Super-resolution imaging as a method to study GPCR dimers and higher-order oligomers

The study of G protein-coupled receptor (GPCR) dimers and higher-order oligomers has unveiled mechanisms for receptors to diversify signaling and potentially uncover novel therapeutic targets. The functional and clinical significance of these receptor–receptor associations has been facilitated by the development of techniques and protocols, enabling researchers to unpick their function from the molecular interfaces, to demonstrating functional significance in vivo, in both health and disease. Here we describe our methodology to study GPCR oligomerization at the single-molecule level via super-resolution imaging. Specifically, we have employed photoactivated localization microscopy, with photoactivatable dyes (PD-PALM) to visualize the spatial organization of these complexes to <10 nm resolution, and the quantitation of GPCR monomer, dimer, and oligomer in both homomeric and heteromeric forms. We provide guidelines on optimal sample preparation, imaging parameters, and necessary controls for resolving and quantifying single-molecule data. Finally, we discuss advantages and limitations of this imaging technique and its potential future applications to the study of GPCR function

Targeting RNS/caveolin-1/MMP signaling cascades to protect against cerebral ischemia-reperfusion injuries: potential application for drug discovery

Reactive nitrogen species (RNS) play important roles in mediating cerebral ischemia-reperfusion injury. RNS activate multiple signaling pathways and participate in different cellular events in cerebral ischemia-reperfusion injury. Recent studies have indicated that caveolin-1 and matrix metalloproteinase (MMP) are important signaling molecules in the pathological process of ischemic brain injury. During cerebral ischemia-reperfusion, the production of nitric oxide (NO) and peroxynitrite (ONOO-), two representative RNS, down-regulates the expression of caveolin-1 (Cav-1) and, in turn, further activates nitric oxide synthase (NOS) to promote RNS generation. The increased RNS further induce MMP activation and mediate disruption of the blood-brain barrier (BBB), aggravating the brain damage in cerebral ischemia-reperfusion injury. Therefore, the feedback interaction among RNS/Cav-1/MMPs provides an amplified mechanism for aggravating ischemic brain damage during cerebral ischemia-reperfusion injury. Targeting the RNS/Cav-1/MMP pathway could be a promising therapeutic strategy for protecting against cerebral ischemia-reperfusion injury. In this mini-review article, we highlight the important role of the RNS/Cav-1/MMP signaling cascades in ischemic stroke injury and review the current progress of studies seeking therapeutic compounds targeting the RNS/Cav-1/MMP signaling cascades to attenuate cerebral ischemia-reperfusion injury. Several representative natural compounds, including calycosin-7-O-β-D-glucoside, baicalin, Momordica charantia polysaccharide (MCP), chlorogenic acid, lutein and lycopene, have shown potential for targeting the RNS/Cav-1/MMP signaling pathway to protect the brain in ischemic stroke. Therefore, the RNS/Cav-1/MMP pathway is an important therapeutic target in ischemic stroke treatment.published_or_final_versio

Expansion microscopy: principles and uses in biological research

Many biological investigations require 3D imaging of cells or tissues with nanoscale spatial resolution. We recently discovered that preserved biological specimens can be physically expanded in an isotropic fashion through a chemical process. Expansion microscopy (ExM) allows nanoscale imaging of biological specimens with conventional microscopes, decrowds biomolecules in support of signal amplification and multiplexed readout chemistries, and makes specimens transparent. We review the principles of how ExM works, advances in the technology made by our group and others, and its applications throughout biology and medicine.NIH (Grants 1R01NS102727, 1R01EB024261, 1R41MH112318, 1R01MH110932, 1RM1HG008525, 1DP1NS087724)NSF (Grant 1734870)IARPA (Grant D16PC00008)US Army Research Laboratory & US Army Research Office (Contract W911NF1510548)US–Israel Binational Science Foundation (Grant 2014509

Insights into Mechanisms of Blood-Brain Barrier Permeability – Roles of Free Radicals, Matrix Metalloproteinsases, and Caveolin-1

Free radicals, including reactive oxygen species (ROS) and reactive nitrogen species (RNS), are important mediators in cerebral ischemia-reperfusion injury and other neurological diseases. Accumulation of toxic free radicals not only increase the susceptibility of brain tissue to ischemic damage but also trigger numerous molecular cascades, leading to increased blood-brain barrier (BBB) permeability, brain edema, hemorrhage and inflammation, and brain death. Matrix metalloproteinases (MMPs) are one of the major targets in BBB breakdown. MMPs are proteolytic zinc-containing enzymes responsible for degradation of the extracellular matrix around cerebral blood vessels and neurons. Free radicals can activate MMPs and induce the degradations of tight junctions (TJs), leading to BBB breakdown. Recent studies indicate that caveolin-1, a 22 kDa membrane integral protein located at caveolae, can inhibit RNS production and MMPs activity, protect TJ proteins from degradation, and reduce the BBB permeability in cerebral ischemia-reperfusion injury. The interaction of RNS, caveolin-1, and MMPs forms a positive feedback loop which provides amplified impacts on BBB dysfunction during cerebral ischemia-reperfusion injury. Herein, we review the recent progress in the interaction of RNS, caveolin-1, and MMPs and the impact of the interaction on BBB permeability. For drug discovery, we summarize current evidence about antioxidant therapy in regulations of MMPs and caveolin-1 and anticipate the potential of developing antioxidants for the treatment of stroke and other neurological diseases. In conclusion, the interaction of RNS, caveolin-1, and MMPs could be a critical signal pathway in BBB disruption and infarction enlargement during cerebral ischemia-reperfusion injury and other neurological diseases