[Show abstract][Hide abstract] ABSTRACT: We used actin tagged with enhanced green fluorescent protein (EGFP-actin) to characterize the distribution and dynamics of actin in living presynaptic terminals in rat CNS neurons. Actin was preferentially concentrated around--and appeared to surround--the presynaptic vesicle cluster. In resting terminals, approximately 30% of actin was found to be in a polymerized but dynamic state, with a remodeling time scale of approximately 20 s. During electrical activity, actin was further polymerized and recruited from nearby axonal regions to the regions surrounding vesicles. Treatment of terminals with the actin monomer-sequestering agent latrunculin-A completely dispersed the actin network and abolished activity-dependent actin dynamics. We used a variety of methods to examine the role of actin in the presynaptic vesicle cycle. These data rule out a propulsive role for actin, either in maintaining the vesicle cluster or in guiding vesicle recycling. Instead, we propose that actin acts as a scaffolding system for regulatory molecules in the nerve terminal.
[Show abstract][Hide abstract] ABSTRACT: The function of the clathrin coat in synaptic vesicle endocytosis is assisted by a variety of accessory factors, among which amphiphysin (amphiphysin 1 and 2) is one of the best characterized. A putative endocytic function of amphiphysin was supported by dominant-negative interference studies. We have now generated amphiphysin 1 knockout mice and found that lack of amphiphysin 1 causes a parallel dramatic reduction of amphiphysin 2 selectively in brain. Cell-free assembly of endocytic protein scaffolds is defective in mutant brain extracts. Knockout mice exhibit defects in synaptic vesicle recycling that are unmasked by stimulation and suggest impairments at multiple stages of the cycle. These defects correlate with increased mortality due to rare irreversible seizures and with major learning deficits, suggesting a critical role of amphiphysin for higher brain functions.
[Show abstract][Hide abstract] ABSTRACT: A pH-sensitive form of green-fluorescent protein (GFP) fused to the lumenal domain of VAMP (synapto-pHluorin) provides a sensitive optical probe to track the net balance between exocytosis and endocytosis of this protein at small synaptic terminals of the central nervous system. Here we used a reversible proton-pump blocker that prevents vesicle re-acidification upon endocytosis to trap vesicles in the alkaline state during recycling. In combination with optical measurements of synapto-pHluorin, we used alkaline trapping to examine the kinetic components of exocytosis and endocytosis separately at synaptic terminals. Using this approach, we show that, in addition to controlling exocytosis, intracellular calcium levels tightly regulate the speed of endocytosis, increasing it to a maximal speed of approximately one vesicle per second.
[Show abstract][Hide abstract] ABSTRACT: Genetically encoded reporters for optical measurements of presynaptic activity hold significant promise for measurements of neurotransmission within intact or semi-intact neuronal networks. We have characterized pH-sensitive green fluorescent protein-based sensors (pHluorins) of synaptic vesicle cycling at nerve terminals. pHluorins have a pK approximately 7.1, which make them ideal for tracking synaptic vesicle lumen pH upon cycling through the plasma membrane during action potentials. A theoretical analysis of the expected signals using this approach and guidelines for future reporter development are provided.
[Show abstract][Hide abstract] ABSTRACT: Following the fusion of synaptic vesicles with the presynaptic plasma membrane of nerve terminals by the process of exocytosis, synaptic-vesicle components are recycled to replenish the vesicle pool. Here we use a pH-sensitive green fluorescent protein to measure the residence time of VAMP, a vesicle-associated SNARE protein important for membrane fusion, on the surfaces of synaptic terminals of hippocampal neurons following exocytosis. The time course of VAMP retrieval depends linearly on the amount of VAMP that is added to the plasma membrane, with retrieval occurring between about 4 seconds and 90 seconds after exocytosis, and newly internalized vesicles are rapidly acidified. These data are well described by a model in which endocytosis appears to be saturable, but proceeds with an initial maximum velocity of about one vesicle per second. We also find that, following exocytosis, a portion of the newly inserted VAMP appears on the surface of the axon.