Single vesicle imaging indicates distinct modes of rapid membrane retrieval during nerve growth
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Hines et al. BMC Biology 2012, 10:4 http://www.biomedcentral.com/1741-7007/10/4
RESEARCH ARTICLE
Open Access
Single vesicle imaging indicates distinct modes of rapid membrane retrieval during nerve growth Jacob H Hines1,2, Steven J Henle1, Lucas P Carlstrom1, Mohammad Abu-Rub1,3 and John R Henley1,4*
Abstract Background: During 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. Results: To 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. Livecell 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. Conclusions: This 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.
Background During the construction of neural circuits, growing axons of developing neurons extend long distances en route to the appropriate synaptic targets. New membrane and materials are added to extending axons and increase the plasma membrane surface area by 10 to 1,000-fold [1]. A typical 1-μm-diameter axon extends at the rate of 0.5 mm per day, necessitating the insertion of new membrane at the rate of 1.1 μm 2 per minute. The body of evidence indicates that new membrane is incorporated primari
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