Vacuole dynamics in growth cones: correlated EM and video observations

The neuronal growth cone is a major site of surface membrane dynamics associated with uptake and release of materials, motility, and axon extension. Although intracellular membrane organelles are thought to mediate surface membrane addition and retrieval at the growth cone, membrane events are fleeting and therefore difficult to study directly. In an effort to capture transient interactions between intracellular membrane organelles and the plasmalemma at the growth cone, embryonic rat sympathetic neuron cultures were prepared for whole-mount electron microscopy (EM) by rapid freezing and freeze substitution. We identified a set of vacuole-like organelles (> or = 150 nm in diameter) that appeared to interact directly with the plasmalemma. In stereo-pair EM images the bounding membrane of some of these vacuoles had an orifice at sites where the organelle was adjoining the plasmalemma, suggesting that the organelle and surface membranes were confluent. Since this population of organelles could be labeled with cationized ferritin or HRP when added to living cultures just prior to freezing or chemical fixation, they were probably derived from the plasmalemma. Combined light microscopy and EM of individual growth cones showed that these same vacuoles had a conspicuous reverse shadowcast appearance in differential interference contrast images. Thus, we used real-time video microscopy to follow these organelles in living growth cones. Many of these vacuoles spontaneously appeared, remained visible for several minutes, and then disappeared. Reverse shadowcast vacuoles were formed at various sites throughout the growth cone, including surface membrane ruffles at the leading edge [P (peripheral)-domain] as well as quiescent and retracting regions at the growth cone base [C (central)-domain]. Vacuoles in the P-domain moved centripetally and rarely grew in size. In contrast, those in the C-domain exhibited Brownian-like movements and sometimes appeared to increase in size, raising the possibility that new membrane may be added to these organelles. Vacuoles within both the P- and C-domains shrank before rapidly disappearing, but rarely vesiculated, suggesting that they had fused with the plasmalemma. The results indicate that vacuoles are a highly dynamic population of organelles that directly communicate with the plasma membrane at the growth cone; they provide a major route of surface membrane uptake and may also play a role in membrane recycling.