Munc13-1 is a large multifunctional protein essential for synaptic vesicle fusion and neurotransmitter release. Its dysfunction has been linked to many neurological disorders. Evidence suggests that the MUN domain of Munc13-1 collaborates with Munc18-1 to initiate SNARE assembly, thereby priming vesicles for fast calcium-triggered vesicle fusion. The underlying molecular mechanism, however, is poorly understood. Recently, it was found that Munc18-1 catalyzes neuronal SNARE assembly through an obligate template complex intermediate containing Munc18-1 and 2 SNARE proteins—syntaxin 1 and VAMP2. Here, using single-molecule force spectroscopy, we discovered that the MUN domain of Munc13-1 stabilizes the template complex by ∼2.1 kBT. The MUN-bound template complex enhances SNAP-25 binding to the templated SNAREs and subsequent full SNARE assembly. Mutational studies suggest that the MUN-bound template complex is functionally important for SNARE assembly and neurotransmitter release. Taken together, our observations provide a potential molecular mechanism by which Munc13-1 and Munc18-1 cooperatively chaperone SNARE folding and assembly, thereby regulating synaptic vesicle fusion.

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Synaptic vesicle fusion is mediated by SNARE proteins—VAMP2 on the vesicle and Syntaxin‐1/SNAP25 on the presynaptic membrane. Chaperones Munc18‐1 and Munc13‐1 cooperatively catalyze SNARE assembly via an intermediate ‘template’ complex containing Syntaxin‐1 and VAMP2. How SNAP25 enters this reaction remains a mystery. Here, we report that Munc13‐1 recruits SNAP25 to initiate the ternary SNARE complex assembly by direct binding, as judged by bulk FRET spectroscopy and single‐molecule optical tweezer studies. Detailed structure–function analyses show that the binding is mediated by the Munc13‐1 MUN domain and is specific for the SNAP25 ‘linker’ region that connects the two SNARE motifs. Consequently, freely diffusing SNAP25 molecules on phospholipid bilayers are concentrated and bound in ~ 1 : 1 stoichiometry by the self‐assembled Munc13‐1 nanoclusters.

The cell interior is highly crowded and far from thermodynamic equilibrium. This environment can dramatically impact molecular motion and assembly, and therefore influence subcellular organization and biochemical reaction rates. These effects depend strongly on length-scale, with the least information available at the important mesoscale (10-100 nanometers), which corresponds to the size of crucial regulatory molecules such as RNA polymerase II. It has been challenging to study the mesoscale physical properties of the nucleoplasm because previous methods were labor-intensive and perturbative. Here, we report nuclear Genetically Encoded Multimeric nanoparticles (nucGEMs). Introduction of a single gene leads to continuous production and assembly of protein-based bright fluorescent nanoparticles of 40 nm diameter. We implemented nucGEMs in budding and fission yeasts and in mammalian cell lines. We found that the nucleus is more crowded than the cytosol at the mesoscale, that mitotic chromosome condensation ejects nucGEMs from the nucleus, and that nucGEMs are excluded from heterochromatin and the nucleolus. nucGEMs enable hundreds of nuclear rheology experiments per hour, and allow evolutionary comparison of the physical properties of the cytosol and nucleoplasm.

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Liquid–liquid phase separation (LLPS) can concentrate biomolecules and accelerate reactions within membraneless organelles. For example, the nucleolus and PML-nuclear bodies are thought to create network hubs by bringing signaling molecules such as kinases and substrates together. However, the mechanisms and principles connecting mesoscale organization to signaling dynamics are difficult to dissect due to the pleiotropic effects associated with disrupting endogenous condensates. Here, we recruited multiple distinct kinases and substrates into synthetic LLPS systems to create new phosphorylation reactions within condensates, and generally found increased activity and broadened specificity. Dynamic phosphorylation within condensates could drive cell-cycle-dependent localization changes. Detailed comparison of phosphorylation of clients with varying recruitment valency and affinity into condensates comprised of either flexible or rigid scaffolds revealed unexpected principles. First, high client concentration within condensates is important, but is not the main factor for efficient multi-site phosphorylation. Rather, the availability of a large number of excess client binding sites, together with a flexible scaffold is crucial. Finally, phosphorylation within a suboptimal, flexible condensate was modulated by changes in macromolecular crowding. Thus, condensates readily generate new signaling connections and can create sensors that respond to perturbations to the biophysical properties of the cytoplasm.

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