Identification of Cellular Protein Targets of a Half-Sandwich Iridium(III) Complex Reveals Its Dual Mechanism of Action via Both Electrophilic and Oxidative Stresses.

Identification of intracellular targets of anticancer drug candidates provides key information on their mechanism of action. Exploiting the ability of the anticancer (C∧N)-chelated half-sandwich iridium(III) complexes to covalently bind proteins, click chemistry with a bioorthogonal azido probe was used to localize a phenyloxazoline-chelated iridium complex within cells and profile its interactome at the proteome-wide scale. Proteins involved in protein folding and actin cytoskeleton regulation were identified as high-affinity targets. Upon iridium complex treatment, the folding activity of Heat Shock Protein HSP90 was inhibited in vitro and major cytoskeleton disorganization was observed. A wide array of imaging and biochemical methods validated selected targets and provided a multiscale overview of the effects of this complex on live human cells. We demonstrate that it behaves as a dual agent, inducing both electrophilic and oxidative stresses in cells that account for its cytotoxicity. The proposed methodological workflow can open innovative avenues in metallodrug discovery.

Fibroblasts mediate endothelium response to angiogenic cues in a newly developed 3D stroma engineered model.

Three-dimensional stroma engineered models would enable fundamental and applicative studies of human tissues interaction and remodeling in both physiological and pathological conditions. In this work, we propose a 3D vascularized stroma model to be used as in vitro platform for drug testing. A pullulan/dextran-based porous scaffold containing pre-patterned microchannels of 100 µm diameter is used for co-culturing of fibroblasts within the matrix pores and endothelial cells to form the lumen. Optical clearing of the constructs by hyperhydration allows for in-depth imaging of the model up to 1 mm by lightsheet and confocal microscopy. Our 3D vascularized stroma model allows for higher viability, metabolism and cytokines expression compared to a monocultured vascular model. Stroma-endothelium cross-talk is then investigated by exposing the system to pro and anti-angiogenic molecules. The results highlight the protective role played by fibroblasts on the vasculature, as demonstrated by decreased cytotoxicity, restoration of nitric oxide levels upon challenge, and sustained expression of endothelial markers CD31, vWF and VEGF. Our tissue model provides a 3D engineered platform for in vitro studies of stroma remodeling in angiogenesis-driven events, known to be a leading mechanism in diseased conditions, such as metastatic cancers, retinopathies and ischemia, and to investigate related potential therapies.

Mitophagy of polarized sperm-derived mitochondria after fertilization.

Loss of membrane potential of sperm mitochondria has been regarded as the first step preceding mitophagy degradation after their entry into the C. elegans oocyte at fertilization. This is in line with the classical view of mitophagy of defective or abnormal mitochondria and could serve as a recognition signal for their specific and quick autophagy degradation. Here, using TMRE (tetramethylrhodamine ethyl ester) and live imaging we show that this is not the case. Instead, sperm inherited mitochondria show a stable labeling with TMRE before and at the time of autophagosomes formation. Interestingly, this labeling remains in late-stage-embryos of autophagy-defective-mutants suggesting that the loss of membrane potential occurs upon the entry of the mitochondria into the autophagy pathway. These stabilized and still polarized sperm mitochondria remain distinct but associated with the maternal-derived mitochondrial network suggesting a mechanism that prevents their fusion and represents an efficient additional protective system against fertilization-induced heteroplasmy.

Endocytosis-mediated siderophore uptake as a strategy for Fe acquisition in diatoms.

Phytoplankton growth is limited in vast oceanic regions by the low bioavailability of iron. Iron fertilization often results in diatom blooms, yet the physiological underpinnings for how diatoms survive in chronically iron-limited waters and outcompete other phytoplankton when iron becomes available are unresolved. We show that some diatoms can use siderophore-bound iron, and exhibit a species-specific recognition for siderophore types. In Phaeodactylum tricornutum, hydroxamate siderophores are taken up without previous reduction by a high-affinity mechanism that involves binding to the cell surface followed by endocytosis-mediated uptake and delivery to the chloroplast. The affinity recorded is the highest ever described for an iron transport system in any eukaryotic cell. Collectively, our observations suggest that there are likely a variety of iron uptake mechanisms in diatoms besides the well-established reductive mechanism. We show that iron starvation-induced protein 1 (ISIP1) plays an important role in the uptake of siderophores, and through bioinformatics analyses we deduce that this protein is largely diatom-specific. We quantify expression of ISIP1 in the global ocean by querying the Tara Oceans atlas of eukaryotic genes and show a link between the abundance and distribution of diatom-associated ISIP1 with ocean provinces defined by chronic iron starvation.

Coordination between Intra- and Extracellular Forces Regulates Focal Adhesion Dynamics.

Focal adhesions (FAs) are important mediators of cell-substrate interactions. One of their key functions is the transmission of forces between the intracellular acto-myosin network and the substrate. However, the relationships between cell traction forces, FA architecture, and molecular forces within FAs are poorly understood. Here, by combining Förster resonance energy transfer (FRET)-based molecular force biosensors with micropillar-based traction force sensors and high-resolution fluorescence microscopy, we simultaneously map molecular tension across vinculin, a key protein in FAs, and traction forces at FAs. Our results reveal strong spatiotemporal correlations between vinculin tension and cell traction forces at FAs throughout a wide range of substrate stiffnesses. Furthermore, we find that molecular tension within individual FAs follows a biphasic distribution from the proximal (toward the cell nucleus) to distal end (toward the cell edge). Using super-resolution imaging, we show that such a distribution relates to that of FA proteins. On the basis of our experimental data, we propose a model in which FA dynamics results from tension changes along the FAs.

Super-resolution for everybody: An image processing workflow to obtain high-resolution images with a standard confocal microscope.

In the presented work we aimed at improving confocal imaging to obtain highest possible resolution in thick biological samples, such as the mouse oocyte. We therefore developed an image processing workflow that allows improving the lateral and axial resolution of a standard confocal microscope. Our workflow comprises refractive index matching, the optimization of microscope hardware parameters and image restoration by deconvolution. We compare two different deconvolution algorithms, evaluate the necessity of denoising and establish the optimal image restoration procedure. We validate our workflow by imaging sub resolution fluorescent beads and measuring the maximum lateral and axial resolution of the confocal system. Subsequently, we apply the parameters to the imaging and data restoration of fluorescently labelled meiotic spindles of mouse oocytes. We measure a resolution increase of approximately 2-fold in the lateral and 3-fold in the axial direction throughout a depth of 60µm. This demonstrates that with our optimized workflow we reach a resolution that is comparable to 3D-SIM-imaging, but with better depth penetration for confocal images of beads and the biological sample.

Predictive polymer modeling reveals coupled fluctuations in chromosome conformation and transcription.

A new level of chromosome organization, topologically associating domains (TADs), was recently uncovered by chromosome conformation capture (3C) techniques. To explore TAD structure and function, we developed a polymer model that can extract the full repertoire of chromatin conformations within TADs from population-based 3C data. This model predicts actual physical distances and to what extent chromosomal contacts vary between cells. It also identifies interactions within single TADs that stabilize boundaries between TADs and allows us to identify and genetically validate key structural elements within TADs. Combining the model's predictions with high-resolution DNA FISH and quantitative RNA FISH for TADs within the X-inactivation center (Xic), we dissect the relationship between transcription and spatial proximity to cis-regulatory elements. We demonstrate that contacts between potential regulatory elements occur in the context of fluctuating structures rather than stable loops and propose that such fluctuations may contribute to asymmetric expression in the Xic during X inactivation.