We examined the involvement of membrane microdomains during human luteinizing hormone (LH) receptor recovery from receptor desensitization after removal of bound hormone. Lateral motions of individual desensitized LH receptors expressed on the surface of Chinese hamster ovary cells and transient association of these receptors with detergent-resistant membrane (DRM) microdomains isolated using isopycnic sucrose gradient ultracentrifugation were assessed. Single particle tracking experiments showed untreated individual LH receptors to be confined within cell-surface membrane compartments with an average diameter of 199 ± 17 nm and associated with membrane fractions characteristic of bulk plasma membrane. After brief exposure to human chorionic gonadotropin (hCG), LH receptors remained for several hours desensitized to hCG challenge. Throughout this period, significantly increased numbers of LH receptors were confined within smaller diameter (<120 nm) membrane compartments and associated with DRM fragments of characteristically low density. By 5 h, when cells again produced cAMP in response to hCG, unoccupied LH receptors were found in larger 169 ± 22 nm diameter cell-surface membrane compartments and >90 % of LH receptors were again found in high-density membrane fragments characteristic of bulk plasma membrane. Taken together, these results suggest that, during recovery from LH receptor desensitization, LH receptors are both located with DRM lipid environments and confined within small, mesoscale (80–160 nm) cell-surface compartments. This may reflect hormone-driven translocation of receptors into DRM and formation there of protein aggregates too large or too rigid to permit effective signaling. Once bound hormone is removed, receptor structures would have to dissociate before receptors can again signal effectively in response to hormone challenge. Moreover, such larger protein complexes would be more easily constrained laterally by membrane structural elements and so appear resident in smaller cell-surface compartments.

We have examined the association of insulin receptors (IR) and downstream signaling molecules with membrane microdomains in rat basophilic leukemia (RBL-2H3) cells following treatment with insulin or tris(2-pyridinecarbxylato)chromium(III) (Cr(pic)3). Single-particle tracking demonstrated that individual IR on these cells exhibited reduced lateral diffusion and increased confinement within 100 nm-scale membrane compartments after treatment with either 200 nM insulin or 10 μM Cr(pic)3. These treatments also increased the association of native IR, phosphorylated insulin receptor substrate 1 and phosphorylated AKT with detergent-resistant membrane microdomains of characteristically high buoyancy. Confocal fluorescence microscopic imaging of Di-4-ANEPPDHQ labeled RBL-2H3 cells also showed that plasma membrane lipid order decreased following treatment with Cr(pic)3 but was not altered by insulin treatment. Fluorescence correlation spectroscopy demonstrated that Cr(pic)3 did not affect IR cell-surface density or compete with insulin for available binding sites. Finally, Fourier transform infrared spectroscopy indicated that Cr(pic)3 likely associates with the lipid interface in reverse-micelle model membranes. Taken together, these results suggest that activation of IR signaling in a cellular model system by both insulin and Cr(pic)3 involves retention of IR in specialized nanometer-scale membrane microdomains but that the insulin-like effects of Cr(pic)3 are due to changes in membrane lipid order rather than to direct interactions with IR.

We used fluorescence correlation spectroscopy to examine the binding of insulin, insulin-like growth factor 1 (IGF1) and anti-receptor antibodies to insulin receptors (IR) and IGF1 receptors (IGF1R) on individual 2H3 rat basophilic leukemia cells. Experiments revealed two distinct classes of insulin binding sites with KD of 0.11 nM and 75 nM, respectively. IGF1 competes with insulin for a portion of the low-affinity insulin binding sites with KD of 0.14 nM and for the high-affinity insulin binding sites with KD of 10 nM. Dissociation rate constants of insulin and IGF1 were determined to be 0.015 min−1 and 0.013 min−1, respectively, allowing estimation of ligand association rate constants. Combined, our results suggest that, in addition to IR and IGF1R homodimers, substantial numbers of hybrid IR-IGF1R heterodimers are present on the surface of these cells.Fluorescence correlation spectroscopy reveals details of ligand binding to insulin receptors and IGF1 receptors on single cells. (A) Equilibrium binding of FITC-insulin to the surface of live cells demonstrates two distinct classes of ligand binding sites. (B) Complete dissociation of FITC-insulin from ligand binding sites on live cells occurs over approximately two hours.Highlights► FCS permits examination of interactions between biological molecules on individual live cells. ► 2H3-RBL cells express IR and IGF1R homodimers, as well as hybrid IR-IGF1R heterodimers. ► Insulin and IGF1 compete with differing affinities for available binding sites on these cells. ► Hybrid IR-IGF1R heterodimer formation is likely due to random-association of IR and IGF1R monomers.

The Mast cell Function-associated Antigen (MAFA) is a membrane glycoprotein on rat mast cells (RBL-2H3) expressed at a ratio of ∼ 1:30 with respect to the Type I Fcε receptor (FcεRI). Despite this stoichiometry, clustering MAFA by its specific mAb G63 substantially inhibits secretion of both granular and de novo synthesized mediators induced upon FcεRI aggregation. Since the FcεRIs apparently signal from within raft micro-environments, we investigated possible co-localization of MAFA within these membrane compartments containing aggregated FcεRI. We used cholera toxin B subunit (CTB) to cluster the raft component ganglioside GM1 and studied the effects of this perturbation on rotation of FcεRI and MAFA by time-resolved phosphorescence anisotropy of erythrosin-conjugated probes. CTB treatment would be expected to substantially inhibit rotation of raft-associated molecules. Experimentally, CTB has no effect on rotational parameters such as the long-time anisotropy (r∞) of unperturbed FcεRI or MAFA. However, on cells where FcεRI-IgE has previously been clustered by antigen (DNP14-BSA), CTB treatment increases the FcεRI-IgE's r∞ by 0.010 and MAFA's by 0.014. Similarly, CTB treatment of cells where MAFA had been clustered by mAb G63 increases MAFA's r∞ by 0.010 but leaves FcεRI's unaffected. Evaluation of raft localization of FcεRI and MAFA using sucrose gradient ultracentrifugation of Triton X-100 treated membrane fragments demonstrates that a significant fraction of MAFA molecules sediments with rafts when FcεRI is clustered by antigen or when MAFA itself is clustered by mAb G63. The large excess of FcεRI over MAFA explains why clustering MAFA does not substantively affect FcεRI dynamics. Moreover, in single-particle tracking studies of individual FcεRI-IgE or MAFA molecules, these proteins, upon clustering by antigen, move into small membrane compartments of reduced, but similar, dimensions. This provides additional indication of constitutive interactions between FcεRI and MAFA. Taken together, these results of distinct methodologies suggest that MAFA functions within raft microdomains of the RBL-2H3 cell membrane and thus in close proximity to the FcεRI which themselves signal from within the raft environment.

Lateral diffusion measurements on cell membrane molecules, most commonly accomplished through fluorescence photobleaching recovery (FPR or FRAP), provide information on such molecules’ size, environment, and participation in intermolecular interactions. However, difficulties arise in FPR measurements of lateral dynamics of materials, such as visible fluorescent protein (VFP) fusion proteins, where fluorescent intracellular species contribute to the fluorescence recovery signal and thus distort measurements intended to reflect surface molecules only. A new method helps eliminate these difficulties. In total internal reflection interference fringe FPR, interfering laser beams enter a 1.65-numercial aperture (NA) Olympus objective at the periphery of the back focal plane where the NA exceeds 1.38. This creates an extended interference pattern totally internally reflected at the coverslip–medium interface which excites fluorescence only from fluorescent molecules located where the cell contacts the coverslip. The large illuminated area interrogates many more membrane receptors than spot methods and hence obtains more diffusion information per measurement while rejecting virtually all interfering intracellular fluorescence. We report successful measurements of membrane dynamics of both VFP-containing and conventionally labeled molecules by this technique and compare them with results of other FPR methods.

We present, for the red fluorescent protein mCherry acting as both fluorescence resonant energy transfer (FRET) donor and acceptor, Förster critical distance (r0) values with five important visible fluorescent protein (VFP) variants as well as with itself. The pair EYFP–mCherry exhibits an r0 of 5.66 nm, equaling or exceeding any combination of VFPs reported previously. Moreover, mCherry should be an excellent chromophore for homo-FRET with an r0 of 5.10 nm for energy transfer between two mCherry moieties. Finally, mCherry exhibits higher r0 values than does DsRed. These characteristics, combined with mCherry’s rapid folding and excellent spectral properties, suggest that mCherry constitutes a valuable long-wavelength hetero-FRET acceptor and probe for homo-FRET experiments.