Identification of the region required for the association of TPD54 with INVs. (A) Alignment of human TPD54/TPD52L2 (UniProt accession no. O43399), TPD53/TPD52L1 (UniProt accession no. Q16890) and TDP52/TPD52 (UniProt accession no. P55327). Numbers indicate residue positions in TPD54. Pink highlighted area represents the coiled-coil domain (predicted with PCOILS, window size 28). Blue underlining indicates C-terminal conserved region. Gray shadowing shows positively charged residues. Lettering: teal, fully conserved residues; purple, strongly similar (>0.5 in the Gonnet PAM 250 matrix); and yellow, weakly similar (<0.5 in the Gonnet PAM 250 matrix). (B) Representative confocal micrographs of HeLa cells expressing mCherry-FKBP or mCherry-FKBP–tagged TPD54 FL (1–206) or indicated mutants. Blue labels indicate high variance measured in C. Inset: 4× zoom. Scale bar, 10 µm or 1 µm (inset). (C) Schematic diagram to show how spatiotemporal (ST) variance can be used to measure association with motile vesicular structures such as INVs. Spatial (S) and temporal (T) variance is shown for comparison. Scatter dot plot to show the spatiotemporal variance (mean variance per pixel over time) for the indicated constructs. Dots, individual cells; black bars, mean ± SD. The mean ± SD for mCherry-FKBP (control) is also shown as a black line and gray zone, down the plot. Dunnett’s post hoc test was done using mCherry-FKBP as control; blue indicates P < 0.05. Right: Representation of the mCherry-FKBP–tagged TPD54 constructs analyzed. Pink region, coiled-coil domain (CC). Yellow line, position of point mutation. Light blue region, underlined area in A.