Septins spatially guide MT growth and MT–MT interactions. (A) Time-composite images of EB1-dsRed and α-tubulin–GFP after the lapse of 60 s. MDCK-EB1-dsRed cells were transfected with α-tubulin–GFP and control/SEPT2 siRNAs. (B) Graph shows the mean number of intersecting EB1-dsRed trajectories after a lapse of 60 s per control (n = 18) and SEPT2-depleted (n = 20) cells. (C) Schematic depicts the lateral meandering of MT plus ends in the absence of SEPT2. (D) Dual-color time-lapse images of α-tubulin–GFP (grayscale) overlaid with EB1-dsRed (red). Panels depict three types of MT–MT interactions. Yellow arrows point to MT plus ends moving through the cytoplasm. (E and F) Quantification of EB1-dsRed head-on encounters with MTs in MDCKs transfected with α-tubulin–GFP and control (10 cells; n = 99) and SEPT2 siRNAs (12 cells; n = 98). Right histogram shows mean angle width of all EB1-MT encounters. Data were analyzed from 90-s-long videos. (G) MDCK-EB1-dsRed cells were transfected with paxillin-GFP and control and SEPT2 siRNAs. Graph shows mean number of EB1-dsRed particles that enter a focal adhesion per 60 s (five cells; n = 20–25). Error bars represent SEM.