The I282N (Lupo) mutation impairs both membrane sculpting by PSTPIP2 and PSTPIP2 stability. (A) Transmission electron micrographs of Folch liposomes (left panel) and liposomes coincubated with WT PSTPIP2 (middle panel) demonstrate that PSTPIP2 typically generates narrow (∼ 25-nm diameter) tubules. Striations decorate some portions of the tubules (inset), suggesting a PSTPIP2 repeating scaffold. (Right panel) PSTPIP2 l282N mutation does not attenuate tubulation in vitro. (B) Mutation of I282 affects plasma membrane tubule stability. Pseudocolored fluorescence image of COS-7 cells expressing EGFP alone (left), WT EGFP-PSTPIP2 (middle) or EGFP-PSTPIP2 l282N (right) under live cell imaging. The EGFP fluorescence at time = 0 seconds is displayed in green and in the same cell 30 seconds later, in red. The lengths of PSTPIP2 l282N tubules decrease over time (arrows), whereas WT PSTPIP2 tubules appear stable, as indicated by the colocalization of red and green signals (yellow). Note: movement of tubules during the 30 seconds time frame results in separate green and red images for the same tubule. (C) Time-lapse video frames of live cell imaging of a COS-7 cell expressing EGFP-PSTPIP2 I282N at moderate levels. The arrow in the first frame denotes the site of tubule uncoating. (D) At high expression levels EGFP-PSTPIP2 I282N misfolds into large protein aggregates that are tethered to tubules (inset, arrows). Scale bars, 200 nm in panel A and 20 μm in panels B through D.