Pyrene Excimer-Based Fluorescent Labeling of Cysteines Brought into Close Proximity by Protein Dynamics: ASEM-Induced Thiol-Ene Click Reaction for High Spatial Resolution CLEM

Abstract

Fluorescence microscopy (FM) has revealed vital molecular mechanisms of life. Mainly, molecules labeled by fluorescent probes are imaged. However, the diversity of labeling probes and their functions remain limited. We synthesized a pyrene-based fluorescent probe targeting SH groups, which are important for protein folding and oxidative stress sensing in cells. The labeling achieved employs thiol-ene click reactions between the probes and SH groups and is triggered by irradiation by UV light or an electron beam. When two tagged pyrene groups were close enough to be excited as a dimer (excimer), they showed red-shifted fluorescence; theoretically, the proximity of two SH residues within ~30 Å can thus be monitored. Moreover, correlative light/electron microscopy (CLEM) was achieved using our atmospheric scanning electron microscope (ASEM); radicals formed in liquid by the electron beam caused the thiol-ene click reactions, and excimer fluorescence of the labeled proteins in cells and tissues was visualized by FM. Since the fluorescent labeling is induced by a narrow electron beam, high spatial resolution labeling is expected. The method can be widely applied to biological fields, for example, to study protein dynamics with or without cysteine mutagenesis, and to beam-induced micro-fabrication and the precise post-modification of materials.

Ca2+-ATPase Molecules as a Calcium-Sensitive Membrane-Endoskeleton of Sarcoplasmic Reticulum

“…For the further analyses of the precise movement of the ATPase molecules, we are planning to perform statistical image analysis based on machine learning in combination with cryo-electron microscopy (cryo-EM) [ 49 ] and liquid-phase immuno-EM including the use of the atmospheric scanning electron microscope (ASEM) we have developed [ 50 , 51 , 52 , 53 , 54 ].…”

Ca2+ Dependent Formation/Collapse of Cylindrical Ca2+-ATPase Crystals in Scallop Sarcoplasmic Reticulum (SR) Vesicles: A Possible Dynamic Role of SR in Regulation of Muscle Contraction

“…Since cracks are created when a solid 3D structure is crushed by a large pressure, these results prompt the hypothesis that stable ATPase crystal patches gather close to one another to form a tight assembly, transforming the vesicle into a 3D spherical form. However, when the liquid droplet containing the SR vesicles was dried on the carbon film of a TEM grid, the huge water surface tension pressed and crushed the spherical 3D structure of the SR, as suggested by the crushing of filamentous palladium networks [ 14 ] dynamically monitored using our developed Atmospheric SEM (ASEM) [ 15 , 16 , 17 , 18 ] as the water droplet containing them dried.…”

Biofilm Spreading by the Adhesin-Dependent Gliding Motility of Flavobacterium johnsoniae. 1. Internal Structure of the Biofilm

“…Consequently, the thick fiber-containing EPM might include double-stranded DNA filaments and extracellular polysaccharides, as observed in the biofilms of S. aureus and P. acnes immersed in aqueous liquid by atmospheric scanning electron microscopy (ASEM) [45,46]. Further experiments are required to understand the water-rich architecture of F. johnsoniae biofilms, for example, using ASEM that enabled observations of various water-rich phenomena of organic and inorganic substances at high resolution [16,51,52]. The undulations observed for the layered surface coats of F. johnsoniae cells in biofilm has a possibility to be artefacts, due to the dehydration pretreatment of the Epon-embedded TEM.…”