Synaptic Vesicle Endocytosis

National Institute Of Neurological Disorders And Stroke

Investigators

Linked publications & trials

Abstract

Our progress in the last year is described below. 1. Membrane fission is thought to involve helix-forming proteins to constrict the Ω-shaped profile's neck. We provided a review of a recent breakthrough, which suggests that membrane pit-coating proteins, especially clathrin, may also mediate fission via polymerization on the Ω-profile's base or head to generate neck constriction. This mechanism underlies various endocytic modes previously attributed as clathrin (Ω-profile head) independent. 2. Despite decades of pharmacological studies, how the ubiquitous cytoskeletal actin regulates synaptic transmission remains poorly understood. We addressed this issue with a tissue-specific knockout of actin β-isoform or γ-isoform, combined with recordings of postsynaptic EPSCs, presynaptic capacitance jumps or fluorescent synaptophysin-pHluorin changes, and electron microscopy in large calyx-type and small conventional hippocampal synapses. We found that actin restrains basal synaptic transmission during single action potential firings by lowering the readily releasable vesicle’s release probability. Such an inhibition of basal synaptic transmission is turned into facilitation during repetitive firings by slowing down depletion of the readily releasable vesicle pool and, thus, short-term synaptic depression, leading to more effective synaptic transmission for a longer time. These mechanisms, together with the previous finding that actin promotes vesicle replenishment to the readily releasable pool, may control synaptic transmission and short-term synaptic plasticity at many synapses, contributing to neurological disorders caused by actin cytoskeleton impairment. 3. With electron microscopy after cryofixation and fluorescent imaging, we found that dense-core vesicles in adrenal chromaffin cells contain exosomes, which are generally believed to reside within multivesicular bodies and are released into the extracellular space to mediate various forms of cell-to-cell communication. Given that dense-core vesicle fusion mediates many physiological functions, such as stress responses, immune responses, behavior regulation, and blood glucose regulation, exosome release from dense-core vesicle fusion might contribute to mediating these important functions. 4. Exocytosis, which mediates important functions like synaptic transmission and stress responses, has been postulated to release all transmitter molecules in the vesicle in the “all-or-none” quantal hypothesis. Challenging this hypothesis, amperometric current recordings of catecholamine release propose that sub-quantal or partial transmitter release is dominant in various cell types, particularly chromaffin cells. The sub-quantal hypothesis predicts that fusion pore closure (kiss-and-run fusion), a non-canonical form of endocytosis that causes sub-quantal release, is dominant, and blocking endocytic pore closure increases quantal size. We tested these predictions by imaging fusion pore closure and amperometric recording of catecholamine release in chromaffin cells during high potassium application, the most-used stimulation protocol for sub-quantal release study. We found that fusion pore closure is not predominant, and inhibition of the fusion pore closure does not increase the quantal size calculated from the amperometric current charge when a sufficiently long integration time is used. These results suggest that sub-quantal release is not prevalent during prolonged stimulation in adrenal chromaffin cells. 5. We provided a review of a series of studies that examine the membrane’s role as a signaling platform, the proteins that drive fusion and budding, and the physical principles underlying these events. 6. Activation of the innate immune Stimulator of Interferon Genes (STING) pathway potentiates antitumor immunity. However, delivering STING agonists systemically to tumors presents a formidable challenge, and resistance to STING monotherapy has emerged in clinical trials with diminishing natural killer (NK) cell proliferation. Here, we encapsulated the STING agonist diABZI within polymersomes containing a Type I photosensitizer (NBS), creating a nanoagonist (PNBS/diABZI) for highly responsive tumor immunotherapy. This structure promoted H-aggregation and intersystem crossing of NBS, resulting in a ∼ 3-fold amplification in superoxide anion and singlet oxygen generation. The photodynamic therapy directly damaged hypoxia tumor cells and stimulated the proliferation of NK cells and cytotoxic T lymphocytes, thereby sensitizing STING immunotherapy. A single systemic intravenous administration of PNBS/diABZI eradicated orthotopic mammary tumors in murine models, achieving long-term antitumor immune memory to inhibit tumor recurrence and metastasis and significantly improving long-term tumor-free survival. This work provides a design rule for boosting reactive oxygen species production by promoting the intersystem crossing process, highlighting the potential of Type I photosensitizer-polymer vehicles for augmenting STING immunotherapy.