Bright photoactivatable fluorophores for single-molecule imaging

Grimm, Jonathan B.; English, Brian P.; Choi, Heejun; Muthusamy, Anand K.; Mehl, Brian P.; Dong, Peng; Brown, Timothy A.; Lippincott‐Schwartz, Jennifer; Liu, Zhe; Lionnet, Timothée; Lavis, Luke D.

doi:10.1038/nmeth.4034

Cited by 356 publications

(384 citation statements)

References 32 publications

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“…The mean Kullback-Leibler divergence was ~0.3 bits further demonstrating that most clusters are not a photo-blinking artifact. Finally, we note that a recent paper demonstrates that PA-JF549 shows limited photo-blinking (Grimm et al, 2016). …”

Section: Methodsmentioning

confidence: 68%

“…Tracking fast-diffusing molecules has been a major challenge. To overcome this issue, we took advantage of bright new dyes (Grimm et al, 2016) and developed stroboscopic (Elf et al, 2007) photo-activation (Manley et al, 2008) single-molecule tracking (paSMT; Figure 3—figure supplement 1A), which makes tracking unambiguous (Materials and methods). We tracked individual Halo-mCTCF molecules at ~225 Hz and plotted the displacements between frames (Figure 3A).…”

Section: Resultsmentioning

confidence: 99%

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CTCF and cohesin regulate chromatin loop stability with distinct dynamics

Hansen

Pustova

Cattoglio

et al. 2017

eLife

520

593

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Folding of mammalian genomes into spatial domains is critical for gene regulation. The insulator protein CTCF and cohesin control domain location by folding domains into loop structures, which are widely thought to be stable. Combining genomic and biochemical approaches we show that CTCF and cohesin co-occupy the same sites and physically interact as a biochemically stable complex. However, using single-molecule imaging we find that CTCF binds chromatin much more dynamically than cohesin (~1–2 min vs. ~22 min residence time). Moreover, after unbinding, CTCF quickly rebinds another cognate site unlike cohesin for which the search process is long (~1 min vs. ~33 min). Thus, CTCF and cohesin form a rapidly exchanging 'dynamic complex' rather than a typical stable complex. Since CTCF and cohesin are required for loop domain formation, our results suggest that chromatin loops are dynamic and frequently break and reform throughout the cell cycle.DOI: http://dx.doi.org/10.7554/eLife.25776.001

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Section: Methodsmentioning

confidence: 68%

Section: Resultsmentioning

confidence: 99%

CTCF and cohesin regulate chromatin loop stability with distinct dynamics

Hansen

Pustova

Cattoglio

et al. 2017

eLife

520

593

View full text Add to dashboard Cite

show abstract

“…Therefore, in the case of the HaloTag labeling system, decreasing the amount of ligand will achieve the expected outcome, as only a small fraction of the molecules will be labeled with the fluorescent dye (Movie S6). Alternatively, optimal label density can also be obtained by utilizing photoactivatable dyes, such as PA-JF 549 , and activating a small fraction of the population with 405 nm light [34]. …”

Section: Single-molecule Tracking For Extracting Binding Charactermentioning

confidence: 99%

“…In all cases, the goal is to decrease the background signal by eliminating the excitation of out-of-focus molecules. The most common method used to study TF dynamics is HILO [11, 14, 15, 19–21, 34–37], most likely due to its easy implementation. In principle, any microscope capable of a TIRF configuration can do HILO illumination.…”

Section: Single-molecule Tracking For Extracting Binding Charactermentioning

confidence: 99%

Quantifying transcription factor binding dynamics at the single-molecule level in live cells

Presman

Ball

Paakinaho

et al. 2017

Methods

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Progressive, technological achievements in the quantitative fluorescence microscopy field are allowing researches from many different areas to start unraveling the dynamic intricacies of biological processes inside living cells. From super-resolution microscopy techniques to tracking of individual proteins, fluorescence microscopy is changing our perspective on how the cell works. Fortunately, a growing number of research groups are exploring single-molecule studies in living cells. However, no clear consensus exists on several key aspects of the technique such as image acquisition conditions, or analysis of the obtained data. Here, we describe a detailed approach to perform single-molecule tracking (SMT) of transcription factors in living cells to obtain key binding characteristics, namely their residence time and bound fractions. We discuss different types of fluorophores, labeling density, microscope, cameras, data acquisition, and data analysis. Using the glucocorticoid receptor as a model transcription factor, we compared alternate tags (GFP, mEOS, HaloTag, SNAP-tag, CLIP-tag) for potential multicolor applications. We also examine different methods to extract the dissociation rates and compare them with simulated data. Finally, we discuss several challenges that this exciting technique still faces.

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“…Conventional approaches such as biochemistry and structural biology provide little information about in vivo kinetics and stochasticity, and thus cannot resolve this layer of regulation, whereas imaging provides a unique opportunity by directly observing these dynamic processes in real time. Recent improvements in chemical dyes and fast high-resolution imaging platforms have allowed the direct labelling of single molecules and the tracking of their binding, dissociation and diffusion dynamics in live cells [11,34,99,100]. For example, we can directly image the binding of a TF or other DNA-binding protein at its genomic cis -regulatory elements and calculate its resident time [8,101].…”

Section: Advances In Imaging Technology For Probing Dynamic and Stochmentioning

confidence: 99%

Shaping development by stochasticity and dynamics in gene regulation

Dong

Liu

2017

Open Biol.

Self Cite

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Animal development is orchestrated by spatio-temporal gene expression programmes that drive precise lineage commitment, proliferation and migration events at the single-cell level, collectively leading to large-scale morphological change and functional specification in the whole organism. Efforts over decades have uncovered two ‘seemingly contradictory’ mechanisms in gene regulation governing these intricate processes: (i) stochasticity at individual gene regulatory steps in single cells and (ii) highly coordinated gene expression dynamics in the embryo. Here we discuss how these two layers of regulation arise from the molecular and the systems level, and how they might interplay to determine cell fate and to control the complex body plan. We also review recent technological advancements that enable quantitative analysis of gene regulation dynamics at single-cell, single-molecule resolution. These approaches outline next-generation experiments to decipher general principles bridging gaps between molecular dynamics in single cells and robust gene regulations in the embryo.

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Bright photoactivatable fluorophores for single-molecule imaging

Cited by 356 publications

References 32 publications

CTCF and cohesin regulate chromatin loop stability with distinct dynamics

CTCF and cohesin regulate chromatin loop stability with distinct dynamics

Quantifying transcription factor binding dynamics at the single-molecule level in live cells

Shaping development by stochasticity and dynamics in gene regulation

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