The Local Deletion of a Microvillar Cytoskeleton from Photoreceptors of Tipulid Flies During Membrane Turnover

The distal regions of the photoreceptor microvilli of tipulid flies are shed to extracellular space during membrane turnover. Before abscission, the microvillar tips undergo a transformation: they become deformed, and after conventional fixation for electron microscopy are relatively electron-lucent compared to the stable, basal microvillar segments. We now show that the electron-lucent segment is an empty bag of membrane whose P-face after freeze-etch preparation appears as densely particulate as the remainder of the microvillus. Transformation is achieved by the local deletion of a microvillar cytoskeleton which consists of a single, axial filament linked to the plasma membrane by side-arms. The filament may be partially preserved by the chelation of Ca2+; the provision of a divalent cation (Mg2+ or Ba2+) stabilizes the side-arms during subsequent fixation, as has been shown previously for the rhabdomeral cytoskeleton of blowflies. Incubation of the isolated retina in the presence of 0.25 mM Ca2+ at room temperature for 10-20 min causes proteolysis of the cytoskeleton which is blocked by as little as 0.5 mM of the thiol protease inhibitors Ep-475 and Ep-459. Loss of the cytoskeleton is accompanied by deformation of all regions of the microvilli. Local deletion of the cytoskeleton from the transformed zone of the normal rhabdom is sufficient to explain deformation of the microvillar tips, but not their subsequent abscission. The intimate association between a Ca2+-activated thiol protease and the cytoskeleton implied by the great rapidity of proteolysis calls for a reassessment of published studies of membrane turnover by radioautography, and of the nature of light-induced damage to arthropod photoreceptor membranes.