Single Molecule Fluorescence Approaches Shed Light on Intracellular RNAs

Pitchiaya, Sethuramasundaram; Heinicke, Laurie A.; Custer, Thomas C.; Walter, Nils G.

doi:10.1021/cr400496q

Cited by 73 publications

(84 citation statements)

References 497 publications

(1,219 reference statements)

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“…The microscope objective was temperature regulated using an aluminum collar to which a water-cooled Peltier element was mounted 40 . For temperaturedependent control measurements in buffer, a custom-built temperature-controlled sample holder for glass cuvettes employing Peltier elements and a HAT Control-M20 temperature controller with a Pt100 temperature sensor was used as previously described 5 . For both extracellular and intracellular measurements, the temperature at the laser focus was calibrated using the temperature-dependent fluorescence lifetime of rhodamine B 40,59,60 .…”

Section: Instrumentation For Single-molecule Fret In Cellsmentioning

confidence: 99%

“…Owing to its sensitivity, fluorescence has become particularly popular-especially with the broad availability of fluorescent proteins 1 -for investigating cellular localization, biomolecular interactions 2 , and protein stability and folding dynamics 3 in living cells. This sensitivity has enabled the use of single-molecule tracking 4,5 , intracellular fluorescence correlation spectroscopy (FCS) 6 , and image correlation methods 7 for investigating molecular diffusion and transport processes. Super-resolution methods now enable localization and dynamic imaging below the diffraction limit in live cells 8,9 .…”

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confidence: 99%

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Single-molecule spectroscopy of protein conformational dynamics in live eukaryotic cells

et al. 2015

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Single-molecule methods have become widely used for quantifying the conformational heterogeneity and structural dynamics of biomolecules in vitro. Their application in vivo, however, has remained challenging owing to shortcomings in the design and reproducible delivery of labeled molecules, the range of applicable analysis methods, and suboptimal cell culture conditions. By addressing these limitations in an integrated approach, we demonstrate the feasibility of probing protein dynamics from milliseconds down to the nanosecond regime in live eukaryotic cells with confocal single-molecule Förster resonance energy transfer (FRET) spectroscopy. We illustrate the versatility of the approach by determining the dimensions and submicrosecond chain dynamics of an intrinsically disordered protein; by detecting even subtle changes in the temperature dependence of protein stability, including in-cell cold denaturation; and by quantifying the folding dynamics of a small protein. The methodology opens possibilities for assessing the effect of the cellular environment on biomolecular conformation, dynamics and function.

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Section: Instrumentation For Single-molecule Fret In Cellsmentioning

confidence: 99%

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confidence: 99%

Single-molecule spectroscopy of protein conformational dynamics in live eukaryotic cells

et al. 2015

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“…By imaging the mobility of RNA molecules, it was shown that transcript movement is not directional but is influenced by energy 83 . Transcripts leave the nucleus in different ways 84 , typically through nuclear pores. Export was shown to be faster than the nucleoplasmic diffusion rates of mRNA 85 …”

Section: Critical Issues In Aptamer-based Systems Designmentioning

confidence: 99%

RNA imaging in living cells – methods and applications

et al. 2014

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Numerous types of transcripts perform multiple functions in cells, and these functions are mainly facilitated by the interactions of the RNA with various proteins and other RNAs. Insight into the dynamics of RNA biosynthesis, processing and cellular activities is highly desirable because this knowledge will deepen our understanding of cell physiology and help explain the mechanisms of RNA-mediated pathologies. In this review, we discuss the live RNA imaging systems that have been developed to date. We highlight information on the design of these systems, briefly discuss their advantages and limitations and provide examples of their numerous applications in various organisms and cell types. We present a detailed examination of one application of RNA imaging systems: this application aims to explain the role of mutant transcripts in human disease pathogenesis caused by triplet repeat expansions. Thus, this review introduces live RNA imaging systems and provides a glimpse into their various applications.

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“…The first successful labelling and imaging of single mRNPs in vivo was achieved about 10 years ago (Fusco et al, 2003). 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%

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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Single Molecule Fluorescence Approaches Shed Light on Intracellular RNAs

Cited by 73 publications

References 497 publications

Single-molecule spectroscopy of protein conformational dynamics in live eukaryotic cells

Single-molecule spectroscopy of protein conformational dynamics in live eukaryotic cells

RNA imaging in living cells – methods and applications

Labelling and imaging of single endogenous messenger RNA particlesin vivo

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