G-protein-coupled receptors (GPCRs) and G-proteins have a range of roles in many physiological and pathological processes and are among the most studied signaling proteins. A plethora of extracellular stimuli can activate the GPCR and can elicit distinct intracellular responses through the activation of specific transduction pathways. For many years, biologists thought that GPCR signaling occurred entirely on the plasma membrane. However, in recent decades, many lines of evidence have proved that the GPCRs and G-proteins may reside on endomembranes and can start or propagate signaling pathways through the organelles that form the secretory route. How these alternative intracellular signaling pathways of the GPCR and G-proteins influence the physiological and pathological function of the endomembranes is still under investigation. Here, we review the general role and classification of GPCRs and G-proteins with a focus on their signaling pathways in the membrane transport apparatus.

No abstract

Currently, a major technical limitation of microscopy based image analysis is the linkage error – which describes the distance between e.g. the target epitope of cellular protein to the fluorescence emitter, which position is finally detected in a microscope. With continuously improving resolution of today′s (super–resolution) microscopes, the linkage errors can severely hamper the correct interpretation of images and is usually introduced in experiments by the use of standard intracellular staining reagents such as fluorescently labelled antibodies. The linkage error of standard labelled antibodies is caused by the size of the antibody and the random distribution of fluorescent emitters on the antibody surface. Together, these two factors account for a fluorescence displacement of ~40nm when staining proteins by indirect immunofluorescence; and ~20nm when staining with fluorescently coupled primary antibodies. In this study, we describe a class of staining reagents that effectively reduce the linkage error by more than five–fold when compared to conventional staining techniques. These reagents, called Fluo–N–Fabs, consist of an antigen binding fragment of a full-length antibody (Fab / fragment antigen binding) that is selectively conjugated at the N-terminal amino group with fluorescent organic molecules, thereby reducing the distance between the fluorescent emitter and the protein target of the analysis. Fluo–N–Fabs also exhibit the capability to penetrate tissues and highly crowded cell compartments, thus allowing for the efficient detection of cellular epitopes of interest in a wide range of fixed samples. We believe this class of reagents realize an unmet need in cell biological super resolution imaging studies where the precise localization of the target of interest is crucial for the understanding of complex biological phenomena.