Advanced fluorescence microscopy including single-molecule localization-based super-resolution imaging techniques requires bright and photostable dyes orproteins asfluorophores.The photophysical properties of fluoroph...Advanced fluorescence microscopy including single-molecule localization-based super-resolution imaging techniques requires bright and photostable dyes orproteins asfluorophores.The photophysical properties of fluorophores have been proven to be crucial for super-resolution microscopy's localization precision and imaging resolution.Fluorophores TAMRA and Atto Rho6 G,which can interact with macrocyclic host cucurbit[7]uril(CB7) to form host-vip compounds,were found to improve the fluorescence intensity and lifetimes of these dyes.We enhanced the localization precision of direct stochastic optical reconstruction microscopy(dSTORM) by introducing CB7 into the imaging buffer,and showed that the number of photons as well as localizations of both TAMRA and Atto Rho6 G increase over 2 times.展开更多
Most plasmalemmal proteins are organized into clusters to modulate various cellular functions.However,the machineries that regulate protein clustering remain largely unclear.Here,with EGFR as an example,we directly an...Most plasmalemmal proteins are organized into clusters to modulate various cellular functions.However,the machineries that regulate protein clustering remain largely unclear.Here,with EGFR as an example,we directly and in detail visualized the entire process of EGFR from synthesis to secretion onto the plasma membrane(PM)using a high-speed,high-resolution spinning-disk confocal microscope.First,colocalization imaging revealed that EGFR secretory vesicles underwent transport from the ER to the Golgi to the PM,eventually forming different distribution forms on the apical and basal membranes;that is,most EGFR formed larger clusters on the apical membrane than the basal membrane.A dynamic tracking image and further siRNA interference experiment confirmed that fusion of secretory vesicles with the plasma membrane led to EGFR clusters,and we showed that EGFR PM clustering may be intimately related to EGFR signaling and cell proliferation.Finally,we found that the size and origin of the secretory vesicles themselves may determine the difference in the distribution patterns of EGFR on the PM.More importantly,we showed that actin influenced the EGFR distribution by controlling the fusion of secretory vesicles with the PM.Collectively,a comprehensive understanding of the EGFR secretion process helps us to unravel the EGFR clustering process and elucidate the key factors determining the differences in the spatial distribution of EGFR PM,highlighting the correlation between EGFR secretion and its PM distribution pattern.展开更多
基金supported by the National Natural Science Foundation of China(31330082,21373200,21525314)the Instrument Developing Project of the Chinese Academy of Sciences(YZ201455)
文摘Advanced fluorescence microscopy including single-molecule localization-based super-resolution imaging techniques requires bright and photostable dyes orproteins asfluorophores.The photophysical properties of fluorophores have been proven to be crucial for super-resolution microscopy's localization precision and imaging resolution.Fluorophores TAMRA and Atto Rho6 G,which can interact with macrocyclic host cucurbit[7]uril(CB7) to form host-vip compounds,were found to improve the fluorescence intensity and lifetimes of these dyes.We enhanced the localization precision of direct stochastic optical reconstruction microscopy(dSTORM) by introducing CB7 into the imaging buffer,and showed that the number of photons as well as localizations of both TAMRA and Atto Rho6 G increase over 2 times.
基金supported by the National Natural Science Foundation of China(nos.22150003,21727816 and 21721003)the Scientific Instrument Developing Project of the Chinese Academy of Sciences(ZDKYYQ20220005)the Foundation of Science and Technology Department of Jilin Province(YDZJ202201ZYTS530).
文摘Most plasmalemmal proteins are organized into clusters to modulate various cellular functions.However,the machineries that regulate protein clustering remain largely unclear.Here,with EGFR as an example,we directly and in detail visualized the entire process of EGFR from synthesis to secretion onto the plasma membrane(PM)using a high-speed,high-resolution spinning-disk confocal microscope.First,colocalization imaging revealed that EGFR secretory vesicles underwent transport from the ER to the Golgi to the PM,eventually forming different distribution forms on the apical and basal membranes;that is,most EGFR formed larger clusters on the apical membrane than the basal membrane.A dynamic tracking image and further siRNA interference experiment confirmed that fusion of secretory vesicles with the plasma membrane led to EGFR clusters,and we showed that EGFR PM clustering may be intimately related to EGFR signaling and cell proliferation.Finally,we found that the size and origin of the secretory vesicles themselves may determine the difference in the distribution patterns of EGFR on the PM.More importantly,we showed that actin influenced the EGFR distribution by controlling the fusion of secretory vesicles with the PM.Collectively,a comprehensive understanding of the EGFR secretion process helps us to unravel the EGFR clustering process and elucidate the key factors determining the differences in the spatial distribution of EGFR PM,highlighting the correlation between EGFR secretion and its PM distribution pattern.
