A Protocol for Studying Transcription Factor Dynamics Using Fast Single-Particle Tracking and Spot-On Model-Based Analysis

Jha, Asmita; Hansen, Anders S.

doi:10.1007/978-1-0716-2140-0_9

Cited by 4 publications

(2 citation statements)

References 54 publications

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“…We labeled genome-edited cells with a low nanomolar concentration of JF646 and visualized individual molecules of Halo-PARP1/2, as described for other Halo-tagged proteins. 47 , 48 , 50 , 54 , 55 Using single-particle tracking at a high frame rate (97 Hz SPT) with highly inclined and laminated optical sheet (HILO) illumination, 56 we could track individual PARP1/2 molecules inside the nucleus over time ( Figures 1 Aii, 1B, and 1C, Videos S1 and S2 ). While some particles were relatively immobile and therefore presumably chromatin-bound, others displayed rapid diffusion.…”

Section: Resultsmentioning

confidence: 99%

Dynamics of endogenous PARP1 and PARP2 during DNA damage revealed by live-cell single-molecule imaging

et al. 2023

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Section: Resultsmentioning

confidence: 99%

Dynamics of endogenous PARP1 and PARP2 during DNA damage revealed by live-cell single-molecule imaging

et al. 2023

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“…We used our genome-edited Halo-PARP1/2 cell lines to monitor the intranuclear dynamics of individual endogenous PARP1 and PARP2 molecules in the undamaged condition. We labeled genome edited cells with a low nanomolar concentration of JF646 and visualized individual molecules of Halo-PARP1/2, as described for other Halo-tagged proteins (Grimm et al, 2015; Hansen et al, 2017; Jha and Hansen, 2022; Schmidt et al, 2016; Youmans et al, 2018). Using single particle tracking at a high frame rate (97 Hz SPT) with highly inclined and laminated optical sheet (HILO) illumination (Tokunaga et al, 2008), we could track individual PARP1/2 molecules inside the nucleus over time (Figures 1Aii, 1B and 1C).…”

Section: Resultsmentioning

confidence: 99%

Dynamics of endogenous PARP1 and PARP2 during DNA damage revealed by live-cell single-molecule imaging

Mahadevan

Jha

Rudolph

et al. 2022

Preprint

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PARP1 contributes to genome architecture and DNA damage repair through its dynamic association with chromatin. PARP1 and PARP2 (PARP1/2) recognize damaged DNA and recruit the DNA repair machinery. Using single molecule microscopy in live cells, we monitored the movement of PARP1/2 on undamaged and damaged chromatin. We identify two classes of freely diffusing PARP1/2 and two classes of bound PARP1/2. The majority (> 60%) of PARP1/2 diffuse freely in both undamaged and damaged nuclei and in the presence of inhibitors of PARP1/2 used for cancer therapy (PARPi). Laser induced DNA damage results in a small fraction of slowly diffusing PARP1 and PARP2 to become transiently bound. Treatment of cells with PARPi in the presence of DNA damage causes subtle changes in the dynamics of bound PARP1/2, in contrast to bulk studies that suggest PARP trapping. Our results imply that next-generation PARPi could specifically target the small fraction of DNA-bound PARP1/2.

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Impact of temporal resolution in single particle tracking analysis

Schirripa Spagnolo,

Luin

2024

Discover Nano

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Temporal resolution is a key parameter in the observation of dynamic processes, as in the case of single molecules motions visualized in real time in two-dimensions by wide field (fluorescence) microscopy, but a systematic investigation of its effects in all the single particle tracking analysis steps is still lacking. Here we present tools to quantify its impact on the estimation of diffusivity and of its distribution using one of the most popular tracking software for biological applications on simulated data and movies. We found important shifts and different widths for diffusivity distributions, depending on the interplay of temporal sampling conditions with various parameters, such as simulated diffusivity, density of spots, signal-to-noise ratio, lengths of trajectories, and kind of boundaries in the simulation. We examined conditions starting from the ones of experiments on the fluorescently labelled receptor p75NTR, a relatively fast-diffusing membrane receptor (diffusivity around 0.5–1 µm2/s), visualized by TIRF microscopy on the basal membrane of living cells. From the analysis of the simulations, we identified the best conditions in cases similar to these ones; considering also the experiments, we could confirm a range of values of temporal resolution suitable for obtaining reliable diffusivity results. The procedure we present can be exploited in different single particle/molecule tracking applications to find an optimal temporal resolution.

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A Protocol for Studying Transcription Factor Dynamics Using Fast Single-Particle Tracking and Spot-On Model-Based Analysis

Cited by 4 publications

References 54 publications

Dynamics of endogenous PARP1 and PARP2 during DNA damage revealed by live-cell single-molecule imaging

Dynamics of endogenous PARP1 and PARP2 during DNA damage revealed by live-cell single-molecule imaging

Dynamics of endogenous PARP1 and PARP2 during DNA damage revealed by live-cell single-molecule imaging

Impact of temporal resolution in single particle tracking analysis

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