Fig. 9.

A–D: differential cellular distribution of myristoylated alanine-rich protein kinase C substrate-like protein-1 (MLP-1) mutants. To examine the cellular localization of the different MLP-1 constructs, we transfected DCT-15 cells (a distal convoluted tubule clonal cell line) with a phosphatidylinositol 4,5-bisphosphate (PIP₂) reporter and each of the MLP-1 constructs and then used multiphoton microscopy to produce z-axis stacks of the transfected cells. A: an examination of the merged image of the wild-type construct shows that MLP-1 strongly colocalizes with PIP₂ (yellow pixels at the membrane); however, quantitative analysis with ImageJ (Coloc 2 module) shows that only 25% of pixels in the image colocalize. Examination of green pixels shows that 64% colocalize with red pixels, but 62% of red pixels do not colocalize with green pixels. This is consistent with the prediction that most green pixels (PIP₂) are at the membrane and many of them are associated with red pixels (wild-type MLP-1), but there are many red pixels in the cytosol. B: for S3A, 58% of all pixels colocalize. Ninety-nine percent of green pixels are associated with red pixels, and 96% of red pixels are associated with green pixels. This is consistent with the S3A mutant being predominantly associated with the membrane with little in the cytosol compared with wild-type MLP-1. C: for S3D, only 13% of red and green pixels are colocalized with 71% of green pixels unassociated with red pixels. This reflects the distribution of most of S3D in the cytosol with only a small fraction associated with the membrane. D: the GA construct has 22% green and red colocalized pixels. This is close to the colocalization of the wild-type construct, implying that the myristoylation does not strongly influence MLP-1 association with the membrane. Images are typical of 3 similar experiments.