The endocytosis and recycling of synaptic vesicles (SVs) at the presynapse is essential for maintaining neurotransmission and brain function. Activity-dependent bulk endocytosis (ADBE) is the dominant endocytosis mode during high neuronal activity. However little is known regarding its molecular mechanism and specifically where is occurs in relation to other endocytosis modes.
Electron microscopy has provided important information regarding the organization and structure of ADBE-derived endosomes at nanoscale, however it does not allow visualization of the dynamic nature of membrane trafficking in axons. Using a combination of live and fixed-cell super-resolution microscopy, this project will provide a detailed visualization of ADBE at the presynapse. This will range from the initiation of the endocytic invagination at the plasma membrane, cytoskeleton dynamics during bulk endosome formation, and the intracellular dynamics and fate of the mature bulk endosomes.
Endocytosis relies on the interaction of endocytic proteins with various lipid species in the plasma membrane to proceed. PA is a phospholipid precursor enriched in the plasma membrane that is potentially implicated in ADBE due to its proposed role as a regulator of membrane shape. However, the turnover, distribution and concentration of PA in the presynaptic plasma membrane is undetermined, as is whether these parameters are dynamically altered by neuronal activity.
Specifically, this project aims to 1) determine the dynamic nanoscale organization of ADBE in relation to other endocytic modes and 2) the role of the lipid microenvironment in endocytosis, in particular phosphatidic acid (PA).
This project will determine whether ADBE occurs at pre-defined sites at the presynaptic plasma membrane. It will also determine whether the protein and lipid composition of these sites, their relative location to the presynaptic release machinery, and the role of the dynamic instability of actin cytoskeleton in their assembly and disassembly. To achieve this, we will develop new tools and techniques to visualize the organization of ADBE with nanoscale resolution. We will employ live and fixed cell super resolution microscopy (gated-STED and SRRF), and single-particle tracking to obtain a dynamic map of the diffusion properties of ADBE-derived endosomes. We will also examine PA turnover and dynamics at the presynapse at rest and during neuronal activity to determine its contribution to the formation of synaptic endosomes. This will be achieved using fluorescent biosensors for live imaging of PA and pharmacological and genetic manipulations of its expression level. Co-expression with biosensors for other plasma membrane signalling lipids, such as phosphatidylinositol 4,5-biphosphate, will facilitate the visualization of specific lipid microdomains at the presynapse. It will also reveal any potential crosstalk, co-localization and association with both the presynaptic release machinery and other pathways of endocytic membrane retrieval.
This project will reveal how ADBE integrates into the presynaptic architecture and the role of the lipid microenvironment in this process. It will provide a new insight into the fundamental molecular mechanisms controlling presynaptic function and generate a palette of novel tools and techniques to visualise membrane trafficking at the nanoscale.