SEPT2 fibers colocalize with, and are required for, polyglutamylated microtubule tracks. (A and B) Untreated (A) and SEPT2-siRNA-treated (B) MDCK cells were stained with SEPT2 and polyGlu (≥2 glutamate residues) tubulin (mAb B3) antibodies and imaged by confocal microscopy. In A, arrows point to juxtanuclear/Golgi SEPT2 filaments that colocalize with polyGlu-microtubule tracks (see insets for higher magnification). In B, the asterisk marks a SEPT2-depleted cell. (C and D) MDCK cells were transfected with MAP4-GFP and FLAG-tagged MAP1B light chain (MAP1B-FLAG), and stained with SEPT2, polyGlu-tubulin, and FLAG antibodies. Arrows point to polyGlu microtubules and SEPT2 filaments (see insets for higher magnification), and arrowheads point to the juxtanuclear/Golgi regions of MAP-expressing cells. (E) Bar graph shows mean SEPT2 and polyGlu tubulin fluorescence (± SEM) in control (white columns; n = 11) and SEPT2 (black columns; n = 13) siRNA-treated cells. **, P = 0.0042; ***, P < 0.0001. (F) Bar graphs show mean SEPT2 (n = 19) and polyGlu tubulin (n = 25) fluorescence (± SEM) as a function of MAP4-GFP expression after binning GFP fluorescence into low, mid, and high range values. *, P = 0.04; **, P = 0.003. (G) MDCK cells were transfected with MAP4-GFP and ts045-VSV-G-cherry at 41°C and then shifted to 32°C for 1.5 h. Arrowheads (MAP4-GFP-expressing cells) and arrows (nonexpressing cells) point to the plasma membrane at cell–cell contacts. Bar graph shows mean (± SEM) ratios of plasma membrane to intracellular fluorescence for cells (n = 30) with low, middle, and high levels of MAP4-GFP expression from three independent experiments. **; P = 0.001; ***; P < 0.0001.