MVBs/LEs associate preferentially with septin-coated microtubules. (A) Super-resolution confocal microscopy images of an MDCK cell stained for endogenous SEPT7 (inverted burgundy) and α-tubulin (inverted blue). The microtubule subset that colocalizes with SEPT7 is shown as an individual fluorescent channel (SEPT7-coated microtubules; inverted forest green). Insets show in higher magnification the perinuclear regions outlined with a dashed rectangle. Scale bar, 10 and 5 μm (insets). (B–E) Confocal microscopy images show single optical sections from subconfluent monolayers of MDCK cells stained with antibodies against SEPT7 and EEA1 (B), LAMTOR4 (C), TSG101 (D), and LBPA (E). Scale bars, 10 μm. (F) Subconfluent monolayers of MDCK cells that stably express GFP-CD63 (inverted magenta) were stained with antibodies to SEPT7 (inverted burgundy) and α-tubulin (inverted blue). Image (right panel) shows a maximum intensity projection of GFP-CD63 (inverted magenta) and the subset of microtubules that colocalizes with SEPT7 (SEPT7-coated microtubules; inverted forest green) from a z-series stack of optical sections acquired with super-resolution microscopy. Scale bars, 10 and 5 μm (inset). (G) Quantification of the mean (±SEM, error bars) percentage of EEA1 (n = 14 cells), LAMTOR4 (n = 10 cells), TSG101 (n = 12 cells), LBPA (n = 18 cells), and GFP-CD63 (n = 19 cells) membranes that overlap with septin filaments per MDCK cell. Statistical analysis of pairwise comparisons was performed with an unpaired t test with Welch’s correction. n.s., not significant; *** P < 0.001, **** P < 0.0001.