Abstract
In compensatory endocytosis, scission of vesicles from the plasma membrane to the cytoplasm is a prerequisite for intravesicular reacidification and accumulation of neurotransmitter molecules. Here, we provide time-resolved measurements of the dynamics of the alkaline vesicle population which appears upon endocytic retrieval. Using fast perfusion pH-cycling in live-cell microscopy, synapto-pHluorin expressing rat hippocampal neurons were electrically stimulated. We found that the relative size of the alkaline vesicle population depended significantly on the electrical stimulus size: With increasing number of action potentials the relative size of the alkaline vesicle population expanded. In contrast to that, increasing the stimulus frequency reduced the relative size of the population of alkaline vesicles. Measurement of the time constant for reacification and calculation of the time constant for endocytosis revealed that both time constants were variable with regard to the stimulus condition. Furthermore, we show that the dynamics of the alkaline vesicle population can be predicted by a simple mathematical model. In conclusion, here a novel methodical approach to analyze dynamic properties of alkaline vesicles is presented and validated as a convenient method for the detection of intracellular events. Using this method we show that the population of alkaline vesicles is highly dynamic and depends both on stimulus strength and frequency. Our results implicate that determination of the alkaline vesicle population size may provide new insights into the kinetics of endocytic retrieval.
Highlights
Synaptic vesicle recycling is crucial for sustained neurotransmitter release [1]
We found that the time constants for reacidification and endocytosis depend on the stimulation paradigms
Synapto-pHluorin is a pH-sensitive mutant of green fluorescent protein tagged to the luminal site of the synaptic vesicle transmembrane protein synaptobrevin-2 [4,21]
Summary
Synaptic vesicle recycling is crucial for sustained neurotransmitter release [1]. Synaptic vesicles fuse with the plasma membrane to release neurotransmitters into the synaptic cleft. This is followed by compensatory endocytic retrieval of membranes which is clathrin-mediated [2,3]. The intravesicular pH is acidic in contrast to the extracellular fluid [4]. This proton gradient is maintained by the vesicular proton pump. As reacidification occurs very fast after formation of a clathrin-coated vesicle [5,6,7], alkaline vesicles pinpoint synaptic vesicle scission which is an indispensable step upon endocytic retrieval [8]
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