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Journal of Neuroscience, Vol 13, 3375-3393, Copyright © 1993 by Society for Neuroscience
Vacuole dynamics in growth cones: correlated EM and video observations
ME Dailey and PC Bridgman
Department of Anatomy and Neurobiology, Washington University School of Medicine, St. Louis 63110.
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.
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