Extended Data Fig. 2. Controls for FtsZ lifetime measurements.

a FtsZ subunit lifetime is consistent across experimental replicates. To measure lifetimes, cells expressing FtsZ-HaloTag were induced with 20 μM IPTG for 2 hours, labelled with 40 pM JF549-HTL, and then imaged by TIRFM. Light curves: 4 experimental replicates for which N>200. Points represent combined data from all experimental replicates (17 replicates). Error bars: weighted standard deviation of distributions for all replicates. b FtsZ subunit lifetime is consistent across measurement techniques. Lifetime distributions were measured using an automated hidden Markov model (HMM) based analysis pipeline and manually for N=265 particles (dashed line). c FtsZ subunit lifetime is not affected by Pbp2B tagging. The 1-colour strain (bAB309) contains labelled FtsZ-HaloTag, induced as a second copy with 20 μM IPTG for 2 hours; the 2-colour strain (bGS104) contain both this FtsZ-HaloTag construct and a native mNeonGreen-Pbp2B fusion, which was used to localize the division site. d FtsZ subunit lifetime is not affected by photobleaching. If the measurements were affected by photobleaching, the measured lifetimes would increase when we decrease the imaging interval; however, we see that the lifetime distributions are consistent for images taken at 0.5-second intervals and 1-second intervals. For images at 0.5-second intervals, images were acquired continuously with 0.5-second exposures. For images at 1-second intervals, images were acquired with the same settings, with 0.5 seconds of exposure and 0.5-second intervals without illumination. e Co-overexpression of FtsA and FtsZ increases the number of FtsZ filaments in the cell (left) but does not change FtsZ subunit lifetime (right). The increased number of filaments that form upon FtsAZ overexpression is consistent with steady-state treadmilling of FtsZ. The fact that the subunit lifetime does not change when FtsAZ is overexpressed further indicates that the additional FtsZ forms new filaments rather than elongating existing filaments. A second copy of ftsAZ is expressed from an IPTG-inducible promoter with 100 μM IPTG for 2 hours. Filament density is visualized by TIRFM for at least two replicates of each condition. Scale bar: 2 μm.