Single-Molecule Studies for the Characterization of Synaptic Vesicles

Abstract

Synaptic vesicles are subcellular organelles that are found in the synaptic bouton and are responsible for the propagation of signals between neurons. Synaptic vesicles undergo a fast and tightly regulated cycle in which exocytosis to release neurotransmitters and endocytosis to recycle empty vesicles take place for efficient neuronal communication. This dissertation reports the development and application of analytical techniques for the characterization of various physical properties of synaptic vesicles at the single vesicle/molecule level. First, two complementary techniques, single-particle tracking and fluorescence correlation spectroscopy (FCS), for sizing nanoparticles in free solution by confinement within picoliter volume aqueous droplets will be described. Single-particle tracking works well with bright particles that can be continuously illuminated and imaged and was used to size single fluorescent beads. While FCS detects small intensity bursts from particles or molecules diffusing through the confocal probe volume, which works well with dim and rapidly diffusing particles or molecules, was used to size individual synaptic vesicles. Next, a method to determine both the average number and variance of proteins in the few to tens of copies in isolated synaptic vesicles will be described. Other currently available protein quantification techniques provide an average number but lack information on the intervesicler variability of protein number, and thus on the precision with which proteins are sorted to vesicles. The technique we present entails labeling the synaptic vesicles with fluorescent primary-secondary antibody complexes, TIRF (total internal reflection fluorescence) microscopy imaging of the vesicles, digital image analysis, and deconvolution of the fluorescence intensity data. With this technique, 7 major membrane proteins were quantified and we found that the proteins fell into 2 classes; proteins that were monodispersed and polydispersed number. The use of photobleaching as an alternative to obtaining an accurate calibration intensity distribution from the sample directly will be discussed. Finally, the initial steps towards the utilization of the exo- and endocytosis of synaptic vesicles to load nanoparticles as a means of probing the interior of the vesicles will be reported. We have developed a highly sensitive pH-sensing polymer dot, which we plan to use to probe the intravesicular pH of synaptic vesicles.