Synthesis, insertion, and characterization of SARS-CoV-2 membrane protein within lipid bilayers.

Throughout history, coronaviruses have posed challenges to both public health and the global economy; nevertheless, methods to combat them remain rudimentary, primarily due to the absence of experiments to understand the function of various viral components. Among these, membrane (M) proteins are one of the most elusive because of their small size and challenges with expression. Here, we report the development of an expression system to produce tens to hundreds of milligrams of M protein per liter of Escherichia coli culture. These large yields render many previously inaccessible structural and biophysical experiments feasible. Using cryo-electron microscopy and atomic force microscopy, we image and characterize individual membrane-incorporated M protein dimers and discover membrane thinning in the vicinity, which we validated with molecular dynamics simulations. Our results suggest that the resulting line tension, along with predicted induction of local membrane curvature, could ultimately drive viral assembly and budding.

Investigation of HIV-1 Gag binding with RNAs and lipids using Atomic Force Microscopy.

Atomic Force Microscopy was utilized to study the morphology of Gag, ΨRNA, and their binding complexes with lipids in a solution environment with 0.1Å vertical and 1nm lateral resolution. TARpolyA RNA was used as a RNA control. The lipid used was phospha-tidylinositol-(4,5)-bisphosphate (PI(4,5)P2). The morphology of specific complexes Gag-ΨRNA, Gag-TARpolyA RNA, Gag-PI(4,5)P2 and PI(4,5)P2-ΨRNA-Gag were studied. They were imaged on either positively or negatively charged mica substrates depending on the net charges carried. Gag and its complexes consist of monomers, dimers and tetramers, which was confirmed by gel electrophoresis. The addition of specific ΨRNA to Gag is found to increase Gag multimerization. Non-specific TARpolyA RNA was found not to lead to an increase in Gag multimerization. The addition PI(4,5)P2 to Gag increases Gag multimerization, but to a lesser extent than ΨRNA. When both ΨRNA and PI(4,5)P2 are present Gag undergoes comformational changes and an even higher degree of multimerization.

Crystal-to-Gel Transformation Stimulated by a Solid-State E→Z Photoisomerization.

The molecule (E)-(5-(3-anthracen-9-yl-allylidene)-2,2-dimethyl-[1,3] dioxane-4,6-dione) (E-AYAD) undergoes E→Z photoisomerization. In the solid state, this photoisomerization process can initiate a physical transformation of the crystal that is accompanied by a large volume expansion (ca. 10 times), loss of crystallinity, and growth of large pores. This physical change requires approximately 10 % conversion of the E isomer to the Z isomer and results in a gel-like solid with decreased stiffness that still retains its mechanical integrity. The induced porosity allows the expanding gel to engulf superparamagnetic nanoparticles from the surrounding liquid. The trapped superparamagnetic nanoparticles impart a magnetic susceptibility to the gel, allowing it to be moved by a magnetic field. The photoinduced phase transition, starting with a compact crystalline solid instead of a dilute solution, provides a new route for in situ production of functional porous materials.

Measurement of entropic force from polymers attached to a pyramidal tip.

The measurement of the boundary shape dependence of the entropic force from long polymers was attempted. The pyramidal cone-plate geometry was chosen. The polymer molecules were covalently bound to a well-defined Au patch at the apex of a pyramidal cantilever tip of the atomic force microscope (AFM). A smooth hydrophobic plate was used as the second boundary to confine the polymer molecules. The use of the hydrophobic plate allows neglect of polymer adhesion forces. The measurements were made in salt water solution to decrease the effect of electrostatic forces from any uncompensated charges on the boundary. As the functionalized AFM tip approaches the flat hydrophobic surface, the induced entropic forces were measured as a function of the separation distance. The measured force-distance curves are compared with a model of polymer-mediated entropic force between scale-free objects and the Alexander-de Gennes (AdG) theory for a polymer brush.

Senescence Increases Choroidal Endothelial Stiffness and Susceptibility to Complement Injury: Implications for Choriocapillaris Loss in AMD.

PURPOSE: Age-related macular degeneration (AMD) commonly causes blindness in the elderly. Yet, it is untreatable in the large fraction of all AMD patients that develop the early dry form. Dry AMD is marked by the deposition of membrane attack complex (MAC) on choriocapillaris (CC), which is implicated in CC degeneration and subsequent atrophy of overlying retinal pigment epithelium. Since MAC is also found on the CC of young eyes, here we investigated whether and how aging increases choroidal endothelial susceptibility to MAC injury. METHODS: Monkey chorioretinal endothelial cells (ECs, RF/6A) were cultured to high passages (>P60) to achieve replicative senescence. We treated ECs with complement-competent human serum to promote MAC deposition and injury, which were assessed by flow cytometry and trypan blue exclusion assay, respectively. Stiffness of EC was measured by atomic force microscopy indentation while Rho GTPase activity was quantified by Rho G-LISA assay. RESULTS: Our findings reveal that senescent ECs are significantly stiffer than their normal counterparts, which correlates with higher cytoskeletal Rho activity in these cells and their greater susceptibility to complement (MAC) injury. Importantly, inhibition of Rho activity in senescent ECs significantly reduced cell stiffness and MAC-induced lysis. CONCLUSIONS: By revealing an important role of senescence-associated choroidal EC stiffening in complement injury, these findings implicate CC stiffening as an important determinant of age-related CC atrophy seen in dry AMD. Future studies are needed to validate these findings in appropriate animal models so new therapeutic targets can be identified for treatment of dry AMD.

Basement membrane stiffening promotes retinal endothelial activation associated with diabetes.

Endothelial activation is a hallmark of the high-glucose (HG)-induced retinal inflammation associated with diabetic retinopathy (DR). However, precisely how HG induces retinal endothelial activation is not fully understood. We hypothesized that HG-induced up-regulation of lysyl oxidase (LOX), a collagen-cross-linking enzyme, in retinal capillary endothelial cells (ECs) enhances subendothelial basement membrane (BM) stiffness, which, in turn, promotes retinal EC activation. Diabetic C57BL/6 mice exhibiting a 70 and 50% increase in retinal intercellular adhesion molecule (ICAM)-1 expression and leukocyte accumulation, respectively, demonstrated a 2-fold increase in the levels of BM collagen IV and LOX, key determinants of capillary BM stiffness. Using atomic force microscopy, we confirmed that HG significantly enhances LOX-dependent subendothelial matrix stiffness in vitro, which correlated with an ∼2.5-fold increase in endothelial ICAM-1 expression, a 4-fold greater monocyte-EC adhesion, and an ∼2-fold alteration in endothelial NO (decrease) and NF-κB activation (increase). Inhibition of LOX-dependent subendothelial matrix stiffening alone suppressed HG-induced retinal EC activation. Finally, using synthetic matrices of tunable stiffness, we demonstrated that subendothelial matrix stiffening is necessary and sufficient to promote EC activation. These findings implicate BM stiffening as a critical determinant of HG-induced retinal EC activation and provide a rationale for examining BM stiffness and underlying mechanotransduction pathways as therapeutic targets for diabetic retinopathy.

Nanoliposomal Nitroglycerin Exerts Potent Anti-Inflammatory Effects.

Nitroglycerin (NTG) markedly enhances nitric oxide (NO) bioavailability. However, its ability to mimic the anti-inflammatory properties of NO remains unknown. Here, we examined whether NTG can suppress endothelial cell (EC) activation during inflammation and developed NTG nanoformulation to simultaneously amplify its anti-inflammatory effects and ameliorate adverse effects associated with high-dose NTG administration. Our findings reveal that NTG significantly inhibits human U937 cell adhesion to NO-deficient human microvascular ECs in vitro through an increase in endothelial NO and decrease in endothelial ICAM-1 clustering, as determined by NO analyzer, microfluorimetry, and immunofluorescence staining. Nanoliposomal NTG (NTG-NL) was formulated by encapsulating NTG within unilamellar lipid vesicles (DPhPC, POPC, Cholesterol, DHPE-Texas Red at molar ratio of 6:2:2:0.2) that were ~155 nm in diameter and readily uptaken by ECs, as determined by dynamic light scattering and quantitative fluorescence microscopy, respectively. More importantly, NTG-NL produced a 70-fold increase in NTG therapeutic efficacy when compared with free NTG while preventing excessive mitochondrial superoxide production associated with high NTG doses. Thus, these findings, which are the first to reveal the superior therapeutic effects of an NTG nanoformulation, provide the rationale for their detailed investigation for potentially superior vascular normalization therapies.

A novel minimal in vitro system for analyzing HIV-1 Gag-mediated budding.

A biomimetic minimalist model membrane was used to study the mechanism and kinetics of cell-free in vitro HIV-1 Gag budding from a giant unilamellar vesicle (GUV). Real-time interaction of Gag, RNA, and lipid, leading to the formation of mini-vesicles, was measured using confocal microscopy. Gag forms resolution-limited punctae on the GUV lipid membrane. Introduction of the Gag and urea to a GUV solution containing RNA led to the budding of mini-vesicles on the inside surface of the GUV. The GUV diameter showed a linear decrease in time due to bud formation. Both bud formation and decrease in GUV size were proportional to Gag concentration. In the absence of RNA, addition of urea to GUVs incubated with Gag also resulted in subvesicle formation. These observations suggest the possibility that clustering of GAG proteins leads to membrane invagination even in the absence of host cell proteins. The method presented here is promising, and allows for systematic study of the dynamics of assembly of immature HIV and help classify the hierarchy of factors that impact the Gag protein initiated assembly of retroviruses such as HIV.

Fluctuation-induced forces between inclusions in a fluid membrane under tension.

We develop an exact method to calculate thermal Casimir forces between inclusions of arbitrary shapes and separation, embedded in a fluid membrane whose fluctuations are governed by the combined action of surface tension, bending modulus, and Gaussian rigidity. Each object's shape and mechanical properties enter only through a characteristic matrix, a static analog of the scattering matrix. We calculate the Casimir interaction between two elastic disks embedded in a membrane. In particular, we find that at short separations the interaction is strong and independent of surface tension.

Single Molecule Probing of Exocytotic Protein Interactions Using Force Spectroscopy.

Relatively recently, the Atomic Force Microscope (AFM) emerged as a powerful tool for single molecule nanomechanical investigations. Parameters that can be measured by force spectroscopy using AFM, such as the force and total mechanical extension required to break bonds between various proteins can yield valuable insights into the nature of the bond (zippering vs. highly localized binding site), the sequence of its interactions and the energy landscape along the length of the interaction. In this review we discuss the use of AFM in force spectroscopy mode to study intermolecular interactions between the exocytotic proteins of the core SNARE complex. Information gathered by force spectroscopy of protein-protein interactions of this complex supplement previous results acquired with other techniques, and allows a deeper understanding of SNARE protein interactions and their role in exocytosis.