Raster Image Correlation Spectroscopy and Number and Brightness Analysis

Digman, Michelle A.; Stakic, Milka; Gratton, Enrico

doi:10.1016/b978-0-12-388422-0.00006-6

Cited by 56 publications

(75 citation statements)

References 27 publications

(24 reference statements)

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“…5 A and B). The presence of these two different populations was further confirmed using raster scan image correlation spectroscopy (RICS) (29,33) in several subnuclear territories (Fig. S7).…”

Section: Resultsmentioning

confidence: 72%

“…NADH 2P-FLIM cellular map provides sensitive measurements of local activity associated with NADH metabolism (22-28). Fluorescence correlation spectroscopy analyzes the fluctuation of fluorescent molecules in a small illuminated spot, providing spatiotemporal maps of concentration, interaction, or diffusion parameters of molecules (29)(30)(31)(32)(33).In this study, we used 2P-FLIM and fluctuation techniques to decipher the dynamics of NADH metabolism in live cells (details are in SI Introduction to the Techniques). Both 2P-FLIM and fluctuation-based diffusion measurements have pixel resolution to determine SIRT1 dynamics and related NADH metabolism.…”

mentioning

confidence: 99%

“…NADH 2P-FLIM cellular map provides sensitive measurements of local activity associated with NADH metabolism (22)(23)(24)(25)(26)(27)(28). Fluorescence correlation spectroscopy analyzes the fluctuation of fluorescent molecules in a small illuminated spot, providing spatiotemporal maps of concentration, interaction, or diffusion parameters of molecules (29)(30)(31)(32)(33).…”

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confidence: 99%

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Spatial dynamics of SIRT1 and the subnuclear distribution of NADH species

Aguilar-Arnal

Ranjit

Stringari

et al. 2016

Proc. Natl. Acad. Sci. U.S.A.

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Sirtuin 1 (SIRT1) is an NAD + -dependent deacetylase that functions as metabolic sensor of cellular energy and modulates biochemical pathways in the adaptation to changes in the environment. SIRT1 substrates include histones and proteins related to enhancement of mitochondrial function as well as antioxidant protection. Fluctuations in intracellular NAD + levels regulate SIRT1 activity, but how SIRT1 enzymatic activity impacts on NAD + levels and its intracellular distribution remains unclear. Here, we show that SIRT1 determines the nuclear organization of protein-bound NADH. Using multiphoton microscopy in live cells, we show that free and bound NADH are compartmentalized inside of the nucleus, and its subnuclear distribution depends on SIRT1. Importantly, SIRT6, a chromatin-bound deacetylase of the same class, does not influence NADH nuclear localization. In addition, using fluorescence fluctuation spectroscopy in single living cells, we reveal that NAD + metabolism in the nucleus is linked to subnuclear dynamics of active SIRT1. These results reveal a connection between NAD + metabolism, NADH distribution, and SIRT1 activity in the nucleus of live cells and pave the way to decipher links between nuclear organization and metabolism.S irtuins (SIRTs) are a conserved family of deacetylases that target a variety of proteins located in virtually all cellular compartments (1). Deacetylation by SIRTs may control many functional aspects of target proteins (2). Because SIRT deacetylase activity depends on the energy carrier NAD + , these enzymes are thought to operate as cellular metabolic sensors. In addition, because histones are targeted by nuclear SIRT, these enzymes could link variations in cellular metabolism to chromatin function.There are seven mammalian SIRTs (SIRT1 to SIRT7) with distinct subcellular locations. SIRT2 is mainly cytoplasmic; SIRT3, SIRT4, and SIRT5 are found in the mitochondrial compartment; and SIRT1, SIRT6, and SIRT7 are located in the cell nucleus (1). In mammals, SIRT1 contributes to development and protects from metabolic and cardiovascular disease, neurodegeneration, and cancer (3). SIRT1 has been reported to promote healthy aging and regulate lifespan (4, 5). At the cellular level, SIRT1 regulates lipid and glucose homeostasis, apoptosis, DNA repair, and mitochondrial function. Variations in NAD + levels control SIRT1 activity (6-9), a relevant finding in the regulation of circadian rhythms (10). Circadian rhythms in NAD + levels have been observed (11,12), which lead to fluctuating SIRT1 deacetylase activity (9) that, in turn, results into cyclic acetylation of specific SIRT1 targets (6, 9, 13). SIRT1 and SIRT6 segregate circadian metabolism by driving transcription of a differential subset of circadian genes (14). SIRT6 is a chromatinbound protein that was first characterized as a regulator of genome stability (15). The other nuclear SIRT, SIRT7, appears to be highly localized in the nucleolus and possibly involved in Pol-I-dependent transcription (16), and it has been shown to regula...

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Section: Resultsmentioning

confidence: 72%

mentioning

confidence: 99%

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confidence: 99%

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Spatial dynamics of SIRT1 and the subnuclear distribution of NADH species

Aguilar-Arnal

Ranjit

Stringari

et al. 2016

Proc. Natl. Acad. Sci. U.S.A.

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“…19,31,32 In this chapter, we will discuss the application of the fluctuation methods that are more relevant for the studies of membrane systems, which have particular requirements.…”

Section: The Autocorrelation Functionmentioning

confidence: 99%

“…In this contribution, we discuss fluctuation methods based on spatial correlation that reveal the dynamics of membrane lipids and proteins at the nanoscale. [16][17][18][19][20][21][22][23][24][25][26][27] We show here that spatial correlations intrinsically contain more information than the classical temporal correlation first introduced with the single-point FCS. Spatiotemporal correlation approaches have the CONTENTS…”

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confidence: 99%

Domains of Phosphoinositides in the Plasma Membrane

Bogaart

Beest

2014

Cell Membrane Nanodomains

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In the field of membrane lipid and protein dynamics, fluorescence recovery after photobleaching (FRAP) and single-particle tracking methods have provided ample evidence that lipids and proteins could have their motion restricted by interaction with other lipids and with the cell cytoskeleton. 1-8 Fluorescence correlation spectroscopy (FCS) is a relatively new method in this field that has received particular attention recently because of the possible combination with super-resolution microscopy methods. [9][10][11][12][13][14] In this context, the use of very small volumes of excitation or variable volumes of excitation 15 was considered necessary to unravel to transport of molecules at the nanoscale. However, most of the FCS studies done so far are based on the original idea of measuring temporal correlation at a single point in the membrane. Measuring a single location in the membrane is restrictive since the temporal fluctuations at one point cannot reveal local microstructures or the anisotropic molecular transport in membranes. In this contribution, we discuss fluctuation methods based on spatial correlation that reveal the dynamics of membrane lipids and proteins at the nanoscale. [16][17][18][19][20][21][22][23][24][25][26][27] We show here that spatial correlations intrinsically contain more information than the classical temporal correlation first introduced with the single-point FCS. Spatiotemporal correlation approaches have the CONTENTS

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Characterizing proteins in their cellular environment: Examples of recent advances in quantitative fluorescence microscopy

Royer

2019

Protein Science

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Quantitative characterization of protein interactions, both intramolecular and intermolecular, is crucial in understanding the mechanisms and regulation of their function. In recent years, it has become possible to obtain such information on protein systems in live cells, from bacteria to mammalian cell lines. This review discusses recent advances in measuring protein folding, absolute concentration, oligomerization, diffusion, transport, and organization at super-resolution.

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Raster Image Correlation Spectroscopy and Number and Brightness Analysis

Cited by 56 publications

References 27 publications

Spatial dynamics of SIRT1 and the subnuclear distribution of NADH species

Spatial dynamics of SIRT1 and the subnuclear distribution of NADH species

Domains of Phosphoinositides in the Plasma Membrane

Characterizing proteins in their cellular environment: Examples of recent advances in quantitative fluorescence microscopy

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