A gain series method for accurate EMCCD calibration.

Calibration of the gain and digital conversion factor of an EMCCD is necessary for accurate photon counting. We present a new method to quickly calibrate multiple gain settings of an EMCCD camera. Acquiring gain-series calibration data and analyzing the resulting images with the EMCCD noise model more accurately estimates the gain response of the camera. Furthermore, we develop a method to compare the results from different calibration approaches. Gain-series calibration outperforms all other methods in this self-consistency test.

Insulin receptors and downstream substrates associate with membrane microdomains after treatment with insulin or chromium(III) picolinate.

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.

Actin-dependent clustering of insulin receptors in membrane microdomains.

Recent evidence suggests that, after binding insulin, insulin receptors (IR) interact with specialized, cholesterol-containing, membrane microdomains and components of the actin cytoskeleton. Using single particle tracking techniques, we examined how binding of insulin, depletion of membrane cholesterol and disruption of actin filaments affect the lateral diffusion of individual quantum dot-labeled native IR on live rat basophilic leukemia 2H3 cells. We also examined the effects of similar treatments on IR clustering and multivalent insulin binding on these cells using both photon counting histogram analysis and polarization-based fluorescence resonance energy homo-transfer imaging. Our analyses indicate that binding of insulin to IR on these cells is multivalent, involving at least two insulin molecules per IR as labeling concentrations approach 1µM. Insulin binding also reduces lateral diffusion of IR and the size of membrane compartments accessed by IR. For IR that have not bound insulin, lateral diffusion of IR and the size of membrane compartments accessed by IR increase after disrupting actin filaments or depleting membrane cholesterol. However, clustering of insulin-occupied IR is reduced only by disrupting actin filaments or by fixing cells with paraformaldehyde prior to exposure to insulin, but not by depleting membrane cholesterol. Thus, it appears that, although restriction of IR lateral diffusion on these cells is sensitive to both actin filament dynamics and membrane cholesterol content, clustering of insulin-occupied IR primarily involves an actin-dependent mechanism.

Fluorescence correlation spectroscopic examination of insulin and insulin-like growth factor 1 binding to live cells.

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 K(D) of 0.11 nM and 75 nM, respectively. IGF1 competes with insulin for a portion of the low-affinity insulin binding sites with K(D) of 0.14 nM and for the high-affinity insulin binding sites with K(D) 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.

Restricted lateral diffusion of luteinizing hormone receptors in membrane microdomains.

Single particle tracking was used to evaluate lateral motions of individual FLAG-tagged human luteinizing hormone (LH) receptors expressed on CHO cells and native LH receptors on both KGN human granulosa-derived tumor cells and M17 human neuroblastoma cells before and after exposure to human chorionic gonadotropin (hCG). Compared with LH receptors on untreated cells, LH receptors on cells treated with 100 nm hCG exhibit restricted lateral diffusion and are confined in small, nanometer-scale, membrane compartments. Similar to LH receptors labeled with Au-hCG, LH receptors labeled with gold-deglycosylated hCG, an hCG antagonist, also exhibit restricted lateral diffusion and are confined in nanoscale membrane compartments on KGN cells treated with 100 nm hCG. LH receptor point mutants lacking potential palmitoylation sites remain in large compartments despite treatment with 100 nm hCG as do LH receptors on cells treated with cytochalasin D. Finally, both polarization homotransfer fluorescence resonance energy transfer imaging and photon counting histogram analysis indicate that treatment with hCG induces aggregation of YFP-coupled LH receptors stably expressed on CHO cells. Taken together, our results demonstrate that binding of hCG induces aggregation of LH receptors within nanoscale, cell surface membrane compartments, that hCG binding also affects the lateral motions of antagonist binding LH receptors, and that receptor surface densities must be considered in evaluating the extent of hormone-dependent receptor aggregation.