One Sx(I/L)P motif mediates most MT plus-end tracking in tissue culture cells. S2 cells expressing different GFP-tagged Pigs constructs were imaged live. Left panels are single frames from a time-lapse, right panels are maximum intensity projections of pseudocoloured frames from time-lapse movies to make a 15s composite image, with six images taken at 3 s intervals. Boxes indicate comets that are shown magnified to the right. (A,B) Wild-type GFP–PigsFL tracks MT plus-ends. See Movie 1. (C,D) Plus-end tracking is largely abolished in S2 cells treated with RNAi against EB1. See Movie 3. (E,F) GFP–PigsFL with Sx(I/L)P1 mutated (SxIP1mut) still tracks MT plus-ends. (G,H) GFP–PigsFL with Sx(I/L)P2 mutated (SxLP2mut) still tracks MT plus-ends. (I,J) GFP–PigsFL with Sx(I/L)P3 mutated (SxIP3mut) has lost the ability to track MT plus-ends. See Movie 4. (K,L) Mutation of all 3 SxIP motifs in GFP–PigsFL [Sx(I/l)P1/2/3mut] abolishes plus-tip tracking. See Movie 5. Scale bar is 5 μm in A and A', and applies to all images. (M) Quantitative line scans of MT plus ends performed on individual time frames of movies obtained from live cells coexpressing EB1–RFP and GFP–PigsFL (16 comets from 7 cells), GFP–PigsSxIP3mut (9 comets from 3 cells) or GFP–PigsSxIP1/2/3mut (14 comets from 4 cells). Results are mean±S.D.