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

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

We discuss the fluctuation-induced force, a finite-temperature analog of the Casimir force, between two inclusions embedded in a fluid membrane under tension. We suggest a method to calculate this Casimir interaction in the most general case, where membrane fluctuations are governed by the combined action of surface tension, bending modulus, and the Gaussian rigidity. We find that the surface tension strongly modifies the power law in the separation dependence of the Casimir interaction. This results in a strong suppression of the Casimir force at separations beyond a characteristic length, which could affect protein aggregation dynamics in cell membranes.Comment: 4 pages, 1 figur

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, {\Psi}RNA, and their binding complexes with lipids in a solution environment with 0.1{\AA} 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-{\Psi}RNA, Gag-TARpolyA RNA, Gag-PI(4,5)P2 and PI(4,5)P2-{\Psi}RNA-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 {\Psi}RNA 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 {\Psi}RNA. When both {\Psi}RNA and PI(4,5)P2 are present Gag undergoes comformational changes and an even higher degree of multimerization

A Discrete Two-Dimensional Model of a Loaded Cantilever Influenced by Time-Dependent Forces

We developed a discrete two-dimensional model of a cantilever which incorporates the effects of inhomogeneity, the geometry of an attached particle, and the influence of external time-dependent forces. We provide a comparison between the solutions for our discrete model and its continuous limit. The rotational-vibrational mode is studied in detail. The results of numerical simulations demonstrate usefulness of our model for many applications when a cantilever has a complicated geometry and is affected by time-depended and distributed external forces.Comment: Pages 15, Figures 1

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 but exterior to the surface. 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.Comment: 27 pages, 9 Figures and 0 Table

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

Quantum and thermal Casimir interaction between a sphere and a plate: Comparison of Drude and plasma models

We calculate the Casimir interaction between a sphere and a plate, both described by the plasma model, the Drude model, or generalizations of the two models. We compare the results at both zero and finite temperatures. At asymptotically large separations we obtain analytical results for the interaction that reveal a non-universal, i.e., material dependent interaction for the plasma model. The latter result contains the asymptotic interaction for Drude metals and perfect reflectors as different but universal limiting cases. This observation is related to the screening of a static magnetic field by a London superconductor. For small separations we find corrections to the proximity force approximation (PFA) that support correlations between geometry and material properties that are not captured by the Lifshitz theory. Our results at finite temperatures reveal for Drude metals a non-monotonic temperature dependence of the Casimir free energy and a negative entropy over a sizeable range of separations.Comment: 11 pages, 5 figure

A Brief Review of Some Recent Precision Casimir Force Measurements

Here, we review recent advances in precision Casimir force measurements with both non-magnetic and magnetic materials. In addition, the measurement of the geometric dependence of the Casimir force, both lateral and normal, using uniformly corrugated surfaces is briefly presented. Finally, the measurement of the thermal Casimir force in graphene is discussed