Close to the edge

The idea that Ca²⁺ microdomains near the synaptic membrane are crucial for vesicle fusion is not new, but the precise relationship between such microdomains (which can be rather small, extending just over a few hundred nanometres) and the fusion of single vesicles has not been established. A recent article in Nature Neuroscience addresses this issue in chromaffin cells, showing that the relative proximity between Ca²⁺ microdomains and docked vesicles regulates exocytosis at the level of individual vesicles.

In this study, Becherer et al. measured Ca²⁺ microdomains using dual colour evanescent field microscopy — a form of microscopy that selectively probes the near-membrane space by generating a thin short-lived luminous field at the cytosol/glass interface of a cell in culture. Combining this technique with the tracking of single fluorescent chromaffin vesicles and with amperometric measurements of catecholamine release, the authors showed that the interaction between a vesicle and a Ca²⁺ microdomain requires their strict colocalization within 300 nm. Remarkably, only about 10% of the vesicles that were exposed to Ca²⁺ within the microdomain fused immediately with the plasma membrane, indicating that most vesicles might not be ready for exocytosis. In addition, the authors found that lower, near-membrane Ca²⁺ elevations caused the vesicles to move closer to the Ca²⁺ entry sites, transiently increasing the probability of fusion in response to subsequent Ca²⁺ increases.

Although it will be important to test whether this strict requirement for colocalization and this new facilitatory effect of Ca²⁺ on release are also relevant at the synapse, the data of Becherer et al. highlight the power of evanescent field microscopy to question the vesicle fusion process at an unprecedented level of resolution.