Fig. 2

Progranulin-null human neurons have altered lysosomal membrane and interacting proteins and elevated lysosome pH. A Schematic of Lyso-APEX in WT and isogenic GRN KO i³Neurons. B Fluorescence imaging showing the colocalization of PGRN with lysosomes in WT i³Neurons and loss of PGRN signal in GRN KO i³Neurons. Scale bar is 10 μm. C Volcano plot of Lyso-APEX proteomics in GRN KO vs. WT i³Neurons (N = 4 for each group). Cytosolic enriched proteins and nonspecific labeling were removed from the volcano plot based on WT Lyso-APEX vs. Cytosolic-APEX comparison. Red and blue colored proteins belong to lysosomal pH and protein transport GO-terms, respectively. D GO enrichment analysis of significantly upregulated biological processes in GRN KO vs. WT Lyso-APEX proteomics. E Protein network analysis of increased vacuolar-ATPase subunits and their interactors in PGRN-null neurons. F Live cell ratiometric lysosome pH assay. pH calibration curve is generated based on the ratio of pH-sensitive Oregon Green-488 dextran signal and pH-insensitive/loading control Alexa Fluor-555 red dextran in WT i³Neurons. Scale bar is 10 μm. Other linear and nonlinear curve fitting models are provided in Supplementary Figure S2E. G Lysosome pH measurements in WT vs. GRN KO i.³Neurons; three independent experiments are represented with different shapes, each with 5–10 wells of neuron culture replicates (**** denotes p-value < 0.0001)