Figure 8. Evidence for BAR domain activation in PICK1 by membrane localization.

A) COS-7 cells were transiently trans-fected with PICK1 containing an artificial N-terminal myristoylation site (MyriPICK1) (top left panels) or MyriPICK1 3KE to disrupt the function of the BAR domain (top right panels). The constructs are schematically illustrated above the confocal microscopy pictures of representative cells. The clustering was quantified by determining the standard deviation (SD) of a line scan through the cells as a measure of clustering. Representative line scan along indicated green line is shown below the confocal pictures. The SD for the shown scans were 59.0 for MyriPICK1 and 35.7 for MyriPICK1 3KE. The SD values for 64 cells expressing MyriPICK1 and for 63 cells expressing myriPICK1 3KE are indicated in the lower panel. Mean SD was 54.7 ± 1.5 for myriPICK1 and 30.1 ± 1.7 for myriPICK1 3KE. These values were significantly different (p < 0.0001, unpaired t-test). B) COS-7 cells expressing MyriPICK1 G2A to prevent myristoylation (left panel) or MyriPICK1 A87L to prevent ligand binding to the PDZ domain (right panel). The mutations are schematically illustrated above the images. Cells were fixed, permeabilized and stained with a C-terminal PICK1 antibody for confocal microscopy. White bar = 10 μm. The PDZ domain is shown in red and the BAR domain in blue. The black bar illustrates the artificial myristoylation sequence, whereas the purple tilde illustrates the myristoyl chain. The stars indicate where mutations are introduced, and the orientation of the BAR domain relative to the PDZ domain suggests whether the BAR domain is activated or not.