Single-molecule tracking (SMT): a window into live-cell transcription biochemistry

Dahal, Liza; Walther, Nike; Tjian, Robert; Darzacq, Xavier; Graham, Thomas G.W.

doi:10.1042/bst20221242

Cited by 18 publications

(13 citation statements)

References 124 publications

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“…Regarding the occupancy of chromatin analyzed by ChIP-seq and CUT&RUN, the nature of both assays requiring highly specific antibodies and other steps is unlikely to lead to single-cell data. However, precise in vivo imaging of proteins in chromatin of living or fixed individual cells at specific regions of chromatin is technically feasible with appropriate protein labels and instrumentation [ 209 , 210 ].…”

Section: Discussionmentioning

confidence: 99%

Multiomics Analysis Reveals Novel Genetic Determinants for Lens Differentiation, Structure, and Transparency

et al. 2023

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Recent advances in next-generation sequencing and data analysis have provided new gateways for identification of novel genome-wide genetic determinants governing tissue development and disease. These advances have revolutionized our understanding of cellular differentiation, homeostasis, and specialized function in multiple tissues. Bioinformatic and functional analysis of these genetic determinants and the pathways they regulate have provided a novel basis for the design of functional experiments to answer a wide range of long-sought biological questions. A well-characterized model for the application of these emerging technologies is the development and differentiation of the ocular lens and how individual pathways regulate lens morphogenesis, gene expression, transparency, and refraction. Recent applications of next-generation sequencing analysis on well-characterized chicken and mouse lens differentiation models using a variety of omics techniques including RNA-seq, ATAC-seq, whole-genome bisulfite sequencing (WGBS), chip-seq, and CUT&RUN have revealed a wide range of essential biological pathways and chromatin features governing lens structure and function. Multiomics integration of these data has established new gene functions and cellular processes essential for lens formation, homeostasis, and transparency including the identification of novel transcription control pathways, autophagy remodeling pathways, and signal transduction pathways, among others. This review summarizes recent omics technologies applied to the lens, methods for integrating multiomics data, and how these recent technologies have advanced our understanding ocular biology and function. The approach and analysis are relevant to identifying the features and functional requirements of more complex tissues and disease states.

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Section: Discussionmentioning

confidence: 99%

Multiomics Analysis Reveals Novel Genetic Determinants for Lens Differentiation, Structure, and Transparency

et al. 2023

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“…This intrinsic photostability of the signal is highly valuable in most of the tracking experiments where the photobleaching strongly limits the duration of the observed processes and requires more sophisticated approaches to be overcome. 26 Moreover, the SHG signal evolves as the square of the excitation laser power and does not saturate at large excitation power, contrary to fluorescence, 27 as shown on Figure 1d. No electrostriction effect that could alter the SHG signal by modifying the crystal lattice is observed within the available incident power range.…”

Section: Microscopy For Single Particle Trackingmentioning

confidence: 92%

“…This is illustrated in Figure c, in which the SHG of a single NC has been recorded during more than 20 min, showing no alteration of the intensity level. This intrinsic photostability of the signal is highly valuable in most of the tracking experiments where the photobleaching strongly limits the duration of the observed processes and requires more sophisticated approaches to be overcome . Moreover, the SHG signal evolves as the square of the excitation laser power and does not saturate at large excitation power, contrary to fluorescence, as shown on Figure d.…”

Section: Digital Holography and Two-photon Microscopy For Single Part...mentioning

confidence: 99%

3D Real-Time Two-Photon Microscopy Device for Single-Particle Holographic Tracking (3D-Red Shot)

Semmer,

Emperauger,

Lopez

et al. 2023

ACS Photonics

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Three-dimensional real-time tracking of single light emitters is an emerging tool for the assessment of biological processes such as axonal transport, for which high spatiotemporal resolution can provide invaluable insights into molecular motor detailed dynamics. We report the use of second harmonic generation from nonlinear nanocrystals in conjunction with holograms dynamically displayed on a digital micromirror device to steer the excitation laser focus in 3D around the particle on a specific pattern. The particle position is inferred from the collected intensities by using a maximum likelihood approach. The holograms are also used to compensate for the optical aberrations of the optical system. Our device achieves superlocalization precision of nanocrystals down to 5 nm. Experiments were performed at sampling rates up to ≈700 Hz, limited by the digital micromirror device refreshing rate. We also demonstrate the device ability to track the free diffusion of nonlinear nanocrystals over tens of micrometers with velocities as high as 30 μm•s −1 , and the directed motion of particles endocytosed by neuronal cells and transported within neurites.

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“…In SMT, individual proteins are detected, localized, and tracked over thousands of imaging frames at an interval of only a few milliseconds (fast SMT). Diffusion spectra derived from single-molecule trajectories provide the diffusion coefficients of differentially mobile subpopulations and the fraction of immobile molecules, which, in the case of TFs, are largely chromatin-bound (fraction bound) 40,41 .…”

Section: Introductionmentioning

confidence: 99%

Automated live-cell single-molecule tracking in enteroid monolayers reveals transcription factor dynamics probing lineage-determining function

Walther,

Anantakrishnan,

Dailey

et al. 2024

Preprint

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SummaryLineage transcription factors (TFs) provide one regulatory level of differentiation crucial for the generation and maintenance of healthy tissues. To probe TF function by measuring their dynamics during adult intestinal homeostasis, we established HILO-illumination-based live-cell single-molecule tracking (SMT) in mouse small intestinal enteroid monolayers recapitulating tissue differentiation hierarchiesin vitro. To increase the throughput, capture cellular features, and correlate morphological characteristics with diffusion parameters, we developed an automated imaging and analysis pipeline, broadly applicable to 2D culture systems. Studying two absorptive lineage-determining TFs, we find an expression level-independent contrasting diffusive behavior: While Hes1, key determinant of absorptive lineage commitment, displays a large cell-to-cell variability and an average fraction of DNA-bound molecules of ∼32%, Hnf4g, conferring enterocyte identity, exhibits more uniform dynamics and a bound fraction of ∼56%. Our results suggest that TF diffusive behavior can indicate the progression of differentiation and modulate earlyversuslate differentiation within a lineage.Highlights- Automated live-cell single-molecule tracking records hundreds of cells in enteroid monolayers- Cellular diffusion clustering and morphological feature correlation reveals subpopulations- Transcription factor dynamics regulate differentiation independent of expression level- Hes1 and Hnf4g display contrasting dynamics assisting earlyvs.late absorptive differentiation

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Single-molecule tracking (SMT): a window into live-cell transcription biochemistry

Cited by 18 publications

References 124 publications

Multiomics Analysis Reveals Novel Genetic Determinants for Lens Differentiation, Structure, and Transparency

Multiomics Analysis Reveals Novel Genetic Determinants for Lens Differentiation, Structure, and Transparency

3D Real-Time Two-Photon Microscopy Device for Single-Particle Holographic Tracking (3D-Red Shot)

Automated live-cell single-molecule tracking in enteroid monolayers reveals transcription factor dynamics probing lineage-determining function

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