Fluorescence correlation spectroscopy in vivo

Mütze, Jörg; Ohrt, Thomas; Schwille, Petra

doi:10.1002/lpor.200910041

Cited by 64 publications

(51 citation statements)

References 119 publications

(120 reference statements)

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“…2). We quantified translational diffusion by correlating the fluorescence signal from the confocal measurements 6,26 . As expected, the intracellular diffusion time of ProTα increased relative to the diffusion time of the extracellular medium ( Fig.…”

Section: Protein Diffusivity Structure and Conformational Dynamicsmentioning

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 .…”

mentioning

confidence: 99%

“…5), anisotropies (Supplementary Fig. 6) and, most importantly, dynamics in the millisecond range and even below 6,17,[25][26][27] .…”

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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: Protein Diffusivity Structure and Conformational Dynamicsmentioning

confidence: 99%

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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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“…Developed in the early 1970s (27), fluorescence correlation spectroscopy (FCS) and fluorescence cross-correlation spectroscopy (FCCS; Fig. 1A) measure and correlate the temporal intensity fluctuations emitted from fluorescent proteins moving in and out of a small focal volume on a microsecond to millisecond time scale (31). Diffusion coefficients (D) quantify the rates of movement for individual proteins and are derived from the time they dwell within the focal volume and the consequent autocorrelation curves.…”

Section: Methodsmentioning

confidence: 99%

Shank2 contributes to the apical retention and intracellular redistribution of NaPiIIa in OK cells

Dobrinskikh

Lanzanò

Rachelson

et al. 2013

American Journal of Physiology-Cell Physiology

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In renal proximal tubule (PT) cells, sodium-phosphate cotransporter IIa (NaPiIIa) is normally concentrated within the apical membrane where it reabsorbs ∼70% of luminal phosphate (Pi). NaPiIIa activity is acutely regulated by moderating its abundance within the apical membrane. Under low-Pi conditions, NaPiIIa is retained within the apical membrane. Under high-Pi conditions, NaPiIIa is retrieved from the apical membrane and trafficked to the lysosomes for degradation. The present study investigates the role of Shank2 in regulating the distribution of NaPiIIa. In opossum kidney cells, a PT cell model, knockdown of Shank2 in cells maintained in low-Pi media resulted in a marked decrease in NaPiIIa abundance. After being transferred into high-Pi media, live-cell imaging showed that mRFP-Shank2E and GFP-NaPiIIa underwent endocytosis and trafficked together through the subapical domain. Fluorescence cross-correlation spectroscopy demonstrated that GFP-NaPiIIa and mRFP-Shank2 have indistinguishable diffusion coefficients and migrated through the subapical domain in temporal synchrony. Raster image cross-correlation spectroscopy demonstrated these two proteins course through the subapical domain in temporal-spatial synchrony. In the microvilli of cells under low-Pi conditions and in the subapical domain of cells under high-Pi conditions, fluorescence lifetime imaging microscopy-Forster resonance energy transfer analysis of Cer-NaPiIIa and EYFP-Shank2E found these fluors reside within 10 nm of each other. Demonstrating a complexity of functions, in cells maintained under low-Pi conditions, Shank2 plays an essential role in the apical retention of NaPiIIa while under high-Pi conditions Shank2 remains associated with NaPiIIa and escorts NaPiIIa through the cell interior.

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“…Despite its relatively small time resolution and excitation volume, the combination of FCS with a confocal setup is still the most popular among the here discussed techniques [88]. It is not only non invasive and easy to implement using epi-fluorescence microscopes.…”

Section: Application Domain and Invasivenessmentioning

confidence: 99%