Septin-coated actin filaments localize to the cellular periphery and have minimal overlap with GFP-CD63. (A) Maximum intensity projections of super-resolution confocal microscopy images of MDCK cells stained for endogenous SEPT7 and actin in MDCK cells that express GFP-CD63. Arrowheads point to perinuclear septin filaments (SEPT7) that do not overlap with actin filaments. Scale bars, 5 μm. (B and C) Maximum intensity projections of super-resolution confocal microscopy images of the same MDCK (A) show the localization of GFP-CD63 with respect to actin (B) or SEPT7 (C). Arrowheads point to perinuclear septin filaments (SEPT7). Scale bars, 5 μm. (D and E) Maximum intensity projections of super-resolution confocal microscopy images of the same MDCK (A–C) show the localization of GFP-CD63 with respect to SEPT7-coated actin (D), a fluorescent channel that was artificially generated. The outlined perinuclear region of the merged image of GFP-CD63 and SEPT7-coated actin is shown in higher magnification (E). Arrowheads point to perinuclear septin filaments (SEPT7). Scale bars, 5 μm. (F) Bar graph shows the mean (±SEM, error bars) percentage of GFP-CD63 particles that overlap with septin-coated actin filaments (n = 9 cells).