Fig. 2.

PI(3,4)P₂-rich compartment forms the invaginated membrane bud and tube along cortical microtubule tracks. (A) Time-projected images of GFP-PH-Tapp1 and F-actin in MEF-Src (40 to 300 s, color coded). Over time, there are numerous PH-Tapp1–positive membrane invaginations behind the invadopodium arc. (B) Time-lapse images of the boxed region in A (6 × 8 µm²). PH-Tapp1 puncta (membrane buds, arrowheads) initially appear at the trailing edge of the invadopodium arc, elongate inwardly, and become straight tubular membrane structures (membrane tubes). See Movie S1. (C) The histogram of the membrane tube elongation rate (n = 43). (D) Membrane marker GPI-mCherry colocalizes with GFP-PH-Tapp1 in the membrane tube. See Movie S2. (E) Time-lapse images of the boxed region of D (1 × 6 µm²; the arrow indicates the base of the tube). Membrane tubes are repeatedly formed and then detached from the same location behind the invadopodium arc (arrowheads). (F) The intensity profiles of PH-Tapp1 and GPI at t₁ (the fully grown tube) and t₂ (the detached tube) in E. (G) Early endosome marker GFP-Rab5 accumulates in the vesicular compartment behind the mCherry-PH-Tapp1–labeled invadopodium arc. See Movie S3. (H) The kymograph along the boxed region of G (the arrow indicates the base of the tube). Rab5 dynamically associates with the detached membrane compartment (arrowheads). (I) The temporal intensity changes of PH-Tapp1 and Rab5 in the boxed region of H. (J) The GFP-PH-Tapp1–positive membrane bud elongates, breaks away from the invadopodium arc and moves along EMTB-mCherry–labeled microtubule track (arrowheads). The rectangular Inset shows the time-lapse images of the boxed region (6 × 18 μm²). See Movie S4. (K) After 15 min of the nocodazole treatment (10 µM), the formation of the GFP-PH-Tapp1–positive membrane tubes is greatly suppressed compared to the DSMO control. (L) The number of membrane tubes within the 15 min before and after the treatment as indicated (n = 16 and 15). The statistical information is in SI Appendix, Table S1C. The invadopodium arc is labeled by F-actin marker LifeAct-mRuby (in A and B) or EBFP2-UtrCH (in D, E, G, H, J, and K). All experiments have been independently repeated three times. Unpaired two-tailed Student’s t test is used for the statistical analysis. Ns, P > 0.1234; ***P < 0.0002 (Scale bars, 5 μm).