Dynamics of single potassium channel proteins in the plasma membrane of migrating cells

Cell migration is an important cell physiological process, which is among others controlled by regulated ion channel activity. It was revealed that potassium channels, in particular calcium-activated potassium channels (KCa3.1), are required for optimal cell migration. In order to study the dynamics of individual channel proteins in the plasma membrane, single channel proteins were identified and tracked during cell migration. The identification was based on dual-colour labeling with quantum dots (QD) and it was proven that more than 90% of the observed QDs correspond to single potassium channel proteins. In migrating MDCK-F cells (Ncells = 10) single QD-labeled channels (NQD = 534) were visualised and tracked using time lapse total internal reflection fluorescence (TIRF) microscopy. Analysis of the trajectories of hKCa3.1 channels allowed the classification of their dynamics. KCa3.1 channel proteins moved subdiffusively in the plasma membrane with a mean diffusion coefficient Dα = 0.0673 ± 0.0005 μm2/sα and a mean subdiffusion exponent α = 0.824 ± 0.003. Ion channel proteins had a lower displacement at the lamellipodium and at the uropod than in the body of the cell which was due to a smaller subdiffusion coefficient α at these cell parts

Trypanosoma brucei BRCA2 acts in a life cycle-specific genome stability process and dictates BRC repeat number-dependent RAD51 subnuclear dynamics

Trypanosoma brucei survives in mammals through antigenic variation, which is driven by RAD51-directed homologous recombination of Variant Surface Glycoproteins (VSG) genes, most of which reside in a subtelomeric repository of >1000 silent genes. A key regulator of RAD51 is BRCA2, which in T. brucei contains a dramatic expansion of a motif that mediates interaction with RAD51, termed the BRC repeats. BRCA2 mutants were made in both tsetse fly-derived and mammal-derived T. brucei, and we show that BRCA2 loss has less impact on the health of the former. In addition, we find that genome instability, a hallmark of BRCA2 loss in other organisms, is only seen in mammal-derived T. brucei. By generating cells expressing BRCA2 variants with altered BRC repeat numbers, we show that the BRC repeat expansion is crucial for RAD51 subnuclear dynamics after DNA damage. Finally, we document surprisingly limited co-localization of BRCA2 and RAD51 in the T. brucei nucleus, and we show that BRCA2 mutants display aberrant cell division, revealing a function distinct from BRC-mediated RAD51 interaction. We propose that BRCA2 acts to maintain the huge VSG repository of T. brucei, and this function has necessitated the evolution of extensive RAD51 interaction via the BRC repeats, allowing re-localization of the recombinase to general genome damage when needed

Tumor necrosis factor inhibits spread of hepatitis C virus among liver cells, independent from interferons

BACKGROUND & AIMS: Tumor necrosis factor (TNF) an inflammatory cytokine expressed by human fetal liver cells (HFLCs) following infection with cell culture-derived hepatitis C virus. TNF has been reported to increase entry of HCV pseudoparticles into hepatoma cells and inhibit signaling by interferon alpha (IFNA), but have no effect on replication of HCV RNA. We investigated the effects of TNF on HCV infection of and spread among Huh-7 hepatoma cells and primary HFLCs.  METHODS: Human hepatoma (Huh-7 and Huh-7.5) and primary HFLCs were incubated with TNF and/or recombinant IFNΑ2Α, IFNB, IFNL1, and IFNL2 before or during HCV infection. We used 2 fully infectious HCV chimeric viruses of genotype 2A in these studies: J6/JFH (Clone 2) and Jc1(p7-nsGluc2A) (Jc1G), which encodes a secreted luciferase reporter. We measured HCV replication, entry, spread, production, and release in hepatoma cells and HFLCs. RESULTS: TNF inhibited completion of the HCV infectious cycle in hepatoma cells and HFLC in a dose-dependent and time-dependent manner. This inhibition required TNF binding to its receptor. Inhibition was independent of IFNA, IFNB, IFNL1, IFNL2, or JAK signaling via STAT. TNF reduced production of infectious viral particles by Huh-7 and HFLC, and thereby reduced numbers of infected cells and size of foci. TNF had little effect on HCV replicons and increased entry of HCV pseudoparticles. When cells were incubated with TNF before infection, the subsequent anti-viral effects of IFNs were increased. CONCLUSION: In a cell culture system, we found TNF to have antiviral effects independently of, as well as in combination with, IFNs. TNF inhibits HCV infection despite increased HCV envelope glycoprotein-mediated infection of liver cells. These findings contradict those from other studies, which reported that TNF blocks signal transduction in response to IFNs. The destructive inflammatory effects of TNF must be considered along with its antiviral effects

Role of the primer activation signal in tRNA annealing onto the HIV-1 genome studied by single-molecule FRET microscopy

HIV-1 reverse transcription is primed by a cellular tRNAlys3 molecule that binds to the primer binding site (PBS) in the genomic RNA. An additional interaction between the tRNA molecule and the primer activation signal (PAS) is thought to regulate the initiation of reverse transcription. The mechanism of tRNA annealing onto the HIV-1 genome was examined using ensemble and single-molecule Förster Resonance Energy Transfer (FRET) assays, in which fluorescent donor and acceptor molecules were covalently attached to an RNA template mimicking the PBS region. The role of the viral nucleocapsid (NC) protein in tRNA annealing was studied. Both heat annealing and NC-mediated annealing of tRNAlys3 were found to change the FRET efficiency, and thus the conformation of the HIV-1 RNA template. The results are consistent with a model for tRNA annealing that involves an interaction between the tRNAlys3 molecule and the PAS sequence in the HIV-1 genome. The NC protein may stimulate the interaction of the tRNA molecule with the PAS, thereby regulating the initiation of reverse transcription. Copyrigh