Figure 6. PLD modulates mechanosensitivity in Drosophila.

(A) Cholera toxin B (CTxB) robustly labels GM1 lipids (GM1, green) throughout the brain of Drosophila (left). The zoomed section (right) shows that most of the labeling is found on the membrane. There are notable variations in the amount of CTxB labeling, with some cells expressing GM1 over the entire membrane (black arrows) while others only have labeling in small puncta (white arrow). (B) Shear (3 dynes/cm²) robustly activates PLD2 in a live PLD assay with cultures neuronal insect cells. (C) Measurements of Drosophila mechanosensation in vivo. Animals with or without the pld^null gene were stimulated by increasing amounts of mechanical vibration (see Figure 6—figure supplement 1). Flies lacking PLD2 had a decreased threshold (i.e., more sensitivity to mechanical stimulation) compared to genetically matched controls (w1118) (p=0.02, n = 28–29), consistent with the prediction that PA decreases excitability of nerves. (D) The same result was observed in a PLD^RNAi line which results in PLD knockdown only in the neurons of Drosophila (p=0.002, n = 28–29), Mann–Whitney test. (E, F) Flies were subjected to increasing voltages of electrical shock in a two-choice assay. PLD-KD flies showed an increased sensitivity to shock when compared with wild-type flies. PLD^RNAi flies had a higher aversion to shock at 10 V (p=0.0213, n = 21) and 20 V (p=0.0492, n = 27–30), but not at 30 V (p=0.672, n = 12). (G) Proposed role of PLD2 in regulating mechanical thresholds. PA is a signaling lipid in the membrane that activates hyperpolarizing channels and transporters. When PA is low the membrane is less polarized, and cells are more sensitive to mechanical activation. The downstream targets are unknown (shown with a ‘?’). Flies lack a known mechanosensitive TREK-1 homolog.

Figure 6—figure supplement 1. Pan-neuronal knockdown of pld in Drosophila alters sensitivity to electrical shock.

(A) Illustration of the experimental setup for applying mechanical stimulation to Drosophila flies. A series of six increasing vibrations (top) used to stimulate the flies. A vibration motor was attached to the back of the chamber containing the flies, and their responses were monitored using a web camera to assess stimulation-induced arousal (bottom). (B) Illustration of shock avoidance assay.