Investigation of the biophysical properties of a fluorescently modified ceramide-1-phosphate

Abstract

Ceramide-1-phosphate (C1P) is an important signaling sphingolipid and a metabolite of ceramide. C1P contains an anionic phosphomonoester head group and has been shown to regulate physiological and pathophysiological processes such as cell proliferation, inflammation, apoptosis, phagocytosis, and macrophage chemotaxis. Despite this mechanistic information on its role in intra- and intercellular communication, little information is available on the biophysical properties of C1P in biological membranes and how it interacts with effector proteins. Fluorescently labeled lipids have been a useful tool to understand the membrane behavior properties of lipids such as phosphatidylserine, cholesterol, and some phosphoinositides. However, to the best of our knowledge, fluorescently labeled C1P hasn’t been implemented to investigate its ability to serve as a mimetic of endogenous C1P in cells or untagged C1P in in vitro experiments. Cellular and in vitro assays demonstrate TopFluor-C1P harbors a fluorescent group that is fully buried in the hydrocarbon core and fluoresces across the spectrum of physiological pH values. Moreover, TopFluor-C1P didn’t affect cellular toxicity at concentrations employed, was as effective as unlabeled C1P in recruiting an established protein effector to intracellular membranes, and its subcellular localization recapitulated what is known for endogenous C1P. Notably, the diffusion coefficient of TopFluor-C1P was slower than that of TopFluor-phosphatidylserine or TopFluor-cholesterol in the plasma membrane and similar to that of other fluorescently labeled sphingolipids including ceramide and sphingomyelin. These studies demonstrate that TopFluor-C1P should be a reliable mimetic of C1P to study C1P membrane biophysical properties and C1P interactions with proteins.

Excitation of Fluorescent Lipid Probes Accelerates Supported Lipid Bilayer Formation via Photosensitized Lipid Oxidation

“…Illumination for 30 min accelerated the SLB formation time from 440 to 365 s. However, rupture of these vesicles never reaches the single-step profile observed with DOPC/TopFluor-PC vesicles. With TopFluor-PC, the fluorophore is embedded in the hydrophobic core of the membrane, , whereas with BODIPY-PE, the fluorophore is positioned in the aqueous environment. Thus, the position of these closely related fluorophores in relation to the bilayer plays a role in how illumination alters SLB formation.…”

A comparison of lipid diffusive dynamics in monolayers and bilayers in the context of interleaflet coupling

“…All GUVs were labelled with 0.005 mol% TF-PC 89,90 to enable both membrane visualisation and fluorescence fluctuation measurements 49 . Specifically, we performed lsFCS to determine the τ d and number ( N ) of TF-PC molecules within the observation volume, in a single experiment 49,55,56 .…”

Investigation of nano- and microdomains formed by ceramide 1 phosphate in lipid bilayers

“…Our investigation revealed significant findings for giant unilamellar vesicles (GUVs) incorporating saturated C1P species, notably C1P16, previously documented to induce gel domains 13 , 30 . The introduction of fluorescently tagged C1P16 has been shown to enhance rigidity and order within the lipid bilayer 31 . Unlike prior observations, autocorrelation curve morphology implies the presence of dual populations, possibly reflecting fluid and gel phases (Fig.…”