Subcellular Min Oscillations as a Single-Cell Reporter of the Action of Polycations, Protamine, and Gentamicin on Escherichia coli

BACKGROUND: In Escherichia coli, MinD-GFP fusion proteins show rapid pole to pole oscillations. The objective was to investigate the effects of extracellular cations on the subcellular oscillation of cytoplasmic MinD within Escherichia coli. METHODOLOGY/PRINCIPAL FINDINGS: We exposed bacteria to the extracellular cations Ca(++), Mg(++), the cationic antimicrobial peptide (CAP) protamine, and the cationic aminoglycoside gentamicin. We found rapid and substantial increases in the average MinD oscillation periods in the presence of any of these polyvalent cations. For Ca(++) and Mg(++) the increases in period were transient, even with a constant extracellular concentration, while increases in period for protamine or gentamicin were apparently irreversible. We also found striking interdependence in the action of the small cations with protamine or gentamicin, distorted oscillations under the action of intermediate levels of gentamicin and Ca(++), and reversible freezing of the Min oscillation at high cationic concentrations. CONCLUSIONS/SIGNIFICANCE: Intracellular Min oscillations provide a fast single-cell reporter of bacterial response to extracellular polycations, which can be explained by the penetration of polycations into cells

Combining confocal and atomic force microscopy to quantify single-virus binding to mammalian cell surfaces

Over the past five years, atomic force microscopy (AFM)-based approaches have evolved into a powerful multiparametric tool set capable of imaging the surfaces of biological samples ranging from single receptors to membranes and tissues. One of these approaches, force–distance curve-based AFM (FD-based AFM), uses a probing tip functionalized with a ligand to image living cells at high-resolution and simultaneously localize and characterize specific ligand–receptor binding events. Analyzing data from FD-based AFM experiments using appropriate probabilistic models allows quantification of the kinetic and thermodynamic parameters that describe the free-energy landscape of the ligand–receptor bond. We have recently developed an FD-based AFM approach to quantify the binding events of single enveloped viruses to surface receptors of living animal cells while simultaneously observing them by fluorescence microscopy. This approach has provided insights into the early stages of the interaction between a virus and a cell. Applied to a model virus, we probed the specific interaction with cells expressing viral cognate receptors and measured the affinity of the interaction. Furthermore, we observed that the virus rapidly established specific multivalent interactions and found that each bond formed in sequence strengthened the attachment of the virus to the cell. Here we describe detailed procedures for probing the specific interactions of viruses with living cells; these procedures cover tip preparation, cell sample preparation, step-by-step FD-based AFM imaging and data analysis. Experienced microscopists should be able to master the entire set of protocols in 1 month

The complex cascade of cellular events governing inflammasome activation and IL-1β processing in response to inhaled particles

The innate immune system is the first line of defense against inhaled particles. Macrophages serve important roles in particle clearance and inflammatory reactions. Following recognition and internalization by phagocytes, particles are taken up in vesicular phagolysosomes. Intracellular phagosomal leakage, redox unbalance and ionic movements induced by toxic particles result in pro-IL-1β expression, inflammasome complex engagement, caspase-1 activation, pro-IL-1β cleavage, biologically-active IL-1β release and finally inflammatory cell death termed pyroptosis. In this review, we summarize the emerging signals and pathways involved in the expression, maturation and secretion of IL-1β during these responses to particles. We also highlight physicochemical characteristics of particles (size, surface and shape) which determine their capacity to induce inflammasome activation and IL-1β processing