FLUORESCENCE CORRELATION SPECTROSCOPY TO MEASURE MEMBRANE PERTURBATIONS THAT AFFECT RECEPTOR-MEDIATED SIGNALING
Diffusion constants (D) and transient confinement times (τ0) of membrane bound fluorescent probes thought to partition into liquid-ordered (Lo) or liquid-disordered (Ld) inner / outer leaflet membrane regions were measured by imaging fluorescence correlation spectroscopy. Resting rat basophilic leukemia cells were used under a variety of pharmacological perturbations. Yellow Fluorescent Protein-Glycan-Glycosylphosphatidylinositol (YFP-gl-GPI) was used as the outer-leaflet Lo-like region marker, palmitoyl and myristoyl-Enhanced Green Fluorescent Protein (PM-EGFP) was used as the inner-leaflet Lo-like region marker, and geranylgeranylated EGFP (EGFP-GG) was used as an Ld-like lipid region marker on the inner leaflet. Treatments included C2- & C6-ceramide, cholesterol depletion by methyl-beta-cyclodextrin, 7-ketocholesterol addition, and cytochalasin D. These membrane perturbing treatments were done in efforts to identify a condition in which Lo-like regions would be disrupted for further study regarding IgE high affinity receptor signaling. C6-ceramide treatments showed a propensity to disorder Lo-like regions symmetrically for both leaflets of Lo-like domain markers, but only resulted in a ~10% decrease in τ0 for Alexa488-IgE-FcεRI without significant change in D. Aside from short-chain ceramide treatment, all other treatments resulted in either no change, or some degree of increase in τ0 for these fluorescent reporters, indicating the local lipid environment as not having changed or increased respectively. Asymmetric effects under treatment conditions between lipid monolayer Lo-like preferring markers were also observed.
Biochemistry; Cellular biology; Biophysics
Baird, Barbara Ann
Zipfel, Warren R.; Baskin, Jeremy M.
Chemistry and Chemical Biology
M.S., Chemistry and Chemical Biology
Master of Science
dissertation or thesis