Myosin V functions as a vesicle tether at the plasma membrane to control neurotransmitter release in central synapses

Abstract

Synaptic vesicle fusion occurs at specialized release sites at the active zone. How refilling of release sites with new vesicles is regulated in central synapses remains poorly understood. Using nanoscale-resolution detection of individual release events in rat hippocampal synapses we found that inhibition of myosin V, the predominant vesicle-associated motor, strongly reduced refilling of the release sites during repetitive stimulation. Single-vesicle tracking revealed that recycling vesicles continuously shuttle between a plasma membrane pool and an inner pool. Vesicle retention at the membrane pool was regulated by neural activity in a myosin V dependent manner. Ultrastructural measurements of vesicle occupancy at the plasma membrane together with analyses of single-vesicle trajectories during vesicle shuttling between the pools suggest that myosin V acts as a vesicle tether at the plasma membrane, rather than a motor transporting vesicles to the release sites, or directly regulating vesicle exocytosis.

A novel role for Myosin-Va in mitochondrial fission

“…Among other mechanisms, it has been shown that myosin-Va associates to the ER and binds to the ryanodine calcium receptor channels on the ER membrane, and also responds to ER-derived calcium by associating with vesicles and directing them to specific sites 50 . More recently, myosin-Va was shown to tether synaptic vesicles to the plasma membrane near Ca +2 influx sites and help to increase the retention of vesicles at the release site 51 . The decrease in the ER-mitochondrial contact surface, observed here in myosin-Va knockdown cells, is also consistent with a calcium triggered-role for myosin-Va at the ER-mitochondrial interface.…”

Spatiotemporal dynamics of multi-vesicular release is determined by heterogeneity of release sites in central synapses

“…Can factors other than desynchronization contribute to the difference in amplitude within MVR events and its spatial arrangement relative to the AZ center, such as differences in vesicle size or cleft pH along the AZ plane? We used the Large-Area Scanning Electron Microscopy (LaSEM) micrographs of our cultures (Maschi et al, 2018) to determine the size of all vesicles positioned next to the AZ (within 100nm, defined previously as tethered vesicles (Maschi et al, 2018a)) as a function of distance to the AZ center. Vesicle diameter did not exhibit any measurable changes with distance from the AZ center (Figure S3C,D).…”

“…LASEM methods and data used were published previously (Maschi et al, 2018). Briefly, cells were grown on 12 mm glass coverslips, were aspirated of media and fixed in a solution containing 2.5% glutaraldehyde and 2% paraformaldehyde in 0.15 M cacodylate buffer with 2 mM CaCl2, pH 7.4 that had been warmed to 37˚C for one hour.…”

“…Definition of release sites. Release sites were defined using hierarchical clustering algorithm using built-in functions in Matlab as described (Maschi and Klyachko 2017;Maschi et al 2018;Wang et al, 2016). We have previously compared the results of this clustering analysis obtained with experimentally observed distribution of fusion events vs. the same number of simulated events distributed randomly across the same AZs (Maschi and Klyachko, 2017).…”

“…Another possibility is that release site properties are determined, in part, by extrinsic factors. For example, release site refilling and vesicle retention at release sites depends on actin and myosins (Maschi et al, 2018;Miki et al, 2016). Thus a non-homogeneous spatial distribution of actin cytoskeleton could contribute to differential release site usage across a bouton.…”