Opioid receptors are important pharmacological targets for the management of numerous medical conditions (eg, severe pain), but they are also the gateway to the development of deleterious side effects (eg, opiate addiction). Opioid receptor signaling cascades are well characterized. However, quantitative information regarding their lateral dynamics and nanoscale organization in the plasma membrane remains limited. Since these dynamic properties are important determinants of receptor function, it is crucial to define them. Herein, the nanoscale lateral dynamics and spatial organization of kappa opioid receptor (KOP), wild type mu opioid receptor (MOPwt), and its naturally occurring isoform (MOPN40D) were quantitatively characterized using fluorescence correlation spectroscopy and photoactivated localization microscopy. Obtained results, supported by ensemble‐averaged Monte Carlo simulations, indicate that these opioid receptors dynamically partition into different domains. In particular, significant exclusion from GM1 ganglioside‐enriched domains and partial association with cholesterol‐enriched domains was observed. Nanodomain size, receptor population density and the fraction of receptors residing outside of nanodomains were receptor‐specific. KOP‐containing domains were the largest and most densely populated, with the smallest fraction of molecules residing outside of nanodomains. The opposite was true for MOPN40D. Moreover, cholesterol depletion dynamically regulated the partitioning of KOP and MOPwt, whereas this effect was not observed for MOPN40D.

All breast cancers are assessed for levels of human epidermal growth factor receptor 2 (HER2). Fluorescence in situ hybridization (FISH) and immunohistochemistry are currently used to determine if a patient is eligible for anti-HER2 therapy. Limitations of both tests include variability and relatively long processing times. Additionally, neither test determines whether HER2 contains the extracellular domain. While truncated in some tumors, this domain is required for binding of the therapeutic antibody trastuzumab. Here, trastuzumab was used to directly detect HER2 with quantitative single molecule localization microscopy (qSMLM). In proof of concept studies, our new method rapidly quantified both HER2 density and features of nano-organization. In cultured cells, the method was sensitive to subtle variations in HER2 expression. To assess patient samples, we combined qSMLM with tissue touch preparation (touch prep-qSMLM) and examined large areas of intact membranes. For cell lines and patient samples, HER2 copy numbers from FISH showed a significant positive correlation with detected densities from qSMLM and trended with HER2 cluster occupancy.

BackgroundEthanol affects the signaling of several important neurotransmitter and neuromodulator systems in the CNS. It has been recently proposed that ethanol alters the dynamic lateral organization of proteins and lipids in the plasma membrane, thereby affecting surface receptor-mediated cellular signaling. Our aims are to establish whether pharmacologically relevant levels of ethanol can affect the lateral organization of plasma membrane and cytoskeletal proteins at the nanoscopic level, and investigate the relevance of such perturbations for mu-opioid receptor (MOP) function.Methodology/Principal FindingsWe used Photoactivated Localization Microscopy with pair-correlation analysis (pcPALM), a quantitative fluorescence imaging technique with high spatial resolution (15–25 nm) and single-molecule sensitivity, to study ethanol effects on protein organization in the plasma membrane. We observed that short (20 min) exposure to 20 and 40 mM ethanol alters protein organization in the plasma membrane of cells that harbor endogenous MOPs, causing a rearrangement of the lipid raft marker glycosylphosphatidylinositol (GPI). These effects could be largely occluded by pretreating the cells with the MOP antagonist naltrexone (200 nM for 3 hours). In addition, ethanol induced pronounced actin polymerization, leading to its partial co-localization with GPI.Conclusions/SignificancePharmacologically relevant levels of ethanol alter the lateral organization of GPI-linked proteins and induce actin cytoskeleton reorganization. Pretreatment with the MOP antagonist naltrexone is protective against ethanol action and significantly reduces the extent to which ethanol remodels the lateral organization of lipid-rafts-associated proteins in the plasma membrane. Super-resolution pcPALM reveals details of ethanol action at the nanoscale level, giving new mechanistic insight on the cellular and molecular mechanisms of its action.

Quantitative single molecule localization microscopy (qSMLM) is a powerful approach to study in situ protein organization. However, uncertainty regarding the photophysical properties of fluorescent reporters can bias the interpretation of detected localizations and subsequent quantification. Furthermore, strategies to efficiently detect endogenous proteins are often constrained by label heterogeneity and reporter size. Here, a new surface assay for molecular isolation (SAMI) was developed for qSMLM and used to characterize photophysical properties of fluorescent proteins and dyes. SAMI-qSMLM afforded robust quantification. To efficiently detect endogenous proteins, we used fluorescent ligands that bind to a specific site on engineered antibody fragments. Both the density and nano-organization of membrane-bound epidermal growth factor receptors (EGFR, HER2, and HER3) were determined by a combination of SAMI, antibody engineering, and pair-correlation analysis. In breast cancer cell lines, we detected distinct differences in receptor density and nano-organization upon treatment with therapeutic agents. This new platform can improve molecular quantification and can be developed to study the local protein environment of intact cells.