High-resolution three-dimensional mapping of mRNA export through the nuclear pore

Ma, Junjun; Liu, Zhen; Michelotti, Nicole; Pitchiaya, Sethuramasundaram; Veerapaneni, Ram; Androsavich, John R.; Walter, Nils G.; Yang, Weidong

doi:10.1038/ncomms3414

Cited by 107 publications

(170 citation statements)

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“…In our approach, nanobodies against GFP and labeled with Alexa-Fluor dyes were used to recognize individual GFP-fused Nups within native human NPCs. SPEED microscopy was used to provide a spatial distribution for each tested Nup in both the radial and axial dimension, which was achieved through illumination of Nups within single NPCs at bottom of nuclear envelope (NE) for the radial view, and at the edge of the equator of the NE for the axial view (Ma and Yang, 2010;Ma et al, 2012Ma et al, , 2013Ma et al, , 2016. Single-point laser illuminations were used to illuminate single NPCs in contrast to wide-field setups, such as STORM and PALM, in order to reduce background noise and increase precision in identifying the location of single molecules.…”

Section: Introductionmentioning

confidence: 99%

Super-resolution mapping of scaffold nucleoporins in the nuclear pore complex

Kelich

Junod

et al. 2017

Journal of Cell Science

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The nuclear pore complex (NPC), composed of ∼30 different nucleoporins (Nups), is one of the largest supramolecular structures in eukaryotic cells. Its octagonal ring scaffold perforates the nuclear envelope and features a unique molecular machinery that regulates nucleocytoplasmic transport. However, the precise copy number and the spatial location of each Nup in the native NPC remain obscure due to the inherent difficulty of counting and localizing proteins inside of the sub-micrometer supramolecular complex. Here, we combined super-resolution single-point edge-excitation subdiffraction (SPEED) microscopy and nanobody-specific labeling to reveal the spatial distribution of scaffold Nups within three separate layers in the native NPC with a precision of ∼3 nm. Our data reveal both the radial and axial spatial distributions for Pom121, Nup37 and Nup35 and provide evidence for their copy numbers of 8, 32 and 16, respectively, per NPC. This approach can help pave the path for mapping the entirety of Nups in native NPCs and also other structural components of macromolecular complexes.

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Section: Introductionmentioning

confidence: 99%

Super-resolution mapping of scaffold nucleoporins in the nuclear pore complex

Kelich

Junod

et al. 2017

Journal of Cell Science

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“…In single-point edge-excitation sub-diffraction (SPEED) microscopy, a static point focus at an inclined angle of 45°(D) is used to observe mRNP export through single NPCs. This approach allows fast imaging rates and observation of interactions between mRNPs and a single NPC (Ma et al, 2013). Various other illumination schemes reduce background intensity by limiting fluorescence excitation to a thin sheet.…”

Section: Box 1 Illumination Schemes For Single-molecule Imagingmentioning

confidence: 99%

“…These diffusion coefficients confirm the initially obtained values (Politz et al, 1998). Several studies demonstrate that it is not only proteins that exhibit a complex pattern of mobility (Goulian and Simon, 2000;Kues et al, 2001;Bancaud et al, 2009), but also mRNPs that diffuse in the nucleus; their mobility cannot be described by a single diffusion coefficient (Shav-Tal et al, 2004;Siebrasse et al, 2008;Veith et al, 2010;Ma et al, 2013).…”

Section: Intranuclear Mrna Traffickingmentioning

confidence: 99%

“…Recent results using high-speed single-molecule imaging have demonstrated that RNA particles move as fast as theoretically expected for particles of corresponding size in the effective nuclear viscosity, which is in the range of 3 to 10 centipoise (Siebrasse et al, 2008;Veith et al, 2010;Grünwald and Singer, 2010;Ma et al, 2013). For example, Grünwald and Singer have determined a maximum diffusion coefficient of 1.4 µm 2 /s for an RNA the size of 3.3 kb (Grünwald and Singer, 2010), whereas Veith et al have found values of 0.3 and 0.7 µm 2 /s for the particularly bulky Balbiani ring mRNPs (with a diameter of around 50 nm) in the nuclei of the salivary gland cells of Chironomus tentans .…”

Section: Intranuclear Mrna Traffickingmentioning

confidence: 99%

“…Since then, labelling techniques, optical microscopy, tracking procedures and analysis tools have evolved dramatically (for review, see Weil et al, 2010;Eliscovich et al, 2013;Pitchiaya et al, 2014). Combinations of dedicated imaging technology and in vivo labelling have enabled studies of nuclear export (Grünwald and Singer, 2010;Siebrasse et al, 2012;Ma et al, 2013), and observation of the threedimensional (3D) trajectories of single RNA particles within living eukaryotic cells (Spille et al, 2015;Smith et al, 2015).…”

Section: Introductionmentioning

confidence: 99%

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Labelling and imaging of single endogenous messenger RNA particlesin vivo

Spille

Kubitscheck

2015

Journal of Cell Science

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RNA molecules carry out widely diverse functions in numerous different physiological processes in living cells. The RNA life cycle from transcription, through the processing of nascent RNA, to the regulatory function of non-coding RNA and cytoplasmic translation of messenger RNA has been studied extensively using biochemical and molecular biology techniques. In this Commentary, we highlight how single molecule imaging and particle tracking can yield further insight into the dynamics of RNA particles in living cells. In the past few years, a variety of bright and photo-stable labelling techniques have been developed to generate sufficient contrast for imaging of single endogenous RNAs in vivo. New imaging modalities allow determination of not only lateral but also axial positions with high precision within the cellular context, and across a wide range of specimen from yeast and bacteria to cultured cells, and even multicellular organisms or live animals. A whole range of methods to locate and track single particles, and to analyze trajectory data are available to yield detailed information about the kinetics of all parts of the RNA life cycle. Although the concepts presented are applicable to all types of RNA, we showcase here the wealth of information gained from in vivo imaging of single particles by discussing studies investigating dynamics of intranuclear trafficking, nuclear pore transport and cytoplasmic transport of endogenous messenger RNA.

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First‐Passage Processes in Cellular Biology

Iyer‐Biswas

Zilman

2016

Advances in Chemical Physics

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We first review fundamentals of the theory of stochastic processes. The system dynamics are specified by the set of its states, {S}, and the transitions between them, S → S , where S, S ∈ {S}. For example, the state S can denote the position of a Brownian particle, the numbers of molecules of different chemical species, or any other variable that characterizes the state of the system of interest. Here we restrict ourselves to processes for which the transition rates depend only on the system's instantaneous state, and not the entirety of its history. Such memoryless processes are known as Markovian and are applicable to a wide range of systems. We also assume that the transition rates do not explicitly depend on time, a condition known as stationarity. In this review we make the standard assumption that the transitions between the states are Poisson distributed random processes. In other words, the probability of transitioning from state S to state S in an infinitesimal interval, dt, is α(S, S )dt, where α(S, S ) is the transition rate.Examples. For a Brownian particle diffusing along a line, the state S is defined by the particle position; the transition rate is 2D/d 2 , where D is the diffusion coefficient and d is the step length. For a set of radioactive atoms undergoing decay with rate κ per atom, the state S is defined by the number, n, of atoms that have not decayed yet, and the transition rate from state n to state n − 1 is κ n. For a system with N reacting chemical species, the system state is defined by the concentrations of each reactant, (x 1 . . . x N ), and the transition rates are functions of these concentrations.B. Time evolution equation(s) for the system.We now summarize the equations that govern the dynamical evolution of the probability that the system is in state S at time t, which we denote by P (S, t). Typically, such equations are written in one of three formalisms: the Master Equation, the Fokker-Planck Equation, or the Stochastic Differential Equation; each is summarized below in turn. Details of the

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High-resolution three-dimensional mapping of mRNA export through the nuclear pore

Cited by 107 publications

References 44 publications

Super-resolution mapping of scaffold nucleoporins in the nuclear pore complex

Super-resolution mapping of scaffold nucleoporins in the nuclear pore complex

Labelling and imaging of single endogenous messenger RNA particlesin vivo

First‐Passage Processes in Cellular Biology

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