Real-time visualization of filament severing indicates that fragmentation scales with and occurs preferentially at boundaries between bare and cofilin-decorated filament segments, thereby controlling the overall filament length, depending on cofilin binding density.
Cofilin, one of three ABPs that precisely choreograph actin assembly and organization into comet tails that drive motility in vitro [2], binds and stochastically severs aged ADP actin filament segments of de novo growing actin filaments [3].
Alignment and classification of hDmc1 filament segments were performed as described [51].
Filament segments were selected using only two constrains: (i) they had to be straight and (ii) the length of the segments should be at least 3 helical pitches.
Images of the hDmc1-ssDNA filament segments containing ∼2 helical turns were selected manually and image analysis was performed using IMAGIC-5 [35] (Methods).
Repeats spaced 38.7 nm apart axially and containing a 60.7 nm axial length of the actin filaments, their bound cross-bridges and adjacent thick filament segments were centered on the actin target zones.
Two EM reconstructions of the pilus were generated, which differed in the number of filament segments used, the defocus range, and the approach to contrast transfer function (CTF) correction.
