During intracellular membrane trafficking, N-ethylmaleimide-sensitive factor (NSF) and alpha-soluble NSF attachment protein (α-SNAP) disassemble the soluble NSF attachment protein receptor (SNARE) complex for recycling of the SNARE proteins. The molecular mechanism by which NSF disassembles the SNARE complex is largely unknown. Using single-molecule fluorescence spectroscopy and magnetic tweezers, we found that NSF disassembled a single SNARE complex in only one round of adenosine triphosphate (ATP) turnover. Upon ATP cleavage, the NSF hexamer developed internal tension with dissociation of phosphate ions. After latent time measuring tens of seconds, NSF released the built-up tension in a burst within 20 milliseconds, resulting in disassembly followed by immediate release of the SNARE proteins. Thus, NSF appears to use a “spring-loaded” mechanism to couple ATP hydrolysis and unfolding of substrate proteins.
Calling out, a phenomenon where people publicly broadcast their critiques of someone to a larger audience using, has become increasingly common on social media. However, there has been concerns that it could develop into harassment, deteriorating the quality of public discourse by over-punishing individuals for minor transgressions. To investigate this phenomenon, we interviewed 32 Twitter users who had been called out, had called out, or had witnessed a calling out on Twitter. We found that a key determining factor that distinguishes criticism from harassment was the callee's ability to respond to or engage with the criticism, and that different stakeholders hold different perspectives toward how online harassment is defined. We also discovered that the distinction between callers and callees was not absolute, and that there was high interchangeability of roles both within and across events. Through these findings, we discuss design implications for the platform in promoting healthy discourse while preventing toxic behavior on social media.
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