1. Analyzing intracellular binding and diffusion with continuous fluorescence photobleaching.
- Author
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Wachsmuth M, Weidemann T, Müller G, Hoffmann-Rohrer UW, Knoch TA, Waldeck W, and Langowski J
- Subjects
- Bacterial Proteins, Computer Simulation, DNA-Binding Proteins chemistry, DNA-Binding Proteins metabolism, Diffusion, Green Fluorescent Proteins, HeLa Cells, Histones chemistry, Histones metabolism, Humans, Luminescent Proteins chemistry, Luminescent Proteins metabolism, Models, Biological, Motion, Protein Binding, Statistics as Topic, Transcription Factors, Fluorescence Recovery After Photobleaching methods, Intracellular Fluid chemistry, Intracellular Fluid metabolism, Microscopy, Confocal methods, Proteins chemistry, Proteins metabolism, Spectrometry, Fluorescence methods
- Abstract
Transport and binding of molecules to specific sites are necessary for the assembly and function of ordered supramolecular structures in cells. For analyzing these processes in vivo, we have developed a confocal fluorescence fluctuation microscope that allows both imaging of the spatial distribution of fluorescent molecules with confocal laser scanning microscopy and probing their mobility at specific positions in the cell with fluorescence correlation spectroscopy and continuous fluorescence photobleaching (CP). Because fluorescence correlation spectroscopy is restricted to rapidly diffusing particles and CP to slower processes, these two methods complement each other. For the analysis of binding-related contributions to mobility we have derived analytical expressions for the temporal behavior of CP curves from which the bound fraction and/or the dissociation rate or residence time at binding sites, respectively, can be obtained. In experiments, we investigated HeLa cells expressing different fluorescent proteins: Although enhanced green fluorescent protein (EGFP) shows high mobility, fusions of histone H2B with the yellow fluorescent protein are incorporated into chromatin, and these nuclei exhibit the presence of a stably bound and a freely diffusing species. Nonpermanent binding was found for mTTF-I, a transcription termination factor for RNA polymerase I, fused with EGFP. The cells show fluorescent nucleoli, and binding is transient. CP yields residence times for mTTF-I-EGFP of approximately 13 s.
- Published
- 2003
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