Diffusional spread and confinement of newly exocytosed synaptic vesicle proteins.

Neurotransmission relies on the calcium-triggered exocytic fusion of non-peptide neurotransmitter-containing small synaptic vesicles (SVs) with the presynaptic membrane at active zones (AZs) followed by compensatory endocytic retrieval of SV membranes. Here, we study the diffusional fate of newly exocytosed SV proteins in hippocampal neurons by high-resolution time-lapse imaging. Newly exocytosed SV proteins rapidly disperse within the first seconds post fusion until confined within the presynaptic bouton. Rapid diffusional spread and confinement is followed by slow reclustering of SV proteins at the periactive endocytic zone. Confinement within the presynaptic bouton is mediated in part by SV protein association with the clathrin-based endocytic machinery to limit diffusional spread of newly exocytosed SV proteins. These data suggest that diffusion, and axonal escape of newly exocytosed vesicle proteins, are counteracted by the clathrin-based endocytic machinery together with a presynaptic diffusion barrier.

Vesicular Synaptobrevin/VAMP2 Levels Guarded by AP180 Control Efficient Neurotransmission.

Neurotransmission depends on synaptic vesicle (SV) exocytosis driven by soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) complex formation of vesicular synaptobrevin/VAMP2 (Syb2). Exocytic fusion is followed by endocytic SV membrane retrieval and the high-fidelity reformation of SVs. Syb2 is the most abundant SV protein with 70 copies per SV, yet, one to three Syb2 molecules appear to be sufficient for basal exocytosis. Here we demonstrate that loss of the Syb2-specific endocytic adaptor AP180 causes a moderate activity-dependent reduction of vesicular Syb2 levels, defects in SV reformation, and a corresponding impairment of neurotransmission that lead to excitatory/inhibitory imbalance, epileptic seizures, and premature death. Further reduction of Syb2 levels in AP180(-/-)/Syb2(+/-) mice results in perinatal lethality, whereas Syb2(+/-) mice partially phenocopy loss of AP180, indicating that reduced vesicular Syb2 levels underlie the observed defects in neurotransmission. Thus, a large vesicular Syb2 pool maintained by AP180 is crucial to sustain efficient neurotransmission and SV reformation.

SDmixer-a versatile software tool for spectral demixing of multicolor single molecule localization data.

Spectral demixing (SD) offers multicolor single molecule localization microscopy (SMLM) with low crosstalk and without the need to correct for registration errors. Here, we present SDmixer, a versatile, open-source software tool that enables any laboratory to perform rapid SD-based multicolor SMLM. A graphic user interface allows non-experts to process 2D or 3D data sets from any SML software and to reconstruct the super-resolved multicolor images with flexible output options.

Spectral demixing avoids registration errors and reduces noise in multicolor localization-based super-resolution microscopy.

Multicolor single molecule localization-based super-resolution microscopy (SMLM) approaches are challenged by channel crosstalk and errors in multi-channel registration. We recently introduced a spectral demixing-based variant of direct stochastic optical reconstruction microscopy (SD-dSTORM) to perform multicolor SMLM with minimal color crosstalk. Here, we demonstrate that the spectral demixing procedure is inherently free of errors in multicolor registration and therefore does not require multicolor channel alignment. Furthermore, spectral demixing significantly reduces single molecule noise and is applicable to astigmatism-based 3D multicolor imaging achieving 25 nm lateral and 66 nm axial resolution on cellular nanostructures.