Dynamics of M1–AQP4 translocation to the leading edge in live, migrating astrocytes and glioma cells. (A) AQP4-GFP dynamics in randomly migrating U87-MG cells cotransfected with M1– or M23–AQP4–GFP and mCherry-LifeAct (to visualize F-actin–enriched lamellipodial protrusions). Center panels show single full frames and right panels show images of a sub-region (white rectangular box) at 10-s intervals during a cycle of lamellipodial protrusion and retraction. (B) Quantum dot, single-particle tracking in lamellipodial and somatic regions of AQP4^−/− astrocytes transfected with M1– or M23–AQP4 and labeled with mAb r58 (binds an extracellular epitope on AQP4) and QDot-conjugated secondary Ab. Lamellipodial regions were identified from phase-contrast images taken immediately before QDot tracking and overlayed on reconstructed trajectories, allowing their assignment to lamellipodial (L) or somatic (S) analysis groups (top panels). Bottom panels show representative reconstructed trajectories over 5 s of M1–AQP4 in somatic or lamellipodial regions or of M23–AQP4 in somatic regions oriented so the leading edge is in the direction of the arrow. (C) Pooled analysis of 100–150 separate AQP4 trajectories in somatic and lamellipodial regions of 10–12 astrocytes transfected with either M1– or M23–AQP4, shown as the average mean-squared displacement vs. time (top) and the cumulative distribution function of short-range (0–100 ms) diffusion coefficient (middle) and range at 1 s (bottom).