Real-time visual detection of P-i released by flagellar dynein ATPase

Using a novel fluorescent probe for P-i, a method for the direct visualizat ion of P-i release from reactivated flagellar dynein ATPase has been develo ped. The probe undergoes a fluorescence increase when it binds P-i. The tec hnique involves simultaneous imaging of demembranated sperm tails by epi-fl uorescence and darkfield microscopy, and the use of the caged ATP technique for axoneme reactivation. To limit diffusion and thus maintain the release d P-i within the observed field of view, the assay is carried out within a minute droplet under oil (volume 5-15 pl). The video output of a recursivel y filtered ICCD camera is used to visualize the fluorescence signal, which is subsequently digitized and automatically analysed on a PC. A major advan tage of this technique is that it enables simultaneous analysis of the ATP- utilization rate and the motility of the reactivated axonemes.