Abstract
BackgroundDuring nerve growth, cytoplasmic vesicles add new membrane preferentially to the growth cone located at the distal tip of extending axons. Growth cone membrane is also retrieved locally, and asymmetric retrieval facilitates membrane remodeling during growth cone repulsion by a chemorepellent gradient. Moreover, growth inhibitory factors can stimulate bulk membrane retrieval and induce growth cone collapse. Despite these functional insights, the processes mediating local membrane remodeling during axon extension remain poorly defined.ResultsTo investigate the spatial and temporal dynamics of membrane retrieval in actively extending growth cones, we have used a transient labeling and optical recording method that can resolve single vesicle events. Live-cell confocal imaging revealed rapid membrane retrieval by distinct endocytic modes based on spatial distribution in Xenopus spinal neuron growth cones. These modes include endocytic "hot-spots" triggered at the base of filopodia, at the lateral margins of lamellipodia, and along dorsal ridges of the growth cone. Additionally, waves of endocytosis were induced when individual filopodia detached from the substrate and fused with the growth cone dorsal surface or with other filopodia. Vesicle formation at sites of membrane remodeling by self-contact required F-actin polymerization. Moreover, bulk membrane retrieval by macroendocytosis correlated positively with the substrate-dependent rate of axon extension and required the function of Rho-family GTPases.ConclusionsThis study provides insight into the dynamic membrane remodeling processes essential for nerve growth by identifying several distinct modes of rapid membrane retrieval in the growth cone during axon extension. We found that endocytic membrane retrieval is intensified at specific subdomains and may drive the dynamic membrane ruffling and re-absorption of filopodia and lamellipodia in actively extending growth cones. The findings offer a platform for determining the molecular mechanisms of distinct endocytic processes that may remodel the surface distribution of receptors, ion channels and other membrane-associated proteins locally to drive growth cone extension and chemotactic guidance.
Highlights
During nerve growth, cytoplasmic vesicles add new membrane preferentially to the growth cone located at the distal tip of extending axons
The overall number of self-membrane contacts observed during the focal membrane labeling assay tended to be lower after the cytochalasin D treatment but was statistically insignificant from the non-treated controls (Figure 7F). These findings indicate that actin polymerization plays an important role in dynamic membrane retrieval by endocytic vesicles at sites of self-membrane contact in the growth cone
We found that vesicle formation at self-contact sites was sensitive to cytochalasin D treatment, implicating a role for filamentous actin (F-actin) polymerization in this process
Summary
Cytoplasmic vesicles add new membrane preferentially to the growth cone located at the distal tip of extending axons. Repulsive growth cone turning requires endocytic pathways and correlates with asymmetric endocytosis [12,13,14] These findings have led to the notion that the balance of exocytic and endocytic activities across the growth cone serves to control local membrane protrusion versus membrane removal and drives bidirectional axon guidance [15]. Further support for this idea comes from the finding that growth cone collapse is associated with regulated membrane retrieval [16,17]
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