Figure 1.

Acute manipulation of PI 3-phosphates reveals an endocytic function of PI(3,4)P₂. A, proposed models of phosphoinositide conversion in CME. Model I, CCP maturation is accompanied by lipid conversion from PI(4,5)P₂ to PI(4)P PI(3,4)P₂ generated by PI3KC2α. PI(3,4)P₂ can recruit effector proteins (e.g. SNX9/18) to facilitate CCP maturation. Model II, PI3KC2α synthesizes PI(3)P at the plasma membrane, alleviating the need for PI(3,4)P₂ 4-phosphatases. Effector proteins may bind to PI(3)P to facilitate CCP maturation. The kinase responsible for PI(3,4)P₂ synthesis has not been identified in this study (16). Model III, PI(3,4)P₂ synthesized locally by PI3KC2α is converted rapidly to PI(3)P at CCPs by the PI(3,4)P₂-specific 4-phosphatase INPP4A, resulting in generation of PI(3)P. Effector proteins may bind to PI(3)P to facilitate CCP maturation. B and C, supply of exogenous PI(3,4)P₂/AM or PI(3)P/AM rescues stalled CCP dynamics in PI3KC2α-depleted cells. Kymographs of COS7 cells expressing mRFP-clathrin fluorescence over 180 s by TIRF imaging are shown in B. y axis scale bar = 5 μm. Scr, scrambled. C, fraction of dynamic CCPs analyzed by quantitative automated 2D tracking. Mean ± S.E., *** p < 0.001. D, acute depletion of PI(3,4)P₂ by rapalog-induced recruitment of INPP4B impairs transferrin CME. Depletion of a postulated plasma membrane PI(3)P pool by rapalog-induced recruitment of the PI(3)P 3-phosphatase MTM1 had no effect. Phosphatase inactive variants of INPP4B (C842A) or MTM1 (C375S) did not impair transferrin-CME. Bar diagrams represent the ratio of internalized (10 min, 37 °C) to surface transferrin (45 min, 4 °C). Mean ± S.E., n = 3 independent experiments. 790, 890, 916, 444, 332, and 1097 cells from three independent experiments were analyzed. *, p < 0.05; one-way ANOVA. E, accumulation of AP-2α–containing CCPs in PI(3,4)P₂-depleted cells. Bar diagrams represent the mean intensity of endocytic AP-2α–containing CCPs. Mean ± S.E.; n = 3 independent experiments. 179, 158, 119, 109, 119, and 116 cells from three independent experiments were analyzed. ***, p < 0.001; one-way ANOVA. F, rapalog-induced recruitment of INPP4B to the plasma membrane results in decreased PI(3,4)P₂ levels. Mean ± S.E, n = 3 independent experiments. 69 cells were analyzed from three independent experiments for each condition. *, p < 0.05; unpaired t test. G, rapalog-induced endosomal recruitment of WT but not inactive (C375S) MTM1 depletes PI(3)P from endosomes. Mean ± S.E., n = 3 independent experiments. 54 and 64 cells were analyzed. **, p < 0.01; unpaired t test.