Spatial segregation of septins within the MT network. (A) Maximum intensity projection of confocal microscopy z stacks of MDCKs stained for SEPT2 and α-tubulin. (bottom) A single confocal slice from the outlined area in high magnification. (B) Confocal microscopy image of MDCKs stained for SEPT2, α-tubulin, and paxillin. (right) High magnification images of the outlined region. (C) Distribution of the angles of perinuclear SEPT2 filaments (n = 177) and MT bundles (n = 155) from the long axis of the cells (n = 14). (D) Manders coefficients of SEPT2 colocalization with total MTs and MT bundles (n = 14 cells). (E) Spinning-disk confocal microscopy images of MDCK-SEPT2-mCherry cells transfected with α-tubulin (α-tub)–GFP. Arrows point to SEPT2-coated MTs (pseudocolored yellow in grayscale images). MTs free of SEPT2 were pseudocolored red. (F) Quantification of MT fluctuations using image subtraction analysis (Δt = 15 s). Graph shows changes in α-tubulin–GFP intensity values per micrometers squared for SEPT2-mCherry–coated and –free MTs relative to total MTs (8 cells; n = 50). Error bars represent SEM.