Browsing by Subject "single molecule tracking"

Though single-celled organisms lack the ability to maintain their internal temperature, prokaryotes can grow between temperatures of <0 °C and up to 100 °C. Because the macromolecules of cells may be too stable, or denatured and inactive at non-permissible temperatures, these organisms must be able to adapt their interior to cope. In E. coli, the DNA is maintained by DNA-binding proteins and topology enzymes like DNA gyrase, and these proteins have been tracked and observed in cells at permissible temperatures, though little work had been done to characterise the activity and motion of DNA loci, DNA gyrase and the chromosomal compaction at different temperatures. Here, I used super-resolution fluorescence microscopy to track and image DNA and DNA gyrase at a range of temperatures. It was shown that the time-dependence of the mean square displacement (MSD) of DNA Loci behaves subdiffusively (MSD ~ ta, a <1) and that this subdiffusion coefficient itself is dependent on temperature. Additionally, it appeared that the subdiffusive scaling factor a differs between two timescales (< 500 ms and 0.5 – 10 s). It was also shown that the proportion of bound to unbound DNA gyrase changes from being equally DNA-bound and unbound at 23 °C while mostly DNA-unbound at 30 °C and 37 °C, and being mostly DNA-bound at 42 °C. It was shown that the compaction of the E. coli nucleoid did not change massively between steady-state temperatures, but did increase in size upon a shift from 37 °C to 23 °C. The DNA gyrase population shifting from unbound to mostly bound at 42 °C was interpreted to be due to an increase in cellular ATP concentrations, while the increased binding at 23 °C was thought to be because of a lack of available ATP trapping gyrase on the DNA. These results were lightly correlated with the nucleoid’s compaction across temperatures, where an abundance of free, DNA-unbound gyrase coincided with an increase in relative nucleoid size. The subdiffusive scaling coefficients of DNA loci at each temperature was thought to represent the fluidisation of the nucleoid and cytoplasm at 42 °C, but brings into question how the cell can maintain a similar flexibility and mobility of DNA between cold and warm temperatures.