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.
Fluorescence depletion anisotropy (FDA) measurements of protein rotation combine the long lifetime of chromophore triplet states with the sensitivity of the fluorescence excitation and detection. Frequency domain (FDA) addresses certain practical limitations of time-domain procedures, such as the need for detector gating, but presents its own difficulties. We have combined time- and frequency-domain FDA methods into an efficient continuous technique (CFDA). Intensity and polarization of a single laser beam are modulated continuously according to a complex, repetitive waveform and fluorescence signals are averaged over recurring waveform periods by a low rearm time signal averager. Methods for extracting triplet decay and absorption anisotropy decay kinetics from data traces generated by arbitrary waveforms have been developed. For a sample of eosin-BSA in 86% glycerol at 9 degree(s)C, rotational correlation times of 77 micrometers and 137 microsecond(s) , initial anisotropies of 0.109 and 0.125 and limiting anisotropies of 0.017 and 0.022, were obtained by CFDA and cuvet FDA, respectively. Differences in results apparently arise from the weighing of decay components in CFDA by triplet lifetimes of individual components.
KEYWORDS: Molecules, FDA class II medical device development, Diffusion, Proteins, Anisotropy, Phosphorescence, Luminescence, Molecular aggregates, Molecular interactions, Plasma
Antigen presentation by MHC class II molecules can be enhanced by paraformaldehyde fixation of antigen-presenting cells prior to assay. This treatment might be expected to aggregate membrane proteins and thus stabilize and strengthen transient protein-protein interactions involved in intercellular cooperation. Rotational and lateral dynamics of the MHC class II antigen I-Ad on A20 cells fixed with various concentrations of paraformaldehyde were examined by time- resolved phosphorescence anisotropy and fluorescence photobleaching recovery, respectively. Probes were erythrosin and tetramethylrhodamine conjugates of MKD6 Fab fragments. Increasing concentrations of paraformaldehyde progressively increased I-Ad's limiting anisotropy at 4 degrees Celsius above the value of 0.042 seen in untreated cells while leaving the rotational correlation time of 22 microsecond unchanged. On the other hand, translational diffusion coefficients decreased from about 2 X 10-10 cm2 sec-1 while recovery remained unchanged at 40 - 50%. Together these results suggest that fixation crosslinks class II molecules with each other or with other membrane proteins into structures large enough (greater than 500,000 kDa) to appear rotationally immobile but small enough to diffuse translationally with size-dependent rates. Fixation effects on both class II rotation and lateral diffusion are half-maximal at paraformaldehyde concentrations of approximately 0.2%. Possible relations between biology of class II effector functions and physical sizes of fixation-induced aggregates are discussed.
KEYWORDS: Luminescence, Diffusion, Fringe analysis, Proteins, Interferometry, Microscopes, Molecules, Spherical lenses, FDA class II medical device development, Interferometers
Lateral diffusion of cell surface proteins is commonly measured by spot fluorescence photobleaching recovery (FPR) methods where the 1/e2 radius of the interrogated spot is typically 0.5 micrometers . On an 8 micrometers lymphocyte the effective spot area represents only 1/500 of the total surface. An FPR lateral diffusion measurement of a protein expressed as 50,000 copies per cell thus reflects the dynamics of only 100 molecules and this greatly limits the precision and reproducibility of FPR measurements. A new method for interferometric fringe pattern FPR permits simultaneous interrogation of the entire surface of round cells. Fringe patterns are generated interferometrically within the optical path of a conventional microscope FPR system so that spot photobleaching measurements can be performed interchangeably. Methods for interpreting recovery kinetics on round cells and for determining the fraction of mobile protein are presented. Fringe FPR data of the murine MHC Class II antigen I-Ak (wt) expressed on M12.C3.F6 cells showed fluorescence signals improved 100-fold relative to spot FPR, with corresponding improvements in S/N ratios of recovery traces. Diffusion coefficients of 2.07 +/- 0.37 and 1.79 +/- 0.97 X 10-10 cm2sec-1 were obtained by fringe and spot methods, respectively. The corresponding mobile fractions of I-Ak were 66.1 +/- 7.8% and 63.4 +/- 18.0%. Improved reproducibility of fringe over spot results are slightly less than signal improvements predict. There may thus be substantial variation from cell to cell in proteins dynamics and this method may permit the assessment of such variation.
The utility of fluorescence depletion methods for the measurement of slow protein rotational diffusion has been limited by the lack of a rigorous mathematical model to obtain, from depletion data, anisotropies directly comparable to those obtained from phosphorescence emission or triplet absorption measurements. A generalized theory to meet this need is described. The experimental method requires the acquisition of, at most, three separate measurements to calculate absorption or emission anisotropies. Each measurement is made with a different orientation of either the probe beam polarization, pump beam polarization, or emission polarizer. The results of the theory are applied to two experimental configurations. The first of these involves collecting emission at 90 degree(s) to colinear pump and probe beams. From such data we are able to calculate the absorption anisotropy, the emission anisotropy, and the interdipole angle. The second configuration represents a system where all polarization axes lie in a single plane as would occur in a microscope-based system. For this configuration we are able to calculate, given the interdipole angle derived from the 90 degree(s) case, the true absorption anisotropy, the true emission anisotropy, or both.
The laser microscopic method of polarized fluorescence depletion (PFD) has permitted the rotational dynamics of functional membrane proteins to be measured on single cells under physiological conditions. This provides information comparable to that obtained by the older technique of time-resolved phosphorescence anisotropy (TPA) but with a greater than 1000- fold reduction in sample requirements. A new cuvette implementation of PFD methods, together with a data analysis model appropriate to this new experimental geometry, permits small volumes of dilute cell suspensions to be examined in their entirety. Data can thus be obtained at rates comparable to TPA. The instrument can also be used for TPA measurements and this permits direct comparison of PFD and TPA results obtained under otherwise identical conditions. In addition, a new photomultiplier gating device and system timing strategy reduce the minimum observable rotational correlation times to < 2 microsecond(s) ec, a 10-fold improvement over previous systems. These speed improvements have been examined in studies of BSA rotation in glycerol solution.
We have implemented a new laser microscopic method, polarized fluorescence depletion (PFD), for measuring the rotational dynamics of functional membrane proteins on individual, microscopically selected cells under physiological conditions. This method combines the long lifetimes of triplet-state probes with the sensitivity of fluorescence detection to measure macromolecular rotational correlation times from 10 microsec to > 1 ms. As examples, the rotational correlation time of Fc receptors (FcR) on the surface of 2H3 rat basophilic leukemia cells is 79.9 4.4 microsec at 4°C when labeled with eosin conjugates of IgE. This value is consistent with the known 100 kDa receptor size. When labeled with intact F4 anti-FcR monoclonal antibody, the rotational correlation time for FcER is increased about 2-fold to 170.8 +/- 6.5 microsec, consistent with receptor dimer formation on the plasma membrane and with the ability of this antibody to form FcER dimers on 2H3 cell surfaces. We have also examined the rotational diffusion of the luteinizing hormone receptor on plasma membranes of small ovine luteal cells. Luteinizing hormone receptors (LHR), when occupied by ovine luteinizing hormone (oLH), have a rotational correlation time of 20.5 +/- 0.1 microsec at 4°C. When occupied by human chorionic gonadotropin (hCG), LHR have a rotational correlation time of 46.2 +/- 0.4 microsec suggesting that binding of hCG triggers additional LHR interactions with plasma membrane proteins. Together these studies suggest the utility of PFD measurements in assessing molecular size and molecular association of membrane proteins on individual cells. Relative advantages of time- and frequency-domain implementations of PFD are also discussed.
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