On-microscope staging of live cells reveals changes in the dynamics of transcriptional bursting during differentiation.

Determining the mechanisms by which genes are switched on and off during development is a key aim of current biomedical research. Gene transcription has been widely observed to occur in a discontinuous fashion, with short bursts of activity interspersed with periods of inactivity. It is currently not known if or how this dynamic behaviour changes as mammalian cells differentiate. To investigate this, using an on-microscope analysis, we monitored mouse α-globin transcription in live cells throughout erythropoiesis. We find that changes in the overall levels of α-globin transcription are most closely associated with changes in the fraction of time a gene spends in the active transcriptional state. We identify differences in the patterns of transcriptional bursting throughout differentiation, with maximal transcriptional activity occurring in the mid-phase of differentiation. Early in differentiation, we observe increased fluctuation in transcriptional activity whereas at the peak of gene expression, in early erythroblasts, transcription is relatively stable. Later during differentiation as α-globin expression declines, we again observe more variability in transcription within individual cells. We propose that the observed changes in transcriptional behaviour may reflect changes in the stability of active transcriptional compartments as gene expression is regulated during differentiation.

Rebinding of IgE Fabs at haptenated planar membranes: measurement by total internal reflection with fluorescence photobleaching recovery.

In previous work, a general analytical theory for ligand rebinding at cell surfaces was developed for a reversible bimolecular reaction between ligands in solution and receptors on a membrane surface [Lagerholm, B. C., and Thompson, N. L. (1998) Biophys. J. 74, 1215-1228]. This theory can be used to predict theoretical forms for data obtained by using total internal reflection with fluorescence photobleaching recovery (TIR-FPR) [Thompson, N. L., Burghardt, T. P., and Axelrod, D. (1981) Biophys. J. 33, 435-454]. Thus, one method by which the rebinding theory can be tested is to use TIR-FPR. In the work described herein, the reversible kinetics of mouse monoclonal anti-dinitrophenyl (DNP) IgE Fabs at substrate-supported planar membranes composed of 25 mol % DNP-conjugated phosphatidylethanolamine and 75 mol % dipalmitoylphosphatidylcholine have been examined by using TIR-FPR. Data were obtained as a function of the Fab solution concentration. Higher Fab concentrations reduce rebinding (and increase the fluorescence recovery rate) because different Fab molecules compete for the same surface-binding sites. Data were also obtained for solutions containing different volume fractions of glycerol. In these measurements, higher glycerol concentrations increase rebinding (and decrease the fluorescence recovery rate) because the solution viscosity is increased and the Fab diffusion coefficient in solution is decreased. The TIR-FPR data were quantitatively compared with theoretical predictions which follow from the general theory for rebinding at the membrane surface. The data were consistent with the theoretical predictions and, therefore, provide experimental verification of the previously developed theory.

Theory for ligand rebinding at cell membrane surfaces.

Conditions for which a ligand reversibly bound to a cell surface dissociates and then rebinds to the surface have been theoretically examined. The coupled differential equations that describe reaction at the interface between sites on a plane and three-dimensional solution have been described previously (Thompson, N. L., T. P. Burghardt, and D. Axelrod. 1981. Biophys. J. 33:435-454). Here, we use this theoretical formalism to provide an analytical solution for the spatial and temporal dependence of the probabilities of finding a molecule on the surface or in the solution, given initial placement on the surface at the origin. This general analytical solution is used to derive a simple expression for the probability that a molecule rebinds to the surface at a given position and time after release at the origin and time zero. The probability expressions provide fundamental equations that form a basis for subsequent modeling of ligand-receptor interactions in specific geometries.

Total internal reflection fluorescence: applications in cellular biophysics.

Molecular interactions occurring on or near cell membrane surfaces are expected to have different properties from those occurring in bulk solutions. One particularly useful technique for studying surface-associated processes at the molecular level is total internal reflection fluorescence. In this method, the evanescent field from an internally reflected excitation source selectively excites fluorescent molecules on or near a surface. Evanescent excitation has been used recently with a variety of techniques in fluorescence microscopy and spectroscopy to probe the fundamental physicochemical properties of biochemical reactions at natural or model biological surfaces. These studies are providing enhanced understanding of cellular function. Several recent developments in total internal reflection fluorescence methodology from other fields are likely to find future application in cellular biophysics.