F-actin at newly invaginated membrane in neurons: implications for surface area regulation

Neuronal shape and volume changes require accompanying cell surface adjustments. In response to osmotic perturbations, neurons show evidence of surface area regulation; shrinking neurons invaginate membrane at the substratum, pinch off vacuoles, and lower their membrane capacitance. F-actin is implicated in reprocessing newly invaginated membrane because cytochalasin causes the transient shrinking-induced invaginations, vacuole-like dilations (VLDs), to persist indefinitely instead of undergoing recovery. To help determine if cortical F-actin indeed contributes to cell surface area regulation, we test, here, the following hypothesis: invaginating VLD membrane rapidly establishes an association with F-actin and this association contributes to VLD recovery. Cultured molluscan (Lymnaea) neurons, whose large size facilitates three-dimensional imaging, were used. In fixed neurons, fluorescent F-actin stains were imaged. In live neurons, VLD membrane was monitored by brightfield microscopies and actin was monitored via a fluorescent tag. VLD formation (unlike VLD recovery) is cytochalasin insensitive and consistent with this, VLDs formed readily in cytochalasin-treated neurons but showed no association with F-actin. Normally, however (i.e., no cytochalasin), VLDs were foci for rapid reorganization of F-actin. At earliest detection (1-2 min), nascent VLDs were entirely coated with F-actin and by 5 min, VLD mouths (i.e. , at the substratum) had become annuli of F-actin-rich motile leading edge. Time lapse images from live neurons showed these rings to be motile filopodia and lamellipodia. The retrieval of VLD membrane (vacuolization) occurred via actin-associated constriction of VLD mouths. The interplay of surface membrane and cortical cytoskeleton in osmotically perturbed neurons suggests that cell surface area and volume adjustments are coordinated in part via mechanosensitive F-actin dynamics.