Single Molecule Tracking Inside Individual Living Bacterial Cells

Abstract

Single-Pair Fluorescence Resonance Energy Transfer (FRET) experiments reveal structural and dynamic information about macro-molecules by monitoring the change in FRET efficiency between fluorescent dyes attached to a macromolecule. The Nano-Positioning System (NPS) developed recently [1] uses data from several of such experiments to infer the position of a dye attached to protein sites unresolved by x-ray crystallography. Briefly, we perform probabilistic data analysis that allows us to calculate the distribution of possible dye positions in a simple and objective way without relying on ad-hoc procedures. Up to now NPS was limited to the triangulation of just one fluorescently labelled position based on FRET measurements to several other positions known from crystal structure [1,2]. Here, we discuss ways to extend the present model beyond this basic triangulation principle. In particular, we show how to gain three dimensional distance information by analysing triangulation networks where FRET is measured between arbitrary labelling sites in absence of other structural information.[1] A.

Evaluation of Pulsed-FRAP and Conventional-FRAP for Determination of Protein Mobility in Prokaryotic Cells

“…We demonstrate that both techniques yield very similar distributions of diffusion coefficients of GFP in the cytoplasm under normal and osmotic stress conditions. We conclude that the different D values reported for GFP(-like) proteins in the literature [1], [5], [6], [7], [10], [13], [14] are a result of different handling of the cells and true biological variations rather than differences in the FRAP methods used.…”

“…This approach has been successful implemented by others [3] , [4] , [5] , [6] . Subsequently, other methods have been tailored to probe macromolecule diffusion in bacteria, including FRAP related techniques like pulsed–FRAP [7] and continuous photobleaching, using total internal reflection microscopy [8] , [9] , single molecule tracking [10] and fluorescence correlation spectroscopy [11] . For an overview of those techniques, we refer to Mika and Poolman [12] .…”

“…There are now several studies that report the mobility of GFP and related proteins like YFP and mEos2, yielding diffusion coefficients for these proteins in the E.coli cytoplasm that range from 3 µm 2 /s [7] , [13] through 6–7 µm 2 /s [1] , [6] up to 14 µm 2 /s [5] , [10] . The question arises whether this relatively wide range of values reflects differences in the methods used or biological and/or experimental variations?…”