Dynamics imaging of lipid phases and lipid-marker interactions in model biomembranes

Abstract

Biomembranes are complex systems that regulate numerous biological processes. Lipid phases that constitute these membranes influence their properties and transport characteristics. Here, we demonstrate the potential of short-range dynamics imaging (excited-state lifetime, rotational diffusion, and order parameter) as a sensitive probe of lipid phases in giant unilamellar vesicles (GUVs). Liquid-disordered and gel phases were labeled with Bodipy-PC at room temperature. Two-photon fluorescence lifetime imaging microscopy of single-phase GUVs reveals more heterogeneity in fluorescence lifetimes of Bodipy in the gel phase (DPPC: 3.8+/-0.6 ns) as compared with the fluid phase (DOPC: 5.2+/-0.2 ns). The phase-specificity of excited-state lifetime of Bodipy-PC is attributed to the stacking of ordered lipid molecules that possibly enhances homo-FRET. Fluorescence polarization anisotropy imaging also reveals distinctive molecular order that is phase specific. The results are compared with DiI-C12-labeled fluid GUVs to investigate the sensitivity of our fluorescence dynamics assay to different lipid-marker interactions. Our results provide a molecular perspective of lipid phase dynamics and the nature of their microenvironments that will ultimately help our understanding of the structure-function relationship of biomembranes in vivo. Furthermore, these ultrafast excited-state dynamics will be used for molecular dynamics simulation of lipid-lipid, lipid-marker and lipid-protein interactions.

Molecular Perspective of Antigen-mediated Mast Cell Signaling

“…Confocal Fluorescence Microscopy-The experimental setup and data analysis have been described in more detail elsewhere (25,27). Briefly, a confocal microscope consisting of a fibercoupled laser system, a scanner (Olympus FluoView300), differential interference contrast optics, an inverted microscope (Olympus IX81), and an Olympus 60ϫ, 1.2 NA water-immersion objective was used for three-channel imaging.…”

“…Experiments were performed over several days on different cell preparations. Complementary 1P (480 nm, 4.2 MHz) timeresolved fluorescence measurements were carried out to enhance the time resolution and signal-to-noise ratio as described previously (25,27). In these studies, the laser beam was strategically positioned at areas of interest on the cell membrane.…”

“…For 2P-FLIM, measured at the magic angle (54.7°), and polarization imaging, measured at polarizations parallel and perpendicular relative to the excitation laser, the epi-fluorescence polarization was analyzed using a Glan-Thompson polarizer before each of two detector channels. The signal was amplified, and fluorescence lifetime images were constructed with a time-correlated single-photon counting module (Becker & Hickl SPC830) (27). 2P-FLIM images were recorded using 256 ϫ 256 pixels with 65 time bins per pixel (i.e.…”

“…These results supported the hypothesis that lipid molecules are recruited into more ordered domains that serve as platforms for IgE-Fc⑀RI signaling. Furthermore, the gross perturbations inherent under these nonphysiological cross-linking conditions provided a simple platform on which to test and optimize our fluorescence dynamics imaging assay (25,27) for probing molecule-molecule interactions and structural changes relevant to cell signaling.…”

“…Here, in individual mast cells under more physiological conditions, we test the hypothesis that IgE-Fc⑀RI signaling occurs in specialized, cholesterol-rich regions of the plasma membrane, using the same fluorescence dynamics assay (25,27). Our rationale is that membrane-associated molecular events on the ps to ns time scales translate to interrogating structural changes on the spatial order of 100 nm (at 100-ps resolution) (30), which is below the diffraction limit of traditional optical microscopy.…”