Extended Data Figure 3: TssA localization and dynamics.
From: Priming and polymerization of a bacterial contractile tail structure
a, Mean square displacement (MSD; in arbitrary units (a.u.)) of a representative sfGFP–TssA clusters in a wild-type strain (red line) or its ∆tssBC isogenic derivative (black line) were measured by sub-pixel tracking of fluorescent foci and plotted over time (in sec). b, Kymographic analysis reporting representative sfGFP–TssA (green) and TssB–mCherry (red) positions within the cell as a function of time. c, Representative fluorescence lifetime imaging microscopy (FLIM) of sfGFP–TssA clusters in the sfGFP–TssA/TssB–mCherry strain. A membrane-associated sfGFP–TssA cluster was chosen to define the bleached area (red circle). The laser (488 nm) was set to maximum power and focused for 3 s to ensure complete bleaching of the GFP diffusible pool. Images were taken every 30 s to follow recovery dynamics. The scale bar is 1 μm. d, Quantification of sfGFP–TssA fluorescence dynamics over time after bleaching. The dynamics of fluorescence intensity is shown over time for n = 10 independent sfGFP–TssA foci after FLIM (blue line). The fluorescence intensity of the bleached focus was also followed over time (FRAP, red line). As a control for laser focusing and intensity, membrane-associated clusters were systematically bleached in these experiments and showed no recovery suggesting the total intracellular sfGFP–TssA has been bleached by the laser. e, Representative fluorescence microscopy time-lapse recordings of the indicated ∆tssK, ∆tssE, ∆tssF, ∆tssG or ∆hcp cells producing sfGFP–TssA. Individual images were taken every 30 s. Red arrowheads indicate the localizations of TssA foci. The scale bar is 1 μm. f, Representative large fields of fluorescence microscopy analyses showing localization of sfGFP–TssA in the indicated strains. The scale bars are 1 μm. g, Box-and-whisker plots of the measured number of sfGFP–TssA foci per cell for each indicated strain. The lower and upper boundaries of the boxes correspond to the 25% and 75% percentiles respectively. The black bold horizontal bar represents the median values for each strain and the whiskers represent the 10% and 90% percentiles. Outliers are shown as open circles. A Student’s t-test was used to report significant differences (ns, not significant; ***P < 0.0001). The number of cells studied per strain (n) is indicated on top. h, Statistical analyses of sfGFP–TssA dynamics. sfGFP–TssA dynamics were categorized as ‘fixed’, ‘mobile with unidirectional trajectory’ and ‘mobile with random dynamics’ and the number of sfGFP–TssA (n, on top) foci in each category is represented as a percentage for each indicated strain. Kymographs for the two first categories are shown at the bottom of the panel. i, Schematic representation of the assembly pathway of the T6SS based on this study and available data13,15,19,24,25,55. The biogenesis starts with the initial positioning of the TssJ outer membrane lipoprotein and the sequential recruitment of the indicated subunits (from left to right). The recruitment of TssA is dependent on TssM, and that of TssK is dependent on both TssL and TssA. The exact positions of VgrG and TssE (blue) in the pathway are not known but these two subunits are not required for TssA recruitment but necessary for Hcp and TssBC polymerization.
