents data merged from ≥870 trajectories from ≥13 cells from ≥3 independent replicates. Data acquired for H2B-Halo (11,737 trajectories from ≥40 cells from 10 independent replicates) was used for photobleaching correction and thereby deriving values for τ transient and τ stable (See Table S3 ). (G) Scheme showing 97 Hz and 2 Hz SPT workflow. Three-state model fits to 97 Hz SPT data using Spot-On was used to derive fractions and diffusion coefficients of fast diffusing ( F fast , D fast ), slow diffusing ( F slow , D slow ), and bound PARP ( F bound , D bound ) molecules. Further, 2 Hz SPT data were fit using a two-phase exponential model to derive fractions and duration of transient (Fraction transient, τ transient ) and stable (Fraction stable, τ stable ) PARP binding events. (H and I) Pie chart illustrations summarizing the derivation of overall fractions of Halo-PARP1 (in H) and Halo-PARP2 (in I) engaging in transient and stable binding, slow diffusion, and fast diffusion from 97 to 2 Hz SPT experiments. The bound, slow, and fast-diffusing fractions (in i) were determined using Spot-On’s three-state model fitting to 97 Hz SPT data. The bound fraction (in ii) in Halo-PARP1 and Halo-PARP2 cells was analyzed by 2 Hz SPT and fit to a two-phase exponential model. Data acquired for H2B-Halo were used for photobleaching correction, and a correction factor (see ) was applied to obtain the true fraction of transiently and stably binding Halo-PARP molecules (in iii). These data were compiled together to obtain the overall fractions of endogenous Halo-PARP1 and Halo-PARP2 molecules (in iv). (J) Normalized and photobleaching corrected recovery curves from FRAP experiments performed on Halo-PARP1 (blue circles) and Halo-PARP2 (red circles). H2B-Halo (green circles) was used for photobleaching correction. A two-phase exponential model (solid line) was fit to the FRAP data. Error bars represent SD from 11 to 18 cells from ≥3 independent replicates.
Article Snippet: FRAP data analysis (MatLab script) , Hansen et al. and Sprague et al. , , https://gitlab.com/anders.sejr.hansen/Mahadevan_2022.
Techniques: Microscopy, Stable Transfection, Binding Assay, Imaging, Single Particle, Diffusion-based Assay
Table S3 ). (G) Scheme showing 97 Hz and 2 Hz SPT workflow. Three-state model fits to 97 Hz SPT data using Spot-On was used to derive fractions and diffusion coefficients of fast diffusing ( F fast , D fast ), slow diffusing ( F slow , D slow ), and bound PARP ( F bound , D bound ) molecules. Further, 2 Hz SPT data were fit using a two-phase exponential model to derive fractions and duration of transient (Fraction transient, τ transient ) and stable (Fraction stable, τ stable ) PARP binding events. (H and I) Pie chart illustrations summarizing the derivation of overall fractions of Halo-PARP1 (in H) and Halo-PARP2 (in I) engaging in transient and stable binding, slow diffusion, and fast diffusion from 97 to 2 Hz SPT experiments. The bound, slow, and fast-diffusing fractions (in i) were determined using Spot-On’s three-state model fitting to 97 Hz SPT data. The bound fraction (in ii) in Halo-PARP1 and Halo-PARP2 cells was analyzed by 2 Hz SPT and fit to a two-phase exponential model. Data acquired for H2B-Halo were used for photobleaching correction, and a correction factor (see ) was applied to obtain the true fraction of transiently and stably binding Halo-PARP molecules (in iii). These data were compiled together to obtain the overall fractions of endogenous Halo-PARP1 and Halo-PARP2 molecules (in iv). (J) Normalized and photobleaching corrected recovery curves from 
