Single-molecule tracking in liveYersinia enterocoliticareveals distinct cytosolic complexes of injectisome subunits

Abstract

In bacterial type 3 secretion, substrate proteins are actively transported from the bacterial cytoplasm into the cytoplasm of host cells by a large membrane-embedded machinery called the injectisome. Injectisomes transport secretion substrates in response to specific environmental signals, but the molecular details by which the cytosolic secretion substrates are selected and transported through the type 3 secretion pathway remain unclear. Secretion activity and substrate selectivity are thought to be controlled by a sorting platform consisting of the proteins SctK, SctQ, and SctL, which together localize to the cytoplasmic side of membrane-embedded injectisomes. However, recent work revealed that sorting platform proteins additionally exhibit substantial cytosolic populations and that SctQ reversibly binds to and dissociates from the cytoplasmic side of membrane-embedded injectisomes. Based on these observations, we hypothesized that dynamic molecular turnover at the injectisome and cytosolic assembly among sorting platform proteins is a critical regulatory component of type 3 secretion. To determine whether intact sorting platform complexes exist in the cytosol of living cells, we measured the diffusive properties of thousands of SctQ and SctL proteins using high-throughput 3D singlemolecule tracking microscopy. The obtained single-molecule trajectories reveal that both SctQ and SctL exist in several distinct diffusive states in the cytosol of living Yersinia enterocolitica, indicating that these proteins are able to form stable oligomeric complexes in their native environment. Our findings provide new insights into the dynamic regulatory network that interfaces stationary membrane-embedded injectisomes with the soluble cytosolic components of the T3SS.All rights reserved. No reuse allowed without permission.

Molecular organization of soluble type III secretion system sorting platform complexes

“…In fact, the dynamic exchange of the SpaO homolog YscQ in Yersinia has previously been observed by fluorescence microscopy and found to increase during the active secretion process (12). Together with the observation that the diffusion behavior of cytosolic populations of sorting platform components also changes upon secretion activation, this indicates that soluble sorting platform complexes might play an important role in the function of type 3 secretion (28). Thus, it can be hypothesized that soluble building blocks of SpaO/SpaO C /OrgB/InvC could act as T3SS substrate shuttles that recruit substrate-chaperone complexes in the cytosol and transfer them to the basal-body associated sorting platform for subsequent secretion.…”

Computational Correction of Spatially-Variant Optical Aberrations in 3D Single Molecule Localization Microscopy

“…For samples consisting of calibration beads, we diluted 100 nm tetraspeck fluorospheres (Invitrogen) diluted in PBS at a ratio of 1:3000 (v/v) and then spin-coated them onto a microscope coverglass. To maintain a refractive index similar to water, we covered the beads with a 1.5% (w/v) low-melting point agarose (Fisher Scientific) pad made with PBS buffer to mimic experimental conditions used for live cell imaging [12,51,52]. To generate a sample of beads immobilized away from the microscope coverslip, we suspended fluorescent beads into molten 1.5% agarose and spotted a 10 µl of mixture onto the coverglass which was previously spin-coated with fluorescent beads.…”

“…7CD]. Such a situation is encountered when single-molecules randomly dispersed in a 1000 nm sized bacterial cells are localized relative to the nominal focal plane stably positioned at d = -500 nm [12]. The z-errors obtained by double Gaussian fitting using local look-up tables showed spatially dependent zerrors in the ranges of [0, 300] nm [ Fig.…”

“…Determination of molecular positions with a precision of tens of nanometers enables the sub-cellular localization and co-localization of fluorescently-labeled biomolecules, as well as single-molecule tracking [7]. The accuracy and precision of molecular position estimation is of prime importance, because these metrics affect all subsequent steps in data analysis, such as estimation of cluster formation in pointillistic single-molecule localizations [8,9], or molecular displacement estimation in single-molecule tracking [10][11][12][13]. The accuracy of the estimated position, i.e.…”

Resolving Cytosolic Diffusive States in Bacteria by Single-Molecule Tracking

“…Single-molecule trajectories, if sampled in sufficient numbers, provide the distribution of molecular motion behavior in cells, and statistical analyses of localization and trajectory data has been used to resolve the prevalent diffusive states as well as their population fractions. A key benefit of tracking single molecules is that individual trajectories can be sorted according to predefined (quality) metrics, for example, to include only non-blinking molecules (4), or molecules localized in specific subcellular regions of interest (5). These advantages are not shared by ensemble-averaged measurements such as fluorescence recovery after photobleaching (FRAP) and fluorescence correlation spectroscopy (FCS) (6).…”