Saturday, December 21, 2024
12/21/2024
Project Summary Information in the nervous system is relayed mostly at synapses, where neurotransmitter is released with great temporal precision from a presynaptic terminal via the fusion of membrane-bound synaptic vesicles (SVs) with the plasma membrane (PM), in a process called exocytosis. Components of these SVs are subsequently retrieved via endocytosis and recycled for reuse. This grant aims to understand the interplay between SV recycling and membrane tension gradients and associated membrane flows. In neurons and neuroendocrine cells, both exo- and endocytosis are influenced by osmotic forces, suggesting they are influenced by membrane tension, . Conversely, membrane addition to the PM via exocytosis is expected to lower , while endocytosis should restore it. In addition, has been suggested to be a possible signal for coupling exo- to endocytosis. Despite these key roles, there are no measurements of in synaptic terminals and how changes are related to exo-endocytosis is not known, mainly due to technical difficulties. The best method to probe is to pull a thin membrane tether from the PM using optical tweezers; the tether force reflects . However, most terminals are small and tightly coupled to post-synaptic structures, making tether pulling impractical. We overcome this challenge using fish bipolar neurons which possess giant terminals, in a setup that combines optical tweezers with electrophysiology or photostimulation and with high- resolution fluorescence microscopy. We aim to 1) characterize PM flows at neuronal presynaptic terminals. After stimulation, membrane added at an exocytic site needs to flow (and the associated perturbation propagate) over the cell surface, then through the tether to produce a change in the measured tether force. We will characterize membrane flows. 2) Determine mechanisms of cell membrane flow regulation by the cytoskeleton. We found that F-actin is a major regulator of PM-cytoskeleton drag, but how it interacts with the PM at terminals and activity-dependent changes in its structure are not understood. We will characterize F- actin rearrangements upon stimulation at the optical and electron microscopy levels. 3) Establish the relationship between tension changes in response to stimulation, membrane flow, and exo-endocytosis coupling. We will confirm that changes we observed in preliminary experiments are due to exo-endocytosis and map the spatiotemporal relationship between exo- and endocyosis sites as a function of membrane flow to test the idea that exo-endocytosis coupling may depend on membrane flow. 4) Do electromechanical effect matter for exo- or endocytosis? We observed rapid voltage-induced tether force changes consistent with electromechanical effects. The relevance of these effects to exo-endocytosis is not known. We will characterize electromechanical effects and determine whether they may play a role during exo-endocytosis. Overall, these measurements will help generate a model of feedback between membrane trafficking and membrane flows.
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