Super-resolution fluorescence microscopy is distinct among nanoscale imaging tools in its ability to image protein dynamics in living cells. Structured illumination microscopy (SIM) stands out in this regard because of its high speed and low illumination intensities, but typically offers only a twofold resolution gain. We extended the resolution of live-cell SIM through two approaches: ultrahigh numerical aperture SIM at 84-nanometer lateral resolution for more than 100 multicolor frames, and nonlinear SIM with patterned activation at 45- to 62-nanometer resolution for approximately 20 to 40 frames. We applied these approaches to image dynamics near the plasma membrane of spatially resolved assemblies of clathrin and caveolin, Rab5a in early endosomes, and a-actinin, often in relationship to cortical actin. In addition, we examined mitochondria, actin, and the Golgi apparatus dynamics in three dimensions.

Monomeric (m)Eos2 is an engineered photoactivatable fluorescent protein widely used for super-resolution microscopy. We show that mEos2 forms oligomers at high concentrations and forms aggregates when labeling membrane proteins, limiting its application as a fusion partner. We solved the crystal structure of tetrameric mEos2 and rationally designed improved versions, mEos3.1 and mEos3.2, that are truly monomeric, are brighter, mature faster and exhibit higher photon budget and label density.

UNC-13-Munc13s play a central function in synaptic vesicle priming through their MUN domains. However, it is unclear whether this function arises from the ability of the MUN domain to mediate the transition from the Munc18-1–closed syntaxin-1 complex to the SNARE complex in vitro. The crystal structure of rat Munc13-1 MUN domain now reveals an elongated, arch-shaped architecture formed by α-helical bundles, with a highly conserved hydrophobic pocket in the middle. Mutation of two residues (NF) in this pocket abolishes the stimulation caused by the Munc13-1 MUN domain on SNARE complex assembly and on SNARE-dependent proteoliposome fusion in vitro. Moreover, the same mutation in UNC-13 abrogates synaptic vesicle priming in C. elegans neuromuscular junctions. These results strongly support the notion that orchestration of syntaxin-1 opening and SNARE complex assembly underlies the central role of UNC-13-Munc13s in synaptic vesicle priming.