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Diffusion, transport, and cell membrane organization investigated by imaging fluorescence cross-correlation spectroscopy.

ABSTRACT: Cell membrane organization is dynamic and is assumed to have different characteristic length scales. These length scales, which are influenced by lipid and protein composition as well as by the cytoskeleton, can range from below the optical resolution limit (as with rafts or microdomains) to far above the resolution limit (as with capping phenomena or the formation of lipid "platforms"). The measurement of these membrane features poses a significant problem because membrane dynamics are on the millisecond timescale and are thus beyond the time resolution of conventional imaging approaches. Fluorescence correlation spectroscopy (FCS), a widely used spectroscopic technique to measure membrane dynamics, has the required time resolution but lacks imaging capabilities. A promising solution is the recently introduced method known as imaging total internal reflection (ITIR)-FCS, which can probe diffusion phenomena in lipid membranes with good temporal and spatial resolution. In this work, we extend ITIR-FCS to perform ITIR fluorescence cross-correlation spectroscopy (ITIR-FCCS) between pixel areas of arbitrary shape and derive a generalized expression that is applicable to active transport and diffusion. ITIR-FCCS is applied to model systems exhibiting diffusion, active transport, or a combination of the two. To demonstrate its applicability to live cells, we observe the diffusion of a marker, the sphingolipid-binding domain (SBD) derived from the amyloid peptide Abeta, on live neuroblastoma cells. We investigate the organization and dynamics of SBD-bound lipid microdomains under the conditions of cholesterol removal and cytoskeleton disruption.

SUBMITTER: Sankaran J

PROVIDER: S-EPMC2770616 | biostudies-literature | 2009 Nov

REPOSITORIES: biostudies-literature

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Diffusion, transport, and cell membrane organization investigated by imaging fluorescence cross-correlation spectroscopy.

Sankaran Jagadish J Manna Manoj M Guo Lin L Kraut Rachel R Wohland Thorsten T

Biophysical journal 20091101 9

Cell membrane organization is dynamic and is assumed to have different characteristic length scales. These length scales, which are influenced by lipid and protein composition as well as by the cytoskeleton, can range from below the optical resolution limit (as with rafts or microdomains) to far above the resolution limit (as with capping phenomena or the formation of lipid "platforms"). The measurement of these membrane features poses a significant problem because membrane dynamics are on the m ...[more]

PMID: 19883607

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Project description:The ability to quantify protein concentrations and to measure protein interactions in vivo is key information needed for the understanding of complex processes inside cells, but the acquisition of such information from living cells is still demanding. Fluorescence-based methods like two-color fluorescence cross-correlation spectroscopy can provide this information, but measurement precision is hampered by various sources of errors caused by instrumental or optical limitations such as imperfect overlap of detection volumes or detector cross talk. Furthermore, the nature and properties of used fluorescent proteins or fluorescent dyes, such as labeling efficiency, fluorescent protein maturation, photostability, bleaching, and fluorescence brightness can have an impact. Here, we take advantage of previously published fluorescence lifetime correlation spectroscopy which relies on lifetime differences as a mean to discriminate fluorescent proteins with similar spectral properties and to use them for single-color fluorescence lifetime cross-correlation spectroscopy (sc-FLCCS). By using only one excitation and one detection wavelength, this setup avoids all sources of errors resulting from chromatic aberrations and detector cross talk. To establish sc-FLCCS, we first engineered and tested multiple green fluorescent protein (GFP)-like fluorescent proteins for their suitability. This identified a novel, to our knowledge, GFP variant termed short-lifetime monomeric GFP with the so-far shortest lifetime. Monte-Carlo simulations were employed to explore the suitability of different combinations of GFP variants. Two GFPs, Envy and short-lifetime monomeric GFP, were predicted to constitute the best performing couple for sc-FLCCS measurements. We demonstrated application of this GFP pair for measuring protein interactions between the proteasome and interacting proteins and for measuring protein interactions between three partners when combined with a red florescent protein. Together, our findings establish sc-FLCCS as a valid alternative for conventional dual-color fluorescence cross-correlation spectroscopy measurements.

Dataset Information

Diffusion, transport, and cell membrane organization investigated by imaging fluorescence cross-correlation spectroscopy.

Publications

Diffusion, transport, and cell membrane organization investigated by imaging fluorescence cross-correlation spectroscopy.

Similar Datasets

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