Plasma membrane domains enriched in cholesterol, saturated phospholipids and sphingolipids are hypothesized to play functionally important roles in cell signaling, biomolecular trafficking and disease pathogenesis. However, the precise composition, dimension and life span of these domains in biological membranes are still under debate. Detergent extraction and biochemical characterization of membrane domains, including cholesterol depletion studies, have been widely used but are often plagued by various problems and do not allow for direct visualization of domains in live, intact cell membranes. Various microscopic and spectroscopic methods have been developed to provide the spatial and temporal information required to better understand domain formation and function. Still, many of these methods are not amenable to live cells, do not provide single cell data, are spatially limited by optical diffraction, or provide only seconds to microseconds temporal resolution. Under physiological conditions, membrane domains are likely transient and smaller than the diffraction limit of optical microscopy and therefore defy detection using conventional imaging methods.
To overcome these limitations and obtain molecular-level, spatio-temporal information on membrane domains, we developed an ultrafast fluorescence dynamics imaging assay that includes time-resolved fluorescence lifetime and polarization anisotropy imaging. Supported lipid bilayer model membranes and suspended and adherent RBL mast cells under non-physiological and physiological conditions of IgE receptor cross-linking were used as model systems in these studies. The excited-state dynamics and rotational diffusion (picoseconds to nanoseconds timescale) obtained with this assay are inherently sensitive to the immediate surroundings of a fluorescently labeled molecule and allow for real-time monitoring of membrane structure, organization and heterogeneity in individual, live cells.
In this Dissertation, the feasibility of this ultrafast fluorescence dynamics assay is demonstrated on simple model lipid membranes labeled with a fluorescent phospholipid analog. The fluorescent probe exhibits longer lifetime, higher order and longer overall rotational correlation time in more ordered, gel phase membranes as compared to fluid, or liquid-disordered, phase. These site-specific observations agree with the literature for model membranes analyzed via traditional ensemble spectrofluorimetric lifetime and anisotropy experiments.
The fluorescence dynamics assay is then used to detect more ordered, cholesterol enriched domains and relate their nanostructure and dynamics to IgE receptor signaling, in the plasma membrane of single, live RBL mast cells under non-physiological and physiological conditions. In mast cells, antigen-mediated cross-linking of the high affinity IgE receptor (FcϵRI) results in movement of FcϵRI into cholesterol-rich domains in the plasma membrane, where it is phosphorylated by the Src kinase, Lyn, to initiate the exocytotic release of histamine in the allergic response. Lyn-induced phosphorylation of FcϵRI occurs in a cholesterol-dependent manner, leading to the hypothesis that cholesterol-rich membrane domains may act as functional receptor signaling platforms. When IgE-FcϵRI is non-physiologically cross-linked with anti-IgE at 4°C, molecules associated with cholesterol-rich microdomains (e.g., saturated lipids [the lipid analog 1,1'-dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine (diI-C 18) or glycosphingolipids]) and lipid-anchored proteins co-redistribute with cross-linked IgE-FcϵRI. We find an enhancement in diI-C18 and Alexa Fluor 488-labeled IgE-FcϵRI fluorescence lifetime and anisotropy in optically resolvable microdomains where these molecules co-localize. These results suggest that fluorescence lifetime and, particularly, anisotropy permit us to measure lipid molecule recruitment into more ordered domains that serve as IgE-mediated signaling platforms.
In a collaborative effort, using time-of-flight secondary ion mass spectrometry (ToF-SIMS) imaging of intact, individual mast cells, lipid chemical identity and distribution are also investigated as a function of extensive cross-linking. Multiple lipid species are visualized at the sub-cellular level, with the results suggesting that IgE-FcϵRI cross-linking induces very subtle changes in cholesterol distribution, necessitating exquisitely sensitive analysis methods such as fluorescence lifetime and polarization anisotropy.
Under more challenging physiological conditions, excited-state fluorescence dynamics are used to correlate sub-resolution nanostructural changes in the diI-C18-labeled plasma membrane with IgE-FcϵRI cross-linking in adherent mast cells stimulated with multivalent antigen at ∼20°C. Time-dependent fluorescence lifetime imaging of diI-C18 shows changes in lifetime that agree with the kinetics of stimulated FcϵRI tyrosine phosphorylation under the same conditions. Lifetime imaging of Alexa Fluor 488-labeled IgE-FcϵRI indicates that fluorescence resonance energy transfer (FRET) occurs with diI-C18 with similar kinetics. These live cell studies provide direct evidence for IgE-FcϵRI associations with specialized cholesterol-rich domains within ∼4 nm at maximal association during IgE receptor signaling.
