Figure 3. Leep1 localizes to the leading... | Rockefeller University Press

Figure 3.

Leep1 localizes to the leading edge by association with PIP3. (A and B) GFP-Leep1 translocation in response to folic acid (FA; A) or cAMP in the presence of LatA (B). (C) Lipid dot blot assay using GFP-Leep1 cell lysates. (D) Cells lysates containing GFP-tagged Leep1, TAPP1, or cPH×3 were incubated with PI(3,4)P2- or PIP3-coated agarose beads. Proteins eluted from the beads were probed with anti-GFP antibody. Lysate (2.4%) was loaded as input. (E) GFP-Leep1 translocation in response to cAMP in the presence of LY294002. (F–H) GFP-Leep1 localization in cells lacking the respective phosphoinositide kinases or phosphatases. (I) Sequential accumulation of GFP-Leep1 and TAPP1-RFP. GFP-Leep1 was enriched at membrane ruffles and macropinocytic cups and quickly removed from internalized macropinosomes, whereas the signal of TAPP1 gradually increased until a peak was reached after the macropinosomes detached from the cell surface. (J) A schematic representation of full-length Leep1 and truncation constructs. The green bar represents a putative PH domain-like fold (aa 17–115) and cyan bars a series of LRRs. (K) Localization of the N-terminal (Leep1N), middle (Leep1M), and C-terminal (Leep1C) fragments of Leep1, and Leep1 mutated at lysine 28 and arginine 39 (Leep1AA). (L) Leep1N mediates cAMP-stimulated translocation. Scale bar = 10 µm.

Leep1 localizes to the leading edge by association with PIP₃. (A and B) GFP-Leep1 translocation in response to folic acid (FA; A) or cAMP in the presence of LatA (B). (C) Lipid dot blot assay using GFP-Leep1 cell lysates. (D) Cells lysates containing GFP-tagged Leep1, TAPP1, or cPH×3 were incubated with PI(3,4)P₂- or PIP₃-coated agarose beads. Proteins eluted from the beads were probed with anti-GFP antibody. Lysate (2.4%) was loaded as input. (E) GFP-Leep1 translocation in response to cAMP in the presence of LY294002. (F–H) GFP-Leep1 localization in cells lacking the respective phosphoinositide kinases or phosphatases. (I) Sequential accumulation of GFP-Leep1 and TAPP1-RFP. GFP-Leep1 was enriched at membrane ruffles and macropinocytic cups and quickly removed from internalized macropinosomes, whereas the signal of TAPP1 gradually increased until a peak was reached after the macropinosomes detached from the cell surface. (J) A schematic representation of full-length Leep1 and truncation constructs. The green bar represents a putative PH domain-like fold (aa 17–115) and cyan bars a series of LRRs. (K) Localization of the N-terminal (Leep1^N), middle (Leep1^M), and C-terminal (Leep1^C) fragments of Leep1, and Leep1 mutated at lysine 28 and arginine 39 (Leep1^AA). (L) Leep1^N mediates cAMP-stimulated translocation. Scale bar = 10 µm.