Biological Insights from Single-Particle Tracking in Living Cells

Detta är en avhandling från Uppsala : Acta Universitatis Upsaliensis

Sammanfattning: Single-particle tracking is a technique that allows for quantitative analysis of the localization and movement of particles. In this technique, trajectories are constructed by determining and connecting the positions of individual particles from consecutive images. Recent advances have made it possible to track hundreds of particles in an individual cell by labeling the particles of interest with photoactivatable or photoconvertible fluorescent proteins and tracking one or a few at a time.Single-particle tracking can be used to study the diffusion of particles. Here, we use intracellular single-particle tracking and trajectory simulations to study the diffusion of the fluorescent protein mEos2 in living Escherichia coli cells. Our data are consistent with a simple model in which mEos2 diffuses normally at 13 µm2 s?1 in the E. coli cytoplasm. Our approach can be used to study the diffusion of intracellular particles that can be labeled with mEos2 and are present at high copy numbers.Single-particle tracking can also be used to determine whether an individual particle is bound or free if the free particle diffuses significantly faster than its binding targets and remains bound or free for a long time. Here, we use single-particle tracking in living E. coli cells to determine the fractions of free ribosomal subunits, classify individual subunits as free or mRNA-bound, and quantify the degree of exclusion of bound and free subunits separately. We show that, unlike bound subunits, free subunits are not excluded from the nucleoid. This finding strongly suggests that translation of nascent mRNAs can start throughout the nucleoid, which reconciles the spatial separation of DNA and ribosomes with co-transcriptional translation. We also show that, after translation inhibition, free subunit precursors are partially excluded from the compacted nucleoid. This finding indicates that it is active translation that normally allows ribosomal subunits to assemble on nascent mRNAs throughout the nucleoid and that the effects of translation inhibitors are enhanced by the limited access of ribosomal subunits to nascent mRNAs in the compacted nucleoid.