Organization and Dynamics of NBD-Labeled Lipids in Membranes Analyzed by Fluorescence Recovery after Photobleaching

Abstract

Lateral diffusion of membrane constituents plays an important role in membrane organization and represents a central theme in current models describing the structure and function of biological membranes. Fluorescence recovery after photobleaching (FRAP) is a widely used approach that provides information regarding dynamic properties and spatial distribution of membrane constituents. On the basis of the unique concentration-dependent fluorescence emission properties of a fluorescently labeled cholesterol analogue modified at the tail region, 25-[N-[(7-nitrobenz-2-oxa-1,3-diazol-4-yl)-methyl]amino]-27-norcholesterol (25-NBD-cholesterol), we have previously shown that it exhibits local organization even at very low concentrations in membranes. In this paper, we address aspects regarding the molecular size and dynamics of such an organized assembly of 25-NBD-cholesterol by monitoring its lateral diffusion characteristics using FRAP. To obtain a comprehensive understanding of the organization and dynamics of 25-NBD-cholesterol in the membrane, we compare its diffusion properties to that of a fluorescent phospholipid analogue 1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine-N-(7-nitro-2-(1,3-benzoxadiazol-4-yl)) (NBD-PE). Our results indicate significant differences in the membrane dynamics of these NBD-labeled lipids. Importantly, on the basis of a novel wavelength-selective FRAP approach, our results show that the organization of 25-NBD-cholesterol is heterogeneous, with the presence of fast-and slow-diffusing species which could correspond to predominant populations of monomers and dimers of 25-NBD-cholesterol. The potential application of the wavelength-selective FRAP approach to monitor the organization and dynamics of molecules in membranes therefore represents an exciting possibility.

A real-time high-throughput fluorescence assay for sphingosine kinases

“…Our assay exploits a spectral REES in the environmentally sensitive properties of the NBD fl uor on NBD-Sph compared with NBD-S1P, which was completely dependent on SphK catalyzed phosphorylation of NBDSph to NBD-S1P. Our results suggest that NBD-S1P tends to associate in Triton X-100 micelles and that interactions between NBD groups on NBD-S1P are likely responsible for the red shift, similar to that observed with NBD-cholesterol in artifi cial membranes, where increasing concentrations show REES spectral transitions ( 16 ). Our assay does not require radioactive materials or isolation of products and is suffi ciently sensitive for high-throughput screening of chemical libraries and characterization of lead compounds.…”

“…Fractions corresponding to the monomeric volume by guest, on May 10, 2018 www.jlr.org Downloaded from to a red shift in the excitation wavelength is termed red edge excitation shift (REES). The NBD fl uorophore is highly sensitive to its environment, and the REES of NBDlabeled lipids has been used to monitor a variety of biological and biophysical processes in cellular and/or artifi cial lipid membranes ( 16,17 ). Therefore, we next examined whether the NBD-Sph spectral changes induced by SphK1 or SphK2 activity were accompanied by a corresponding shift in fl uorescence emission.…”

Cholesterol: An evergreen molecule in biology

“…These results assume relevance in the context of increasing membrane thickness from Golgi to the plasma membrane due to the gradient in cholesterol concentration [5]. We later showed, using wavelengthselective FRAP [46] and REES [29] that the dynamics of the cholesterol monomer and dimer population is considerably different. The monomer population exhibited faster lateral dynamics and was found to be less ordered than the dimer (Fig.…”

Adenylyl cyclase AC8 directly controls its micro-environment by recruiting the actin cytoskeleton in a cholesterol-rich milieu

“…Thus, whereas FRAP accurately measures the mobility of a population of molecules within 1.54 mm 2 of a juxtanuclear region of the PM, FCS measures the diffusion of tens of molecules in real time, within a smaller area of the apical membrane (,0.1 mm 2 ) (Edidin, 1992;Kusumi et al, 2005). Furthermore, FCS distinguishes fast moving molecules within a microdomain, whereas FRAP detects long-range diffusion between several microdomains in the PM (Hancock and Parton, 2005;Haustein and Schwille, 2004;Lingwood et al, 2008;Pucadyil et al, 2007). However, unlike FRAP, FCS cannot reliably resolve very slow moving or immobile proteins and hence cannot determine the mobile fraction.…”