Lipid−Protein Interactions Revealed by Two-Photon Microscopy and Fluorescence Correlation Spectroscopy

Sánchez, Susana A.; Gratton, Enrico

doi:10.1021/ar040026l

Cited by 66 publications

(25 citation statements)

References 39 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…Fluorescence correlation spectroscopy (FCS) and related techniques scanning-FCS [64] and image correlation spectroscopy [17,30] are becoming more popular and hold great potential for in vivo mobility measurements (see [68] and references therein). FCS measures the fluctuations in fluorescence in the small excitation volume (~1 femtoliter) obtained in a confocal or two-photon microscope.…”

Section: Introductionmentioning

confidence: 99%

Exploring dynamics in living cells by tracking single particles

Levi

Gratton

2007

Cell Biochem Biophys

Self Cite

134

View full text Add to dashboard Cite

In the last years, significant advances in microscopy techniques and the introduction of a novel technology to label living cells with genetically encoded fluorescent proteins revolutionized the field of Cell Biology. Our understanding on cell dynamics built from snapshots on fixed specimens has evolved thanks to our actual capability to monitor in real time the evolution of processes in living cells. Among these new tools, single particle tracking techniques were developed to observe and follow individual particles. Hence, we are starting to unravel the mechanisms driving the motion of a wide variety of cellular components ranging from organelles to protein molecules by following their way through the cell. In this review, we introduce the single particle tracking technology to new users. We briefly describe the instrumentation and explain some of the algorithms commonly used to locate and track particles. Also, we present some common tools used to analyze trajectories and illustrate with some examples the applications of single particle tracking to study dynamics in living cells.

show abstract

Section: Introductionmentioning

confidence: 99%

Exploring dynamics in living cells by tracking single particles

Levi

Gratton

2007

Cell Biochem Biophys

Self Cite

134

View full text Add to dashboard Cite

show abstract

“…Fluorescence correlation spectroscopy (FCS), f luorescence crosscorrelation spectroscopy (FCCS), and photon-counting histograms (PCH) are techniques that examine fluctuations of fluorescent molecules in and out of a low-intensity laser beam focused to a volume of Ͻ1 fl (1)(2)(3)(4)(5)(6). In FCS, the fluctuations of a single species can be analyzed using statistical means to determine the average number of particles in the focal volume and the relative mobility of the fluorescent particles.…”

mentioning

confidence: 99%

Mapping dynamic protein interactions in MAP kinase signaling using live-cell fluorescence fluctuation spectroscopy and imaging

Slaughter

Schwartz

2007

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

128

124

View full text Add to dashboard Cite

fluorescence correlation spectroscopy ͉ fluorescence cross-correlation spectroscopy ͉ photon-counting histograms ͉ scaffold ͉ fluorescence resonance energy transfer A quantitative understanding of cellular systems requires temporally and spatially resolved characterization of dynamic molecular movement and interactions in live cell settings. Fluorescence correlation spectroscopy (FCS), f luorescence crosscorrelation spectroscopy (FCCS), and photon-counting histograms (PCH) are techniques that examine fluctuations of fluorescent molecules in and out of a low-intensity laser beam focused to a volume of Ͻ1 fl (1-6). In FCS, the fluctuations of a single species can be analyzed using statistical means to determine the average number of particles in the focal volume and the relative mobility of the fluorescent particles. An extension of FCS, fluorescence crosscorrelation spectroscopy (FCCS), is potentially useful for quantitative examination of interactions between specific pairs of proteins in live cells. In FCCS, fluctuations of two different fluorescent signals are recorded simultaneously and are analyzed using a cross-correlation function (6, 7). The amplitude of the crosscorrelation reflects the extent of codiffusion of the fluorescent molecules and hence their presence in the same mobile complex (Fig. 1A) (6, 8-10). PCH, however, is suited for examining the oligomeric status of the proteins of interest (5, 11). Although to date the in vivo application of these techniques is limited, recent progress in commercially available microscopes and the development of red fluorescent proteins (12) to pair with GFP have opened the door to biologists for live cell examination of mobility, association, and oligomeric status of proteins under native expression.Successful FCS requires low molecular concentration and high enough mobility to minimize photobleaching during data acquisition. This requirement makes FCS uniquely suitable for analyzing cytosolic biochemistry where the endogenous concentrations of protein molecules are low, and most molecules are sufficiently mobile. To date, the majority of live-cell FCS studies used exogenously introduced fluorescent molecules, and these analysis were somewhat hampered by the difficulty to control molecular concentrations. We reasoned that yeast may be a good model system for live-cell FCS because of the ease of introducing genetically encoded fluorescent tags to proteins expressed under the endogenous promoter at the native chromosomal loci. Native expression levels may be ideal for fluctuation measurements and allow for a meaningful and relevant account of particle numbers in the mobile pool.We applied FCS and FCCS to the study of protein complex formation in the MAPK cascade, a widely studied signaling pathway whose main components are shared across eukaryotic organisms. Members of the yeast MAPK cascade are involved in distinct morphogenetic and stress response pathways, such as mating in response to pheromones, filamentous growth in response to nutrient conditions, and stress r...

show abstract

“…GUVs constitute an ideal model for microscopic studies because of their size (20-50 mm); this fact permits the direct observation of micrometer-scale domains that can be observed directly by fluorescence microscopy and the study of liquid phases from a more controlled physical perspective than is possible in cells. The GUVs have been used extensively in different studies (18)(19)(20)(21)(22)(23), and we have used them to study the interaction of phospholipase A 2 (24), apoA-I, and rHDL with lipid bilayers (17,25,26). We used LAURDAN generalized polarization (GP) imaging to characterize phase organization in GUVs made of different lipid mixtures containing cholesterol, and we show the usefulness of this technique to quantify the kinetics of cholesterol removal from different membrane pools.…”

mentioning

confidence: 99%

Interaction of high density lipoprotein particles with membranes containing cholesterol

Sánchez

Tricerri

Gratton

2007

Journal of Lipid Research

Self Cite

View full text Add to dashboard Cite

In this study, free cholesterol (FC) efflux mediated by human HDL was investigated using fluorescence methodologies. The accessibility of FC to HDL may depend on whether it is located in regions rich in unsaturated phospholipids or in domains containing high levels of FC and sphingomyelin, known as "lipid rafts." Laurdan generalized polarization and two-photon microscopy were used to quantify FC removal from different pools in the bilayer of giant unilamellar vesicles (GUVs). GUVs made of POPC and FC were observed after incubation with reconstituted particles containing apolipoprotein A-I and POPC [78Å diameter reconstituted high density lipoprotein (rHDL)]. Fluorescence correlation spectroscopy data show an increase in rHDL size during the incubation period. GUVs made of two "raft-like" mixtures [DOPC/DPPC/FC (1:1:1) and POPC/SPM/FC (6:1:1)] were used to model liquidordered/liquid-disordered phase coexistence. Through these experiments, we conclude that rHDL preferentially removes cholesterol from the more fluid phases. These data, and their extrapolation to in vivo systems, show the significant role that phase separation plays in the regulation of cholesterol homeostasis.-Sanchez, S. A., M. A. Tricerri, and E. Gratton. Interaction of high density lipoprotein particles with membranes containing cholesterol. J. Lipid Res.

show abstract

Lipid−Protein Interactions Revealed by Two-Photon Microscopy and Fluorescence Correlation Spectroscopy

Cited by 66 publications

References 39 publications

Exploring dynamics in living cells by tracking single particles

Exploring dynamics in living cells by tracking single particles

Mapping dynamic protein interactions in MAP kinase signaling using live-cell fluorescence fluctuation spectroscopy and imaging

Interaction of high density lipoprotein particles with membranes containing cholesterol

Contact Info

Product

Resources

About