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 ex tension. Although intracellular membrane organelles are thought to med iate surface membrane addition and retrieval at the growth cone, membr ane, events are fleeting and therefore difficult to study directly in an effort to capture transient interactions between intracellular memb rane organelles and the plasmalemma at the growth cone, embryonic rat sympathetic neuron cultures were prepared for whole-mount electron mic roscopy (EM) by rapid freezing and freeze substitution. We identified a set of vacuole-like organelles (greater-than-or-equal-to 150 nm in d iameter) that appeared to interact directly with the plasmalemma. In s tereo-pair EM images the bounding membrane of some of these vacuoles h ad an orifice at sites where the organelle was adjoining the plasmalem ma, 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 th at 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. M any of these vacuoles spontaneously appeared, remained visible for sev eral minutes, and then disappeared. Reverse shadowcast vacuoles were f ormed at various sites throughout the growth cone, including surface m embrane 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 m ovements and sometimes appeared to increase in size, raising the possi bility that new membrane may be added to these organelles. Vacuoles wi thin both the P- and C-domains shrank before rapidly disappearing, but rarely vesiculated, suggesting that they had fused with the plasmalem ma. The results in indicate that vacuoles are a highly dynamic populat ion of organelles that directly communicate with the plasma membrane a t the growth cone; they provide a major route of surface membrane upta ke and may also play a role in membrane recycling,