Synaptic proteins promote calcium-triggered fast transition from point contact to full fusion.

The molecular underpinnings of synaptic vesicle fusion for fast neurotransmitter release are still unclear. Here, we used a single vesicle-vesicle system with reconstituted SNARE and synaptotagmin-1 proteoliposomes to decipher the temporal sequence of membrane states upon Ca(2+)-injection at 250-500 μM on a 100-ms timescale. Furthermore, detailed membrane morphologies were imaged with cryo-electron microscopy before and after Ca(2+)-injection. We discovered a heterogeneous network of immediate and delayed fusion pathways. Remarkably, all instances of Ca(2+)-triggered immediate fusion started from a membrane-membrane point-contact and proceeded to complete fusion without discernible hemifusion intermediates. In contrast, pathways that involved a stable hemifusion diaphragm only resulted in fusion after many seconds, if at all. When complexin was included, the Ca(2+)-triggered fusion network shifted towards the immediate pathway, effectively synchronizing fusion, especially at lower Ca(2+)-concentration. Synaptic proteins may have evolved to select this immediate pathway out of a heterogeneous network of possible membrane fusion pathways.DOI:http://dx.doi.org/10.7554/eLife.00109.001

Smartphone-based image analysis and chemometric recognition of the thin-layer chromatographic fingerprints of herbal materials

Thin-layer chromatography (TLC) is commonly used as a screening method to verify the identity and quality of dried herbal medicinal plant material. While TLC is relatively simple, the method still requires technical experience and relies on the subjective classification of sample TLC profiles as “within-specifications” or “off-specifications.” In this work, we report the development of an objective TLC-based system for the identification and quality assessment of herbal medicinal materials. Our proposed system is a miniaturized Pharmacopeia-based TLC method coupled with a smartphone app that allows for an objective interpretation of TLC profiles via multivariate image analysis and chemometric fingerprinting. An image of the TLC profile is captured using a smartphone camera interfaced with a 3D-printed photo-box, and the analysis is automated using a framework of pre-uploaded algorithms hosted on a cloud server. The TLC profile image is converted to an unfolded red, green, and blue (RGB) channel intensity profile, and classified as “within-specifications” or “off-specifications” using aggregated Soft Independent Modeling of Class Analogy (SIMCA) models. We present the application of our system to two herbal medicinal plants, Blumea balsamifera and Vitex negundo. The proposed system demonstrates 90.2% sensitivity and 86.2% specificity for B. balsamifera classification, and 81.4% sensitivity and 92.0% specificity for V. negundo classification when compared to the respective laboratory-based Pharmacopeia TLC protocols for the ability to distinguish authentic samples from non-authentic and degraded samples. The system developed in this work is a cost-effective, rapid method that can serve as a herbal material quality assessment tool in resource-limited settings

Synaptic proteins promote calcium-triggered fast transition from point contact to full fusion.

The molecular underpinnings of synaptic vesicle fusion for fast neurotransmitter release are still unclear. Here, we used a single vesicle-vesicle system with reconstituted SNARE and synaptotagmin-1 proteoliposomes to decipher the temporal sequence of membrane states upon Ca(2+)-injection at 250-500 μM on a 100-ms timescale. Furthermore, detailed membrane morphologies were imaged with cryo-electron microscopy before and after Ca(2+)-injection. We discovered a heterogeneous network of immediate and delayed fusion pathways. Remarkably, all instances of Ca(2+)-triggered immediate fusion started from a membrane-membrane point-contact and proceeded to complete fusion without discernible hemifusion intermediates. In contrast, pathways that involved a stable hemifusion diaphragm only resulted in fusion after many seconds, if at all. When complexin was included, the Ca(2+)-triggered fusion network shifted towards the immediate pathway, effectively synchronizing fusion, especially at lower Ca(2+)-concentration. Synaptic proteins may have evolved to select this immediate pathway out of a heterogeneous network of possible membrane fusion pathways.DOI:http://dx.doi.org/10.7554/eLife.00109.001

Complexin‑1 Enhances the On-Rate of Vesicle Docking via Simultaneous SNARE and Membrane Interactions

In synaptic terminals, complexin is thought to have inhibitory and activating roles for spontaneous “mini” release and evoked synchronized neurotransmitter release, respectively. We used single vesicle–vesicle microscopy imaging to study the effect of complexin-1 on the on-rate of docking between vesicles that mimic synaptic vesicles and the plasma membrane. We found that complexin-1 enhances the on-rate of docking of synaptic vesicle mimics containing full-length synaptobrevin-2 and full-length synaptotagmin-1 to plasma membrane-mimicking vesicles containing full-length syntaxin-1A and SNAP-25A. This effect requires the C-terminal domain of complexin-1, which binds to the membrane, the presence of PS in the membrane, and the core region of complexin-1, which binds to the SNARE complex