分类: 生物学 >> 生物物理学 >> 生物物理、生物化学与分子生物学 提交时间: 2016-05-12
摘要: UNC-13-Munc13s have 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 the rat Munc13-1 MUN domain now reveals an elongated, arch-shaped architecture formed by a-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 Caenorhabditis elegans neuromuscular junctions. These results 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.
分类: 生物学 >> 生物物理学 >> 生物物理、生物化学与分子生物学 提交时间: 2016-05-11
摘要: Background: Phosphatidylinositides in the plasma membrane (PM) are pivotal for cellular functions. Results: Superresolution imaging reveals homogeneous distribution of PI(4,5)P-2, PI4P, and PI(3,4,5)P-3 in the major area of the PM. Conclusion: Phosphatidylinositides detected by PH domains are uniformly distributed in the major regions of the PM, with limited concentration gradients. Significance: This result may imply a new working model of phosphatidylinositides at nanometer scale. Both phosphatidylinositol 4-phosphate (PI4P) and phosphatidylinositol 4,5-bisphosphate (PI(4,5)P-2) are independent plasma membrane (PM) determinant lipids that are essential for multiple cellular functions. However, their nanoscale spatial organization in the PM remains elusive. Using single-molecule superresolution microscopy and new photoactivatable fluorescence probes on the basis of pleckstrin homology domains that specifically recognize phosphatidylinositides in insulin-secreting INS-1 cells, we report that the PI(4,5)P-2 probes exhibited a remarkably uniform distribution in the major regions of the PM, with some sparse PI(4,5)P-2-enriched membrane patches/domains of diverse sizes (383 +/- 14 nm on average). Quantitative analysis revealed a modest concentration gradient that was much less steep than previously thought, and no densely packed PI(4,5)P-2 nanodomains were observed. Live-cell superresolution imaging further demonstrated the dynamic structural changes of those domains in the flat PM and membrane protrusions. PI4P and phosphatidylinositol (3,4,5)-trisphosphate (PI(3,4,5)P-3) showed similar spatial distributions as PI(4,5)P-2. These data reveal the nanoscale landscape of key inositol phospholipids in the native PM and imply a framework for local cellular signaling and lipid-protein interactions at a nanometer scale.