Munc13-1 acts as a master regulator of neurotransmitter release, mediating docking-priming of synaptic vesicles and diverse presynaptic plasticity processes. It is unclear how the functions of the multiple domains of Munc13-1 are coordinated. The crystal structure of a Munc13-1 fragment including its C1, C2B and MUN domains (C1C2BMUN) reveals a 19.5 nm-long multi-helical structure with the C1 and C2B domains packed at one end. The similar orientations of the respective diacyglycerol- and Ca2+-binding sites of the C1 and C2B domains suggest that the two domains cooperate in plasma-membrane binding and that activation of Munc13-1 by Ca2+ and diacylglycerol during short-term presynaptic plasticity are closely interrelated. Electrophysiological experiments in mouse neurons support the functional importance of the domain interfaces observed in C1C2BMUN. The structure imposes key constraints for models of neurotransmitter release and suggests that Munc13-1 bridges the vesicle and plasma membranes from the periphery of the membrane-membrane interface.
- Josep Rizo
- Josep Rizo
- Christian Rosenmund
- Christian Rosenmund
The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.
Animal experimentation: Animal welfare committees of Charité Medical University and the Berlin state government Agency for Health and Social Services approved all protocols for animal maintenance and experiments (license no. T 0220/09).
- Reinhard Jahn, Max Planck Institute for Biophysical Chemistry, Germany
© 2017, Xu et al.
This article is distributed under the terms of the Creative Commons Attribution License permitting unrestricted use and redistribution provided that the original author and source are credited.
Previously we showed that 2D template matching (2DTM) can be used to localize macromolecular complexes in images recorded by cryogenic electron microscopy (cryo-EM) with high precision, even in the presence of noise and cellular background (Lucas et al., 2021; Lucas et al., 2022). Here, we show that once localized, these particles may be averaged together to generate high-resolution 3D reconstructions. However, regions included in the template may suffer from template bias, leading to inflated resolution estimates and making the interpretation of high-resolution features unreliable. We evaluate conditions that minimize template bias while retaining the benefits of high-precision localization, and we show that molecular features not present in the template can be reconstructed at high resolution from targets found by 2DTM, extending prior work at low-resolution. Moreover, we present a quantitative metric for template bias to aid the interpretation of 3D reconstructions calculated with particles localized using high-resolution templates and fine angular sampling.
To fire action-potential-like electrical signals, the vacuole membrane requires the two-pore channel TPC1, formerly called SV channel. The TPC1/SV channel functions as a depolarization-stimulated, non-selective cation channel that is inhibited by luminal Ca2+. In our search for species-dependent functional TPC1 channel variants with different luminal Ca2+ sensitivity, we found in total three acidic residues present in Ca2+ sensor sites 2 and 3 of the Ca2+-sensitive AtTPC1 channel from Arabidopsis thaliana that were neutral in its Vicia faba ortholog and also in those of many other Fabaceae. When expressed in the Arabidopsis AtTPC1-loss-of-function background, wild-type VfTPC1 was hypersensitive to vacuole depolarization and only weakly sensitive to blocking luminal Ca2+. When AtTPC1 was mutated for these VfTPC1-homologous polymorphic residues, two neutral substitutions in Ca2+ sensor site 3 alone were already sufficient for the Arabidopsis At-VfTPC1 channel mutant to gain VfTPC1-like voltage and luminal Ca2+ sensitivity that together rendered vacuoles hyperexcitable. Thus, natural TPC1 channel variants exist in plant families which may fine-tune vacuole excitability and adapt it to environmental settings of the particular ecological niche.