A major role of cell membranes is to provide an ideal environment for the constituent proteins to perform their biological functions. A deep understanding of the membrane proteins assembly process under physiological conditions is quite important to elucidate both the structure and the function of the cell membranes. Along these lines, in this work, a complete workflow of the cell membrane sample preparation and the correlated AFM and dSTORM imaging analysis methods are presented. A specially designed, angle-controlled sample preparation device was used to prepare the cell membrane samples. The correlated distributions of the specific membrane proteins with the topography of the cytoplasmic side of the cell membranes can be obtained by performing correlative AFM and dSTORM measurements. These methods are ideal for systematically studying the structure of the cell membranes. The proposed method of the sample characterization was not only limited to the measurement of the cell membrane but also can be applied for both biological tissue section analysis and detection.
Fluorescence microscopy and electron microscopy complement each other as the former provides labelling and localisation of specific molecules and target structures while the latter possesses excellent revolving power of fine structure in context. These two techniques can combine as correlative light and electron microscopy (CLEM) to reveal the organisation of materials within the cell. Frozen hydrated sections allow microscopic observations of cellular components in situ in a near-native state and are compatible with superresolution fluorescence microscopy and electron tomography if sufficient hardware and software support is available and a well-designed protocol is followed. The development of superresolution fluorescence microscopy greatly increases the precision of fluorescence annotation of electron tomograms. Here, we provide detailed instructions on how to perform cryogenic superresolution CLEM on vitreous sections. From fluorescence-labelled cells to high pressure freezing, cryo-ultramicrotomy, cryogenic single-molecule localisation microscopy, cryogenic electron tomography and image registration, electron tomograms with features of interest highlighted by superresolution fluorescence signals are expected to be obtained.
Temperature-sensitive ion channels, such as those from the TRP family (thermo-TRPs) present in all animal cells, serve to perceive heat and cold sensations. A considerable number of protein structures have been reported for these ion channels, providing a solid basis for revealing their structure–function relationship. Previous functional studies suggest that the thermosensing ability of TRP channels is primarily determined by the properties of their cytosolic domain. Despite their importance in sensing and wide interests in the development of suitable therapeutics, the precise mechanisms underlying acute and steep temperature-mediated channel gating remain enigmatic. Here, we propose a model in which the thermo-TRP channels directly sense external temperature through the formation and dissociation of metastable cytoplasmic domains. An open–close bistable system is described in the framework of equilibrium thermodynamics, and the middle-point temperature T½ similar to the V½ parameter for a voltage-gating channel is defined. Based on the relationship between channel opening probability and temperature, we estimate the change in entropy and enthalpy during the conformational change for a typical thermosensitive channel. Our model is able to accurately reproduce the steep activation phase in experimentally determined thermal-channel opening curves, and thus should greatly facilitate future experimental verification.
The functions of DNA-binding proteins are dependent on protein-induced DNA distortion, the binding preference to special sequences, DNA secondary structures, the binding kinetics and the binding affinity. Recent rapid progress in single-molecule imaging and mechanical manipulation technologies have made it possible to directly probe the DNA binding by proteins, footprint the positions of the bound proteins on DNA, quantify the kinetics and the affinity of protein–DNA interactions, and study the interplay of protein binding with DNA conformation and DNA topology. Here, we review the applications of an integrated approach where the single-DNA imaging using atomic force microscopy and the mechanical manipulation of single DNA molecules are combined to study the DNA–protein interactions. We also provide our views on how these findings yield new insights into understanding the roles of several essential DNA architectural proteins.
Telomere DNA assumes a high-order G-quadruplex (G4) structure, stabilization of which prevents telomere lengthening by telomerase in cancer. Through applying combined molecular simulation methods, an investigation on the selective binding mechanism of anionic phthalocyanine 3,4ʹ,4ʹʹ,4ʹʹʹ-tetrasulfonic acid (APC) and human hybrid (3 + 1) G4s was firstly performed at the atomic level. Compared to the groove binding mode of APC and the hybrid type I (hybrid-I) telomere G4, APC preferred to bind to the hybrid type II (hybrid-II) telomere G4 via end-stacking interactions, which showed much more favorable binding free energies. Analyses of the non-covalent interaction and binding free energy decomposition revealed a decisive role of van der Waals interaction in the binding of APC and telomere hybrid G4s. And the binding of APC and hybrid-II G4 that showed the highest binding affinity adopted the end-stacking binding mode to form the most extensive van der Waals interactions. These findings add new knowledge to the design of selective stabilizers targeting telomere G4 in cancer.
