3D Protein Dynamics in the Cell Nucleus

Singh, Annette; Galland, Rémi; Finch-Edmondson, Megan; Grenci, Gianluca; Sibarita, Jean‐Baptiste; Studer, Vincent; Viasnoff, Virgile; Saunders, Timothy E.

doi:10.1016/j.bpj.2016.11.3196

Cited by 27 publications

(24 citation statements)

References 43 publications

(59 reference statements)

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“…For instance, by using biologically inert macromolecules and measuring their mobility, it is possible to gather insights about the chromatin architecture at a scale comparable to the size of the macromolecules 3 – 5 . For these reasons many techniques have been applied over the years for studying protein diffusion in the nuclear environment 6 – 11 . Among these techniques, a widely used method for measuring diffusion is single-point fluorescence correlation spectroscopy (FCS) 12 .…”

Section: Introductionmentioning

confidence: 99%

Local raster image correlation spectroscopy generates high-resolution intracellular diffusion maps

et al. 2018

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Raster image correlation spectroscopy (RICS) is a powerful method for measuring molecular diffusion in live cells directly from images acquired on a laser scanning microscope. However, RICS only provides single average diffusion coefficients from regions with a lateral size on the order of few micrometers, which means that its spatial resolution is mainly limited to the cellular level. Here we introduce the local RICS (L-RICS), an easy-to-use tool that generates high resolution maps of diffusion coefficients from images acquired on a laser scanning microscope. As an application we show diffusion maps of a green fluorescent protein (GFP) within the nucleus and within the nucleolus of live cells at an effective spatial resolution of 500 nm. We find not only that diffusion in the nucleolus is slowed down compared to diffusion in the nucleoplasm, but also that diffusion in the nucleolus is highly heterogeneous.

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Section: Introductionmentioning

confidence: 99%

Local raster image correlation spectroscopy generates high-resolution intracellular diffusion maps

et al. 2018

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“…A potential use of this protocol may be for visualizing the mobility of proteins in three dimensions. Recent advances in super-resolution microscopy allow protein mobility to be visualized in vitro in 'x', 'y', and 'z' dimensions 41 42 . Our current method does not account for the mobility of proteins in the 'z' axis.…”

Section: Discussionmentioning

confidence: 99%

<em>In Vivo</em> Single-Molecule Tracking at the Drosophila Presynaptic Motor Nerve Terminal

Bademosi

Lauwers

Amor

et al. 2018

JoVE

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An increasing number of super-resolution microscopy techniques are helping to uncover the mechanisms that govern the nanoscale cellular world. Single-molecule imaging is gaining momentum as it provides exceptional access to the visualization of individual molecules in living cells. Here, we describe a technique that we developed to perform single-particle tracking photo-activated localization microscopy (sptPALM) in Drosophila larvae. Synaptic communication relies on key presynaptic proteins that act by docking, priming, and promoting the fusion of neurotransmitter-containing vesicles with the plasma membrane. A range of protein-protein and protein-lipid interactions tightly regulates these processes and the presynaptic proteins therefore exhibit changes in mobility associated with each of these key events. Investigating how mobility of these proteins correlates with their physiological function in an intact live animal is essential to understanding their precise mechanism of action. Extracting protein mobility with high resolution in vivo requires overcoming limitations such as optical transparency, accessibility, and penetration depth. We describe how photoconvertible fluorescent proteins tagged to the presynaptic protein Syntaxin-1A can be visualized via slight oblique illumination and tracked at the motor nerve terminal or along the motor neuron axon of the third instar Drosophila larva.

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“…The second perspective is to indirectly probe chromatin organization from the point of view of the molecules exploring this complex environment. By this approach, the accessibility of the nuclear landscape toward diffusing molecules is measured using methods such as fluorescence recovery after photobleaching (FRAP) [25][26][27][28][29], single particle tracking (SPT) [30][31][32][33][34] and fluorescence fluctuation spectroscopy (FFS) [35][36][37][38][39][40] (Figure 1). The application of SPT and FRAP to study nuclear factor dynamics within live cell nuclear architecture has recently been reviewed [41].…”

Section: Introductionmentioning

confidence: 99%

“…FFS describes a family of analytical methods that when coupled with live cell microscopy and genetically encoded fluorescent tags, can provide a measurement of nuclear protein diffusion, concentration and oligomerization in the context of live cell chromatin organization [35][36][37][38][39][40]. FFS methods rely on measuring fluctuations in fluorescence intensity arising from a population of fluorescent molecules passing through a small observation volume [e.g.…”

Section: Introductionmentioning

confidence: 99%

Fluorescence fluctuation spectroscopy: an invaluable microscopy tool for uncovering the biophysical rules for navigating the nuclear landscape

Priest

Solano

Lou

et al. 2019

Biochemical Society Transactions

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Nuclear architecture is fundamental to the manner by which molecules traverse the nucleus. The nucleoplasm is a crowded environment where dynamic rearrangements in local chromatin compaction locally redefine the space accessible toward nuclear protein diffusion. Here, we review a suite of methods based on fluorescence fluctuation spectroscopy (FFS) and how they have been employed to interrogate chromatin organization, as well as the impact this structural framework has on nuclear protein target search. From first focusing on a set of studies that apply FFS to an inert fluorescent tracer diffusing inside the nucleus of a living cell, we demonstrate the capacity of this technology to measure the accessibility of the nucleoplasm. Then with a baseline understanding of the exploration volume available to nuclear proteins during target search, we review direct applications of FFS to fluorescently labeled transcription factors (TFs). FFS can detect changes in TF mobility due to DNA binding, as well as the formation of TF complexes via changes in brightness due to oligomerization. Collectively, we find that FFS-based methods can uncover how nuclear proteins in general navigate the nuclear landscape.

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3D Protein Dynamics in the Cell Nucleus

Cited by 27 publications

References 43 publications

Local raster image correlation spectroscopy generates high-resolution intracellular diffusion maps

Local raster image correlation spectroscopy generates high-resolution intracellular diffusion maps

<em>In Vivo</em> Single-Molecule Tracking at the Drosophila Presynaptic Motor Nerve Terminal

Fluorescence fluctuation spectroscopy: an invaluable microscopy tool for uncovering the biophysical rules for navigating the nuclear landscape

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