Integrated Calendar

To overcome the anti-viral effects mediated by type I interferon-induced restriction factors, HIV-1 have evolutionarily acquired viral antagonists. For instance, tetherin (also known as BST2), a well-known protein restricting HIV-1 replication, exerts anti-HIV-1 effect by anchoring released progeny virions on the cell surface, whereas viral protein U (Vpu), an HIV-1-encoding accessory protein, antagonizes the anti-viral action mediated by tetherin. However, its precise role in HIV-1 replication in vivo remains unclear. Here we use a hematopoietic stem cell-transplanted humanized mouse model and several vpu mutants specifically lacking its function and demonstrate that anti-tetherin ability of Vpu is a prerequisite for efficient viral spread during the initial phase of infection. Our results suggest that tetherin is an important intrinsic effector restricting HIV-1 replication in vivo, while Vpu is a key factor to ensure efficient viral spread during the initial phase of infection by antagonizing tetherin.
Furthermore, by using HIV-1-infected humanized mouse model, we have recently launched a new approach to investigate the dynamics of HIV-1 infection through omics analyses. We would like to introduce our recent findings and discuss about them.

Disturbance of glutathione metabolism is a hallmark of numerous diseases, yet glutathione functions are still poorly understood. One key to address this question is to consider its functional compartmentation. In the endoplasmic reticulum (ER), protein folding requires disulfide bond formation catalyzed by the thiol oxidase Ero1 and protein disulfide isomerase (PDI). In the ER, glutathione is thought to counterbalance ER oxidation by the Ero1-PDI redox relay, which explain its relative more oxidized redox state (EGSH -242 mV), relative to the cytoplasm (EGSH = -295 mV). To access the function of GSH in this compartment, we first asked about the mechanism that maintains the ER EGSH homeostatic value. As GSH is exclusively synthesized in the cytoplasm and there is no GSH reductase in the ER to recycle the GSSG produced by the activity of the ER Ero1-PDI oxidative pathway, maintenance of ER EGSH likely depends on an ER import of GSH and export of GSSG. We found that GSH enters the ER by facilitated diffusion through the protein-conducting channel Sec61. We also found that an oxidized form of the chaperone Bip (Kar2 in yeast) inhibits this transport. We show that increased ER transport of GSH triggers Ero1 activation by reduction of its negative regulatory disulfides, which in turn leads to Bip oxidation by the H2O2 by-product of Ero1 activity. This regulated transport is strongly activated during ER stress by the UPR induction of Ero1 and an increase in GSH biosynthesis. Thus, the ER poise is tuned by a reciprocal control of GSH import into the ER and Ero1 activation. Such a reciprocal control is aimed at preventing a “short circuit” between the ER oxidative and reductive pathways, which would lead to Ero1 chronic cycling, cellular GSH consumption and cell death. I will discuss the implications of these findings in human pathology

Next generation sequencing (NGS) is revolutionizing all fields of biological research but it fails to extract the full range of information associated with genetic material. Complementary genomic technologies that analyze individual, unamplified genomic DNA are filling the gaps in the capabilities of NGS. Using such technologies we gain access to the structural variation and long range patterns of genetic and epigenetic information. Recent results from our lab demonstrate our ability to detect and map the epigenetic marks 5-methylacytosine and 5-hydroxymethylcytosine as well as various forms of DNA damage on individual genomic DNA molecules. This new technology allows genetic and epigenetic variation calling on the single cell level without the need to process single cells.

Classical fluorescence microscopy is limited in resolution by the wavelength of light (diffraction limit) restricting lateral resolution to ca. 200 nm, and axial resolution to ca. 500 nm (at typical excitation and emission wavelengths around 500 nm). However, recent years have seen a tremendous development in high- and super-resolution techniques of fluorescence microscopy, pushing spatial resolution to its diffraction-dictated limits and much beyond. One of these techniques is Image Scanning Microscopy (ISM). In ISM, the focus of a conventional laser-scanning confocal microscope (LCSM) is scanned over the sample, but instead of recording only the total fluorescence intensity for each scan position, as done in conventional operation of an LCSM, one records a small image of the illuminated region. The result is a four-dimensional stack of data: two dimensions refer to the lateral scan position, and two dimensions to the pixel position on the chip of the image-recording camera. This set of data can then be used to obtain a super-resolved image with doubled resolution, completely analogously to what is achieved with Structured Illumination Microscopy. However, ISM is conceptually and technically much simpler, suffers less from sample imperfections like refractive index variations, and can easily be implemented into any existing LSCM. I will also present recent results of combining ISM with two-photon excitation, which is important for deep-tissue imaging of e.g. neuronal tissue, and for performing non-linear coherent microscopy such as second-harmonic generation.

A second method which I will present is concerned with achieving nanometer resolution along the optical axis. It is called Metal Induced Energy Transfer or MIET and is based on the fact that, when placing a fluorescent molecule close to a metal, its fluorescence properties change dramatically. In particular, one observes a strongly modified lifetime of its excited state (Purcell effect). This coupling between an excited emitter and a metal film is strongly dependent on the emitter’s distance from the metal. We have used this effect for mapping the basal membrane of live cells with an axial accuracy of ~3 nm. The method is easy to implement and does not require any change to a conventional fluorescence lifetime microscope; it can be applied to any biological system of interest, and is compatible with most other super-resolution microscopy techniques which enhance the lateral resolution of imaging. Moreover, it is even applicable to localizing individual molecules, thus offering the prospect of three-dimensional single-molecule localization microscopy with nanometer isotropic resolution for structural biology.