Abstract

The reserve pool (RP) of synaptic vesicles (SVs) is crucial for sustaining neurotransmission during periods of high-frequency activity, yet its precise structural organization and the molecular mechanisms governing its stability remain incompletely understood. Recent evidence suggests that the clustering of SVs, particularly within the RP, is mediated by protein scaffolds that exhibit properties of liquid-liquid phase separation (LLPS), forming dynamic, non-membrane-bound condensates. This study investigates the organization of the SV RP in nerve terminals genetically modified to lack key protein components known to drive this liquid phase behavior, such as Synapsin. Using advanced electron microscopy and live-cell imaging techniques, we aimed to determine the structural integrity and functional mobilization of the RP in the absence of these condensates. Our findings reveal that while the overall number of SVs in the RP is maintained, their spatial distribution and mobility are significantly altered. Specifically, the characteristic tight clustering is lost, and the kinetics of SV recruitment to the readily releasable pool are impaired under intense stimulation. These results suggest that protein liquid phase components are not strictly essential for the *existence* of the RP, but are indispensable for establishing its *organized* structure and ensuring efficient, activity-dependent mobilization, highlighting a critical role for LLPS in the fundamental cell biology of synaptic transmission.