Live cell imaging and analysis of lipid droplets biogenesis in hepatatis C virus infected cells.

Lipid droplets (LDs) are regulated neutral lipid storage organelles having a central role in numerous cellular processes as well as in various pathologies such as metabolic disorders, immune responses and during pathogen infection. Due to the growing significance of LDs, extensive efforts are made to study the mechanism and the dynamics of their formation and life history and how are these diverted or modified by pathogens. Real-time visualization of lipid droplet biogenesis can assist in clarifying these and other important issues and may have implications towards understanding the pathogenesis of the associated diseases. Typically, LDs are post-experimentally stained using lipophilic dyes and are visualized under a microscope. Alternatively, overexpression of LD-associated proteins or immunofluorescence analyses are used to identify and follow LDs. These experimental approaches only examine a single end point of the experiment and cannot answer questions regarding LD dynamics. Here, we describe a simple and novel experimental setting that allows real-time fluorescence staining and detection of LDs in cultured living as well as infected cells. This method is quick and simple and is not restricted to a specific dye or cell line. Using this system, the biogenesis of LDs and their growth is demonstrated in cells infected with hepatitis C virus (HCV), confirming the strength of this method and the wide range of its applications.

Antibiotic permeation across the OmpF channel: modulation of the affinity site in the presence of magnesium.

We characterize the rate-limiting interaction of the antibiotic enrofloxacin with OmpF, a channel from the outer cell wall of Escherichia coli . Reconstitution of a single OmpF trimer into planar lipid membranes allows measurement of the ion current through the channel. Penetration of antibiotics causes ion current blockages, and their frequency allows a conclusion on the kinetics of channel entry and exit. In contrast to other antibiotics, enrofloxacin is able to block the OmpF channel for several milliseconds, reflecting high affinities comparable to substrate-specific channels such as the maltodextrin-specific maltoporin. Surprisingly, the presence of a divalent ion such as Mg(2+) leads to fast flickering with an increase in the rates of association and dissociation. All-atom computer modeling provides the most probable pathway able to identify the relevant rate-limiting interaction during antibiotic permeation. Mg(2+) has a high affinity for the aspartic acid at the 113 position (D113) in the center of the OmpF intracellular binding site. Therefore, the presence of Mg(2+) reverses the charge and enrofloxacin may cross the constriction region in its favorable orientation with the carboxylic group first.

Interaction of cephalosporins with outer membrane channels of Escherichia coli. Revealing binding by fluorescence quenching and ion conductance fluctuations.

Outer membrane channels in gram-negative bacteria are implicated in the influx of the latest generation of cephalosporins. We have measured the interaction strengths of ceftriaxone, cefpirome and ceftazidime in the two most abundant outer membrane porins of Escherichia coli, OmpF and OmpC, by both ion current fluctuations through single protein channels and fluorescence quenching. Statistical analysis of individual antibiotic entry events in membrane-incorporated porins yielded the kinetic rates and the equilibrium binding constant of each antibiotic-porin pair. Affinity constants were independently obtained by measuring the static quenching of inherent tryptophan fluorescence in the porins in the presence of the antibiotics. Through an empirical inner filter effect correction we have succeeded in measuring the chemical interaction of these strongly absorbing antibiotics, and obtained a qualitative agreement with conductance measurements. The interaction of all three antibiotics is smaller for OmpC than OmpF, and in the case of each porin the interaction strength series ceftriaxone > cefpirome > ceftazidime is maintained.

Molecular basis of enrofloxacin translocation through OmpF, an outer membrane channel of Escherichia coli--when binding does not imply translocation.

The molecular pathway of enrofloxacin, a fluoroquinolone antibiotic, through the outer membrane channel OmpF of Escherichia coli is investigated. High-resolution ion current fluctuation analysis reveals a strong affinity for enrofloxacin to OmpF, the highest value ever recorded for an antibiotic-channel interaction. A single point mutation in the constriction zone of OmpF, replacing aspartic acid at the 113 position with asparagine (D113N), lowers the affinity to a level comparable to other antibiotics. All-atom molecular dynamics simulations allow rationalizing the translocation pathways: wild-type OmpF has two symmetric binding sites for enrofloxacin located at each channel entry separated by a large energy barrier in the center, which inhibits antibiotic translocation. In this particular case, our simulations suggest that the ion current blockages are caused by molecules occupying either one of these peripheral binding sites. Removal of the negative charge on position 113 removes the central barrier and shifts the two peripheral binding sites to a unique central site, which facilitates translocation. Fluorescence steady-state measurements agree with the different location of binding sites for wild-type OmpF and the mutant. Our results demonstrate how a single-point mutation of the porin, and the resulting intrachannel shift of the affinity site, may substantially modify translocation.