Yuchong Li

Allosteric modulation in monomers and oligomers of a G protein-coupled receptor

The M2 muscarinic receptor is the prototypic model of allostery in GPCRs, yet the molecular and the supramolecular determinants of such effects are unknown. Monomers and oligomers of the M2 muscarinic receptor therefore have been compared to identify those allosteric properties that are gained in oligomers. Allosteric interactions were monitored by means of a FRET-based sensor of conformation at the allosteric site and in pharmacological assays involving mutants engineered to preclude intramolecular effects. Electrostatic, steric, and conformational determinants of allostery at the atomic level were examined in molecular dynamics simulations. Allosteric effects in monomers were exclusively negative and derived primarily from intramolecular electrostatic repulsion between the allosteric and orthosteric ligands. Allosteric effects in oligomers could be positive or negative, depending upon the allosteric-orthosteric pair, and they arose from interactions within and between the constituent protomers. The complex behavior of oligomers is characteristic of muscarinic receptors in myocardial preparations. DOI: http://dx.doi.org/10.7554/eLife.11685.001

Synthesis of Stable Multifunctional Perfluorocarbon Nanoemulsions for Cancer Therapy and Imaging

Nanotechnology provides a promising platform for drug-delivery in medicine. Nanostructured materials can be designed with desired superparamagnetic or fluorescent properties in conjunction with biochemically functionalized moieties (i.e., antibodies, peptides, and small molecules) to actively bind to target sites. These multifunctional properties make them suitable agents for multimodal imaging, diagnosis, and therapy. Perfluorohexane nanoemulsions (PFH-NEs) are novel drug-delivery vehicles and contrast agents for ultrasound and photoacoustic imaging of cancer in vivo, offering higher spatial resolution and deeper penetration of tissue when compared to conventional optical techniques. Compared to other theranostic agents, our PFH-NEs are one of the smallest of their kind (<100 nm), exhibit minimal aggregation, long-term stability at physiological conditions, and provide a noninvasive cancer imaging and therapy alternative for patients. Here, we show, using high-resolution imaging and correlative techniques, that our PFH-NEs, when in tandem with silica-coated gold nanoparticles (scAuNPs), can be used as a drug-loaded therapeutic via endocytosis and as a multimodal imaging agent for photoacoustic, ultrasound, and fluorescence imaging of tumor growth.

Single-Molecule Analysis of the Supramolecular Organization of the M2 Muscarinic Receptor and the Gαi1 Protein.

G protein-coupled receptors constitute the largest family of transmembrane signaling proteins and the largest pool of drug targets, yet their mechanism of action remains obscure. That uncertainty relates to unresolved questions regarding the supramolecular nature of the signaling complex formed by receptor and G protein. We therefore have characterized the oligomeric status of eGFP-tagged M2 muscarinic receptor (M2R) and Gi1 by single-particle photobleaching of immobilized complexes. The method was calibrated with multiplexed controls comprising 1-4 copies of fused eGFP. The photobleaching patterns of eGFP-M2R were indicative of a tetramer and unaffected by muscarinic ligands; those of eGFP-Gi1 were indicative of a hexamer and unaffected by GTPγS. A complex of M2R and Gi1 was tetrameric in both, and activation by a full agonist plus GTPγS reduced the oligomeric size of Gi1 without affecting that of the receptor. A similar reduction was observed upon activation of eGFP-Gαi1 by the receptor-mimic mastoparan plus GTPγS, and constitutively active eGFP-Gαi1 was predominantly dimeric. The oligomeric nature of Gi1 in live CHO cells was demonstrated by means of Förster resonance energy transfer and dual-color fluorescence correlation spectroscopy in studies with eGFP- and mCherry-labeled Gαi1; stochastic FRET was ruled out by means of non-interacting pairs. These results suggest that the complex between M2R and holo-Gi1 is an octamer comprising four copies of each, and that activation is accompanied by a decrease in the oligomeric size of Gi1. The structural feasibility of such a complex was demonstrated in molecular dynamics simulations.

Multifunctional perfluorocarbon nanoemulsions for cancer therapy and imaging

There is currently interest in the development of nanoemulsions as imaging and therapeutic agents, particularly perfluorohexane (PFH) droplets, whose amphiphilic shell protects drugs against physico-chemical and enzymatic degradation. When delivered to their target sites, these perfluorocarbon (PFC) droplets can vaporize upon laser excitation, efficiently releasing their drug payload and/or imaging tracers. Due to the optical properties of gold, coupling PFC droplets with gold nanoparticles significantly reduces the energy required for vaporization. In this work, nanoemulsions with a PFC core and Zonyl FSP surfactant shell were produced using sonication. Droplets were characterized in terms of size and morphology using high resolution fluorescence microscopy (i.e. total internal reflection fluorescence microscopy, TIRFM), fluorescence correlation spectroscopy (FCS), transmission electron microscopy (TEM), and light scattering techniques (i.e. dynamic light scattering, DLS). The ability of PFC droplets to vaporize was demonstrated using optical light microscopy.

Exocyst Dynamics During Vesicle Tethering and Fusion

The exocyst is a conserved octameric complex that tethers exocytic vesicles to the plasma membrane prior to fusion. Exocyst assembly and delivery mechanisms remain unclear, especially in mammalian cells. Here we tagged multiple endogenous exocyst subunits with sfGFP or Halo using Cas9 gene editing, to create single and double knock-in lines of mammary epithelial cells, and interrogated exocyst dynamics by high-speed imaging and correlation spectroscopy. We discovered that mammalian exocyst is comprised of tetrameric subcomplexes that, unexpectedly, can associate independently with vesicles and plasma membrane and are in dynamic equilibrium. Membrane arrival times are similar for subunits and vesicles, but with a small delay (~80msec) between subcomplexes. Departure of Sec3 occurs prior to fusion, whereas other subunits depart just after fusion. Single molecule counting indicates ~9 exocyst complexes associated per vesicle. These data reveal the mammalian exocyst as a remarkably dynamic two-part complex and provide important new insights into assembly/disassembly mechanisms.

Ligand-Induced Coupling between Oligomers of the M2 Receptor and the Gi1 Protein in Live Cells

Uncertainty over the mechanism of signaling via G protein-coupled receptors (GPCRs) relates in part to questions regarding their supramolecular structure. GPCRs and heterotrimeric G proteins are known to couple as monomers under various conditions. Many GPCRs form oligomers under many of the same conditions, however, and the biological role of those complexes is unclear. We have used dual-color fluorescence correlation spectroscopy to identify oligomers of the M2 muscarinic receptor and of Gi1 in purified preparations and live Chinese hamster ovary cells. Measurements on differently tagged receptors (i.e., eGFP-M2 and mCherry-M2) and G proteins (i.e., eGFP-Gαi1β1γ2 and mCherry-Gαi1β1γ2) detected significant cross-correlations between the two fluorophores in each case, both in detergent micelles and in live cells, indicating that both the receptor and Gi1 can exist as homo-oligomers. Oligomerization of differently tagged Gi1 decreased upon the activation of co-expressed wild-type M2 receptor by an agonist. Measurements on a tagged M2 receptor (M2-mCherry) and eGFP-Gαi1β1γ2 co-expressed in live cells detected cross-correlations only in the presence of an agonist, which therefore promoted coupling of the receptor and the G protein. The effect of the agonist was retained when a fluorophore-tagged receptor lacking the orthosteric site (i.e., M2(D103A)-mCherry) was co-expressed with the wild-type receptor and eGFP-Gαi1β1γ2, indicating that the ligand acted via an oligomeric receptor. Our results point to a model in which an agonist promotes transient coupling of otherwise independent oligomers of the M2 receptor on the one hand and of Gi1 on the other and that an activated complex leads to a reduction in the oligomeric size of the G protein. They suggest that GPCR-mediated signaling proceeds, at least in part, via oligomers.

Characterization of Fluorescein Arsenical Hairpin (FlAsH ) as a Probe for Single-Molecule Fluorescence Spectroscopy

In recent years, new labelling strategies have been developed that involve the genetic insertion of small amino-acid sequences for specific attachment of small organic fluorophores. Here, we focus on the tetracysteine FCM motif (FLNCCPGCCMEP), which binds to fluorescein arsenical hairpin (FlAsH), and the ybbR motif (TVLDSLEFIASKLA) which binds fluorophores conjugated to Coenzyme A (CoA) via a phosphoryl transfer reaction. We designed a peptide containing both motifs for orthogonal labelling with FlAsH and Alexa647 (AF647). Molecular dynamics simulations showed that both motifs remain solvent-accessible for labelling reactions. Fluorescence spectra, correlation spectroscopy and anisotropy decay were used to characterize labelling and to obtain photophysical parameters of free and peptide-bound FlAsH. The data demonstrates that FlAsH is a viable probe for single-molecule studies. Single-molecule imaging confirmed dual labeling of the peptide with FlAsH and AF647. Multiparameter single-molecule Förster Resonance Energy Transfer (smFRET) measurements were performed on freely diffusing peptides in solution. The smFRET histogram showed different peaks corresponding to different backbone and dye orientations, in agreement with the molecular dynamics simulations. The tandem of fluorophores and the labelling strategy described here are a promising alternative to bulky fusion fluorescent proteins for smFRET and single-molecule tracking studies of membrane proteins.