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Single-Molecule Imaging of mRNA Localization and Regulation during the Integrated Stress Response
摘要： Biological phase transitions form membrane-less organelles that generate distinct cellular environments. How molecules are partitioned between these compartments and the surrounding cellular space and the functional consequence of this localization is not well understood. Here, we report the localization of mRNA to stress granules (SGs) and processing bodies (PBs) and its effect on translation and degradation during the integrated stress response. Using single mRNA imaging in living human cells, we find that the interactions of mRNAs with SGs and PBs have different dynamics, very few mRNAs directly move between SGs and PBs, and that specific RNA-binding proteins can anchor mRNAs within these compartments. During recovery from stress, we show that mRNAs that were within SGs and PBs are translated and degraded at similar rates as their cytosolic counterparts. Our work provides a framework for using single-molecule measurements to directly investigate the molecular mechanisms of phase-separated compartments within their cellular environment.
关键词： P-bodies,integrated stress response,degradation,stress granules,LARP1,mRNA localization,single-molecule imaging,translation
更新于2025-09-23 15:22:29
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Single-molecule imaging of DNA gyrase activity in living <i>Escherichia coli</i>
摘要： Bacterial DNA gyrase introduces negative supercoils into chromosomal DNA and relaxes positive supercoils introduced by replication and transiently by transcription. Removal of these positive supercoils is essential for replication fork progression and for the overall unlinking of the two duplex DNA strands, as well as for ongoing transcription. To address how gyrase copes with these topological challenges, we used high-speed single-molecule fluorescence imaging in live Escherichia coli cells. We demonstrate that at least 300 gyrase molecules are stably bound to the chromosome at any time, with ～12 enzymes enriched near each replication fork. Trapping of reaction intermediates with ciprofloxacin revealed complexes undergoing catalysis. Dwell times of ～2 s were observed for the dispersed gyrase molecules, which we propose maintain steady-state levels of negative supercoiling of the chromosome. In contrast, the dwell time of replisome-proximal molecules was ～8 s, consistent with these catalyzing processive positive supercoil relaxation in front of the progressing replisome.
关键词： Escherichia coli,transcription,DNA gyrase,replication,supercoiling,single-molecule imaging
更新于2025-09-23 15:21:21
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Characterization of functionalized glass and indium tin oxide surfaces as substrates for super-resolution microscopy
摘要： Modern high-throughput biosensors with sensitivity down to a single analyte molecule may be possible with single-molecule localization microscopy (SMLM). Functionalized surfaces can be fabricated with self-assembly monolayer chemistry on indium tin oxide (ITO) substrates but not glass. However, characterizations of SMLM-compatible fluorophores are primarily performed on glass substrates. Here we collect single-molecule kinetics data of isolated Alexa Fluor 647 molecules on bare and functionalized glass and ITO surfaces. Extracting the photophysical dynamics of the fluorophores allows direct comparison of behavior of this dye on these substrates and fitting data to a model that accounts for multiple reversible dark states. All surfaces had sensitivity sufficient to image single fluorophore molecules. Photophysical kinetics observed are similar between the two substrates. The photon yield from individual fluorophores was greatest on bare glass, but functionalized ITO surfaces showed superior yield to functionalized glass surfaces and nearly matched the yield of bare glass. Together these results indicate functionalized ITO as a promising substrate for modern single-molecule biosensors.
关键词： indium tin oxide,super-resolution microscopy,biosensor,single-molecule imaging
更新于2025-09-09 09:28:46