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Reaching sub-millisecond 3D tracking of individual molecules in living cells would enable direct measurements of diffusion-limited macromolecular interactions under physiological conditions. Here, we present a 3D tracking principle that approaches the relevant regime. The method is based on the true excitation point spread function and cross-entropy minimization for position localization of moving fluorescent reporters. Tests on beads moving on a stage reaches 67 nm lateral and 109 nm axial precision with a time resolution of 0.84 ms at a photon count rate of 60 kHz; the measurements agree with the theoretical and simulated predictions. Our implementation also features a method for microsecond 3D PSF positioning and an estimator for diffusion analysis of tracking data. Finally, we successfully apply these methods to track the Trigger Factor protein in living bacterial cells. Overall, our results show that while it is possible to reach sub-millisecond live-cell single-molecule tracking, it is still hard to resolve state transitions based on diffusivity at this time scale.

The bacterial chaperone Trigger factor (TF) binds to ribosome-nascent chain complexes (RNCs) and cotranslationally aids the folding of proteins in bacteria. Decades of studies have given a broad, but often conflicting, description of the substrate specificity of TF, its RNC-binding dynamics, and competition with other RNC-binding factors, such as the Signal Recognition Particle (SRP). Previous RNC-binding kinetics experiments were commonly conducted on stalled RNCs in reconstituted systems, and consequently, may not be representative of the interaction of TF with ribosomes translating mRNA in the cytoplasm of the cell. Here, we used single-particle tracking (SPT) to measure TF binding to actively translating ribosomes inside living Escherichia coli. In cells, TF displays distinct binding modes—longer (ca 1 s) and shorter (ca 50 ms) RNC bindings. Consequently, we conclude that TF, on average, stays bound to the RNC for only a fraction of the translation cycle. Further, binding events are interrupted only by transient excursions to a freely diffusing state (ca 40 ms), suggesting a highly dynamic binding and unbinding cycle of TF in vivo. We also show that TF competes with SRP for RNC binding, and in doing so, tunes the binding selectivity of SRP.

Outer-membrane proteins (OMPs) embedded in the outer membrane (OM) of gram-negative bacteria are important for the rigidity, mobility, pathogenicity, and the selective permeability of the cell. Most OMPs share a common structure and biogenesis pathway, where the unfolded OMP is post-translationally translocated from the cytoplasm to the periplasm, with subsequent folding and insertion into the OM. In vitro studies on the targeting, translocation and folding of OMPs have proven challenging due to difficulties in reconstituting the physical and biological complexity of the biogenesis network, while commonly used in vivo methods suffer from low temporal resolution. Here, we investigate the possibility of using single-molecule FRET to directly observe the folded state of Outer-membrane protein A (OmpA) in live cells. In vitro double-labeled Cy3Cy5-OmpA was incorporated into Escherichia coli by electroporation. Using single-molecule tracking (SMT), we observed both cytosolic and immobilized, membrane-associated particles. On rare occasion, immobile particles displayed FRET, indicative of successful folding and OM incorporation of Cy3Cy5-OmpA. This work lays the foundation for combining SMT and smFRET for direct measurements of the complete targeting, translocation and folding pathway of OMPs at high spatiotemporal resolution in their native environment.