(D) Single molecule tracking of TagT, TagU, and TagV indicates these proteins do not move directionally like MreB. HaloTag fusions to MreB, Pbp2a, TagT, TagU, and TagV, expressed from the native promoter and locus, were sparsely labeled using JF549, then imaged with TIRFM at a 250 msec frame rate. At this acquisition speed directional motion is easily tracked, while particles diffusing on the membrane cannot be tracked for more than a few frames. Trajectories from representative fields are overlayed on a phase image. Trajectories that show directional movement (α >1.25) are colored red, diffusive trajectories (0.75 < α <1.25) purple, and confined/subdiffusive (α <0.75) blue. Trajectories too short for analysis (<10 frames) are colored white. As shown, most MreB molecules move directionally, while Pbp2A has both diffusive and directionally moving molecules. In contrast, no directional motion is observed for TagT, TagU, and TagV, although a small fraction of molecules appear confined for a few seconds. (E) While only 1–5% of the ligase molecules could be tracked for over 10 frames due to their rapid motion, we analyzed the few molecules we could track. We calculated the velocities (V) (left) by fitting MSD versus t to MSD = (Vt)2 + 4Dt+4σ2 of all molecules that moved in a consistent manner during their lifetime [>0.95 r2 fit to log(MSD) versus log(t)]. α values (right) were calculated by linear fits to log(MSD) versus log(t). Shown are only molecules with velocity >5×10−5 nm/s. ‘Relative frequency’ was determined by normalizing to the number of trajectories above 10 frames in length. As expected, most MreB (54%) molecules show directional motion (α >1.25) as do a substantial, but lesser fraction of Pbp2a molecules (20%). In contrast, only a small percentage of the ligase trajectories exceeding 10 frames (which is already a small subfraction, 1–5% of the total) show α >1.25 (TagT = 1.4%, TagU = 2.8% TagV=4.8%). Furthermore, these velocities of these tracks do not appear to be drawn from the same distribution as MreB and Pbp2A. This suggests the molecules in this subset are either (1) independent of MreB, or (2) the small fraction of ‘apparently directional’ motions expected when analyzing a large number of particles undergoing random walks.