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Abstract The Golgi apparatus is a central hub for protein trafficking and signaling, yet its rapid imaging and cell-selective disruption remain challenging. Here, we report cycling molecular assemblies (CyMA) for fast Golgi imaging and cell-selective interference. CyMA precursors are acetylated amphiphilic thiopeptides that traverse plasma membrane and are deacetylated by intracellular thioesterases. This exposes thiols that undergo palmitoylation by Golgi-resident palmitoyl acyltransferases utilizing palmitoyl-CoA. The resulting palmitoylated peptides self-assemble into dynamic nanostructures (i.e., CyMA) localized at the Golgi. Their continuous, reversibleS-acylation enables near-instantaneous Golgi imaging. Replacing fluorophore with a biphenyl motif promotes CyMA accumulation and disrupts functions such as protein modifications, trafficking, and secretion, leading to cell death. This study establishes dynamic supramolecular assembly as an active and selective strategy for Golgi-targeting, pleiotropically interfering with Golgi functions, which may be applicable to targeting other organelles by utilizing alternative enzyme switches to enable kinetic trapping. 

Under super-resolution imaging of probe PCV-1, we developed a new analytical assay named organelle ratiometric probing (ORP), which has successfully achieved quantitative analysis and efficient assessment of the viability of individual cells. 

Single-molecule FRET (smFRET) has become a mainstream technique for studying biomolecular structural dynamics. The rapid and wide adoption of smFRET experiments by an ever-increasing number of groups has generated significant progress in sample preparation, measurement procedures, data analysis, algorithms and documentation. Several labs that employ smFRET approaches have joined forces to inform the smFRET community about streamlining how to perform experiments and analyze results for obtaining quantitative information on biomolecular structure and dynamics. The recent efforts include blind tests to assess the accuracy and the precision of smFRET experiments among different labs using various procedures. These multi-lab studies have led to the development of smFRET procedures and documentation, which are important when submitting entries into the archiving system for integrative structure models, PDB-Dev. This position paper describes the current ‘state of the art’ from different perspectives, points to unresolved methodological issues for quantitative structural studies, provides a set of ‘soft recommendations’ about which an emerging consensus exists, and lists openly available resources for newcomers and seasoned practitioners. To make further progress, we strongly encourage ‘open science’ practices.