PILS-Nir1 is highly sensitive to PA. (A–E) PA biosensors were made from the PABD of Spo20 with added nuclear export sequences (NES) and various linker sequences. Alpha helices are shown by cylinders, while unstructured regions are shown as lines. (F–H) Novel biosensors were designed using the LNS2 domain of the Nir family of proteins. Sensors translocated to the PM after PKC-mediated PLD activation with 100 nM PMA in transfected HEK293A cells. The red inset shows the PM intensity of the sensor before PMA stimulation, and the blue inset shows the PM intensity after PMA stimulation. Data shown are the grand mean of three to four experiments ± SEM. (I) Schematics of full-length Spo20 and Nir proteins. (J) AUC for the biosensor responses in A–H. The small circles indicate the AUC of individual cells (n = 26–52). The large circles show the average AUC for each experimental replicate (n = 3–4). Cells in each replicate are color-coded accordingly. Statistics were calculated with a post hoc one-way ANOVA using the average AUC of each experimental replicate (n = 3–4), and the P values show the comparison of the respective biosensors to PILS-Nir1 (F = 12.74, P < 0.0001, R² = 0.8244). (K) Stimulating HEK293A cells with 100 nM PMA and 750 nM of the PLD1/2 inhibitor FIPI diminished the PM translocation of PILS-Nir1 seen with PMA and cell media. Data shown are the grand mean of three experiments ± SEM. A total of 27–41 cells were analyzed.