Spot-On: robust model-based analysis of single-particle tracking experiments

Hansen, Anders S.; Woringer, Maxime; Grimm, Jonathan B.; Lavis, Luke D.; Tjian, Robert; Darzacq, Xavier

doi:10.1101/171983

Cited by 64 publications

(117 citation statements)

References 41 publications

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“…Single molecule imaging studies of transcription factors in living nuclei have uncovered critical new features of their function that could not have been predicted based on biochemical or genomic approaches (Chen et al, 2014b;Chong et al, 2018;Hansen et al, 2018;Liu and Tjian, 2018;Loffreda et al, 2017;Teves et al, 2016). Here we have presented a series of experiments that investigate in detail the molecular mechanisms that govern the dynamic behavior of MeCP2 in live neurons.…”

Section: Discussionmentioning

confidence: 99%

MeCP2 nuclear dynamics in live neurons results from low and high affinity chromatin interactions

Piccolo

Liu

Dong

et al. 2019

Preprint

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Loss of function mutations in Methyl-CpGbinding Protein 2 (MeCP2) cause the severe neurological disorder Rett Syndrome. MeCP2 is a highly abundant nuclear protein particularly enriched in neurons. Although biochemical and genomic analyses of MeCP2-DNA interaction and genomic distribution demonstrate that MeCP2 binding on chromatin is dependent on DNA modification state, the dynamic behavior of individual MeCP2 proteins in live neurons has not been explored. Here we use live-cell singlemolecule imaging to assess the detailed kinetic features of MeCP2 in distinct sub-nuclear regions at high spatial and temporal resolution. Surprisingly, we found that, in granule cell nuclei, MeCP2 has unique diffusion and chromatin binding kinetics that are distinct from highly mobile sequence-specific transcription factors (TF) and immobile histone proteins. Approximately, half of MeCP2 is bound to DNA in a transiently stable mode that is similar to TF binding to their cognate sites. The binding of meCP2 to DNA requires its methyl-binding domain (MBD) and is sensitive to the levels of both DNA methylation and hydroxymethylation. However, when not stably bound, MeCP2 moves slowly in the nucleus most closely resembling histone H1.0. The rate of MeCP2 diffusion in compact, granule cell nuclei is determined by weak, transient DNA interactions mediated primarily by the MBD and three AT-hook domains located in the C-terminal portion of the protein. Both the fraction of stably bound MeCP2 and its rate of diffusion depend on the level of chromatin compaction and neuronal cell type.Our data reveal new features of MeCP2 that dictate its dynamic behavior in neuronal nuclei and suggest that the limited nuclear diffusion of MeCP2 in live neurons may contribute to its local impact on chromatin structure and gene expression.

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Section: Discussionmentioning

confidence: 99%

MeCP2 nuclear dynamics in live neurons results from low and high affinity chromatin interactions

Piccolo

Liu

Dong

et al. 2019

Preprint

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“…For example, for faster acquisition rates, you may use a model-based fitting to the distributions of displacements to estimate the fraction of the population that is bound and mobile and the diffusion coefficient of these populations. Recently our group has set up a web-based tool called spot-on where you can upload trajectory data to perform such analysis [13]. For residence time analysis on longer exposure time data, you may calculate the distribution of trajectory lengths of binding events, known as the survival probability distribution.…”

Section: Methodsmentioning

confidence: 99%

“…Second you may perform tracking on a fixed sample, calculate a mean-squared displacement curve, and determine the localization error from the intercept of the curve. If you are using spot-on [13] for analysis, it will estimate the localization error based on the data you provide. …”

Section: Notesmentioning

confidence: 99%

Single Molecule Imaging in Live Embryos Using Lattice Light-Sheet Microscopy

Mir

Reimer

Stadler

et al. 2018

Methods in Molecular Biology

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In the past decade, live-cell single molecule imaging studies have provided unique insights on how DNA-binding molecules such as transcription factors explore the nuclear environment to search for and bind to their targets. However, due to technological limitations, single molecule experiments in living specimens have largely been limited to monolayer cell cultures. Lattice light-sheet microscopy overcomes these limitations and has now enabled single molecule imaging within thicker specimens such as embryos. Here we describe a general procedure to perform single molecule imaging in living Drosophila melanogaster embryos using lattice light-sheet microscopy. This protocol allows direct observation of both transcription factor diffusion and binding dynamics. Finally, we illustrate how this Drosophila protocol can be extended to other thick samples using single molecule imaging in live mouse embryos as an example.

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“…Recent years have seen significant advances in both experimental [30][31][32][33][34][35] and data analysis strategies [36][37][38][39][40][41][42][43][44] of SPT, some of which have dramatically improved the information throughput. Among others, spt-PALM combines SPT with photoactivated localization microscopy (PALM) to enable single molecule tracking under dense labeling conditions through stochastic photoswitching 30 .…”

Section: Introductionmentioning

confidence: 99%

“…With spt-PALM, it is routine to acquire tens of thousands of diffusion trajectories from a single cell. A growing list of software tools has also been developed to facilitate spt-PALM data analysis 36,37,39,43,45 . In particular, variational Bayes SPT (vbSPT) package allows construction of a detailed diffusion model from spt-PALM data with parameters such as the number of states, the diffusion coefficient and the occupancy of each state, as well as the state transition rates even when the individual trajectories are short 36 .…”

Section: Introductionmentioning

confidence: 99%

High-throughput single-particle tracking reveals nested membrane nanodomain organization that dictates Ras diffusion and trafficking

Lee

Phelps

Huang

et al. 2019

Preprint

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Membrane nanodomains have been implicated in Ras signaling, but what these domains are and how they interact with Ras remain obscure. Using high throughput single particle tracking with photoactivated localization microscopy and detailed trajectory analysis, here we show that distinct membrane domains dictate KRas diffusion and trafficking in U2OS cells. KRas exhibits an immobile state in domains ~70 nm in size, each embedded in a larger domain (~200 nm) that confers intermediate mobility, while the rest of the membrane supports fast diffusion. Moreover, KRas is continuously removed from the membrane via the immobile state and replenished to the fast state, likely coupled to internalization and recycling. Importantly, both the diffusion and trafficking properties of KRas remain invariant over a broad range of protein expression levels. Our results reveal how membrane organization dictates KRas diffusion and trafficking and offer insight into how Ras signaling may be regulated through spatial mechanisms. Figure 2. KRas diffusion states are associated with distinct membrane domains.A) The three-state model for KRas diffusion with F, N, and I, representing the fast, the intermediate, and the immobile states, respectively. Model parameters were inferred using vbSPT on spt-PALM datasets with at least 30,000 trajectories obtained on cells induced with 2 ng/mL Dox. The arrows indicate state transitions (i.e. the probability of switching to a different state in the next frame) and the area of the circle and the thickness of the arrows are both roughly scaled to reflect their relative values. All parameters were derived from data acquired at 12 ms frame interval except for the diffusion coefficient of the immobile state, which was inferred from data taken at 35 ms frame interval. Error bars are 95% CIs; B)Step size histograms for immobilization events (red), one step before or after the immobilization event (blue), and all other steps (black). A diffusion step was part of an immobilization event if immobile state was assigned to that trajectory segment by vbSPT (see Methods); C) Map of the membrane locations where KRas molecules exhibit specific diffusion states (referred to as state coordinates) within a one-minute duration (taken from a spt-PALM dataset of ~20 min total duration). Red, blue, and green dots represent locations of the immobile, the intermediate, and the fast states, respectively, with each rendered circles scaled proportionally to the mean diffusion coefficient for the state; D) Pair correlation analysis on the averaged state coordinates across multiple, one-minute segments of longer spt-PALM datasets. The same color coding as in B) was used to distinguish the three states. For this analysis, molecules in the same diffusion state in successive frames only contributed a single, averaged state coordinate. The average state coordinates of all molecules captured within a one-minute segment were used for correlation analysis, and the results from multiple one-minute segments were averaged to yield the plot. Th...

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Spot-On: robust model-based analysis of single-particle tracking experiments

Cited by 64 publications

References 41 publications

MeCP2 nuclear dynamics in live neurons results from low and high affinity chromatin interactions

MeCP2 nuclear dynamics in live neurons results from low and high affinity chromatin interactions

Single Molecule Imaging in Live Embryos Using Lattice Light-Sheet Microscopy

High-throughput single-particle tracking reveals nested membrane nanodomain organization that dictates Ras diffusion and trafficking

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