Figure 4. Membrane curvature-dependent... | Rockefeller University Press

Figure 4.

$Figure 4. Membrane curvature-dependent SNX9 binding and INPP4A activity combine to regulate actin polymerization. (A and B) PI(4,5)P2/PI(3)P liposomes (rhodamine-PE labeled) were incubated with Alexa Fluor 647–SNAP-SNX9 from 15 to 250 nM. Concentrations and the fraction of liposomes with SNX9 foci were counted by two-color confocal imaging. Example confocal micrographs of SNX9 (green) bound to 100-nm liposomes (purple, A) and 250-nm liposomes (purple, B). Bars, 3 µm. (C) The number of SNX9 foci per surface area of membrane calculated using the means determined in Fig. S3 C. Data are from three independent experiments. (D) Kinetics of PI(3,4)P2 hydrolysis from 100- or 250-nm vesicles by INPP4A. Initial linear rates over the first 10 min were 0.0028 ± 0.00008 (100-nm vesicles) and 0.0013 ± 0.00021 (250-nm vesicles); P = 0.0037, n = 3 using Student’s t test. Data are from three independent experiments showing the mean and SEM. (E) Cdc42 is not regulated by membrane curvature or PI(3)P. Sedimentation assay of mKate-GBD for Cdc42⋅GTP activation on 100-nm or 250-nm vesicles (various phosphoinositide compositions, control with PC and 30% PS only) shows that Cdc42 activation is curvature independent. Quantification from three independent vesicle batches and Western blots. All error bars show SEM.$

Membrane curvature-dependent SNX9 binding and INPP4A activity combine to regulate actin polymerization. (A and B) PI(4,5)P₂/PI(3)P liposomes (rhodamine-PE labeled) were incubated with Alexa Fluor 647–SNAP-SNX9 from 15 to 250 nM. Concentrations and the fraction of liposomes with SNX9 foci were counted by two-color confocal imaging. Example confocal micrographs of SNX9 (green) bound to 100-nm liposomes (purple, A) and 250-nm liposomes (purple, B). Bars, 3 µm. (C) The number of SNX9 foci per surface area of membrane calculated using the means determined in Fig. S3 C. Data are from three independent experiments. (D) Kinetics of PI(3,4)P₂ hydrolysis from 100- or 250-nm vesicles by INPP4A. Initial linear rates over the first 10 min were 0.0028 ± 0.00008 (100-nm vesicles) and 0.0013 ± 0.00021 (250-nm vesicles); P = 0.0037, n = 3 using Student’s t test. Data are from three independent experiments showing the mean and SEM. (E) Cdc42 is not regulated by membrane curvature or PI(3)P. Sedimentation assay of mKate-GBD for Cdc42⋅GTP activation on 100-nm or 250-nm vesicles (various phosphoinositide compositions, control with PC and 30% PS only) shows that Cdc42 activation is curvature independent. Quantification from three independent vesicle batches and Western blots. All error bars show SEM.