Guy M. Hagen

Constitutively-Active Human LH Receptors are Self-Associated and Located in Rafts

Several naturally occurring mutations in human luteinizing hormone receptors (LHR) at position 578 are associated with constitutive activation of the receptor. To determine whether human LHRs that signal in the absence of ligand are self-associated, fluorescence resonance energy transfer (FRET) between receptors was evaluated. Values for FRET between wild type LHR in the absence of ligand were less than 1% and increased significantly to over 11% after exposure to hCG. Constitutively active receptors exhibited 11-15% FRET efficiency in the absence of hormone and these values did not change with hCG treatment. A large fraction of constitutively active LHR-D578H receptors were also associated with so-called plasma membrane rafts. Disruption of these membrane microdomains reduced FRET efficiency but did not affect signalling through cAMP. Thus, in the absence of ligand, constitutively active receptors are self-associated and located in high buoyancy membrane fractions, both characteristics of the hormone-treated wild type receptor.

Flexible normally on photomultiplier gating strategy for reducing postgate artifacts

A normally on photomultiplier (PMT) gating strategy effectively eliminates postgate signals arising both from ion feedback after pulsing and from capacitive storage of primary photoelectrons. This approach has been applied to gating an Electron Tubes 9816B 14-stage linear focused PMT but should be applicable to a variety of other tubes. A positive potential pulse 10 V smaller than the photocathode (PC)-first dynode (D) potential difference is applied to the PC. This potential prevents ionization of residual gas, while still removing primary photoelectrons to D1. A negative pulse applied to intermediate dynodes D4/D6 increases overall attenuation to 5×106:1 with turn-off and turn-on times of 40 ns. The system adjusts pulse amplitudes to the applied tube HV and so provides consistent gating at all PMT gains. The free-standing, independently powered pulse generators are cabled to a passive tube base and can gate essentially any PMT with a maximum high voltage of 3 kV and a maximum PC-D1 potential of 450 V. A...

Lateral diffusion measurements on genetically introduced fluorescent proteins

Genetic introduction of fluorescent labels such as the Visible Fluorescent Proteins (VFP) has revolutionized the visualization and characterization of cellular proteins. Lateral diffusion measurements, most commonly accomplished through Fluorescence Photobleaching Recovery (FPR or FRAP), provide important information on such molecules’ size, environment and participation in intermolecular interactions. However, serious difficulties arise when these techniques are applied to VFP fusion proteins since cytoplasmic species contribute to the fluorescence recovery signal and thus distort measurements aimed at surface molecules. Two new methods help eliminate these difficulties through distinctly different strategies. In Total Internal Reflection Interference Fringe FPR, interfering laser beams enter a 1.65 NA Olympus objective at the periphery of the back focal plane where the NA exceeds 1.38. This creates an interference pattern totally internally reflected at the coverslip-medium interface. Fluorescence excitation occurs only where the cell contacts the coverslip so no contribution arises from cytoplasmic species. Alternatively, High Probe Intensity (HPI) FPR measurements retain the intrinsic confocality of spot measurements to eliminate interference from fluorescent cytoplasmic species. However, HPI-FPR methods lift the previous requirement that FPR procedures be performed at probe beam intensities low enough to not induce bleaching in samples during measurements. The high probe intensities now employed provide much larger fluorescence signals and thus more information on molecular diffusion from each measurement. We report successful measurements of membrane dynamics of various VFP species obtained by these techniques and compare them with results of earlier FPR methods which previously proved unsatisfactory in these instances.