Evidence that focal adhesion complexes power bacterial gliding motility

Abstract

The bacterium Myxococcus xanthus has two motility systems: S motility, which is powered by type IV pilus retraction, and A motility, which is powered by unknown mechanism(s). We found that A motility involved transient adhesion complexes that remained at fixed positions relative to the substratum as cells moved forward. Complexes assembled at leading cell poles and dispersed at the rear of the cells. When cells reversed direction, the A-motility clusters relocalized to the new leading poles together with S-motility proteins. The Frz chemosensory system coordinated the two motility systems. The dynamics of protein cluster localization suggest that intracellular motors and force transmission by dynamic focal adhesions can power bacterial motility.

Fig. 1

AglZ-YFP localizes to periodic sites that remain fixed relative to the substratum. (A) AglZ-YFP localization in a cell moving at constant velocity. Fluorescence micrographs captured every 30 s are shown. Numbered arrowheads highlight selected bright fluorescence clusters. Scale bar, 1 μm. (B) Line scans of fluorescence intensity as a function of position are shown for each movie frame in (A). For display purposes, individual scans have been shifted horizontally with time. Gray bars represent the three highest peaks in the average line scan, matching the positions of clusters 1 to 3 in (A). Pink bars denote all additional peaks found in the average scan. (C) Quantitative analysis of the AglZ-YFP fluorescence distribution in moving cells. The auto-correlation function of the thresholded line scan from six moving cells was averaged and displayed. (D) Power spectral density of the autocorrelation function.

Fig. 2

AglZ-YFP localizes to transient adhesion sites. (A) AglZ-YFP fluorescence clusters in a cell that bends while in motion. (Top) Cells stained with the membrane dye FM4-64 are shown. (Bottom) An overlay of the membrane signal (gray) and the AglZ-YFP signal (magenta), which is artificially colored for better clarity, are shown. White and black arrowheads point to regions of cell-body curvature and localization of the YFP signal, respectively. Arrows indicate the direction of movement. Scale bar, 1 μm. (B) AglZ-YFP fluorescence clusters in a cell undergoing flailing motion. Fluorescence and overlaid phase micrographs (top and middle rows, respectively) are shown. Time intervals, 1 min. A cartoon representation (bottom row) shows the clusters numbered and color-coded for the analysis shown in (C). The arrow indicates the stuck leading pole. Scale bar, 2 μm. (C) Dynamic behavior of the AglZ-YFP fluorescence clusters in the cell shown in (B). Time intervals, 30 s. (Top) The velocity of the lagging pole over time is shown. Dotted lines mark the times where relaxation of the terminal bend (Relax.) is observed. The leading pole remained immobilized for the entire duration of the time lapse. (Middle) The distance traveled by the AglZ-YFP clusters, color-coded and numbered as in (B), over time is shown. 1, blue triangles; 2, blue diamonds; 3, purple squares; 4, pink squares; 5, green triangles. For each cluster, the distance traveled by the lagging pole (orange diamonds) during the same time interval was plotted to show that the clusters remain mostly fixed relative to the substratum. (Bottom) The relative fluorescence intensity of each cluster over time. The same color code as that used in the middle panel applies.

Fig. 3

AglZ-YFP oscillates from pole to pole upon cellular reversals. (A) AglZ-YFP localized to the new leading pole upon cellular reversals. Fluorescent micrographs of AglZ-YFP (magenta) and a representative reversing cell stained with FM4-64 (gray) were overlaid to show AglZ-YFP dynamics every 30 s. The black arrows inside the panel indicate the direction of movement. The arrowheads in (A) and (B) show the relocalization of AglZ-YFP at the new leading pole. R, reversal. (B) AglZ-YFP dynamics at the time of reversal. Fluorescence micrographs of a reversing cell captured every 5 s are shown. The 10-s delay is indicated by “pause.” Scale bar, 2 μm. (C) AglZ-YFP oscillations in hyper-reversing cells. Fluorescent micrographs of a frzCD^c cell that expresses AglZ-YFP captured every 30 s are shown. The white arrows in (B) and (C) indicate the direction of movement. Scale bar, 2 μm. (D) Quantitative fluorescence analysis of the cell presented in (C). The relative fluorescence intensities of each cell pole were measured in arbitrary units and plotted over time. The black line indicates the initial leading pole, and the gray line indicates the initial trailing pole.

Fig. 4

Dynamics of AglZ-YFP clusters in artificially elongated cells. (A) AglZ-YFP dynamics in A⁺S⁻-motile filamentous cells. Fluorescent micrographs of a representative 20-μm-long cephalexin-treated cell stained with FM4-64 (gray) expressing AglZ-YFP (magenta). AglZ-YFP is only found distributed over the front part of the cell when the cell is in motion. The arrowhead indicates polar condensation of AglZ-YFP. “Pause” indicates times when the cell motion is stopped. Scale bar, 2 μm. (B) Relationship between cluster number and filamentous cell length. (C) Relationship between relative drag force overcome and cluster number in filamentous cells.