Pivotal Role for Cxcr2 in Regulating Tumor-Associated Neutrophil in Breast Cancer.

Chemokines present in the tumor microenvironment are essential for the control of tumor progression. We show here that several ligands of the chemokine receptor Cxcr2 were up-regulated in the PyMT (polyoma middle T oncogene) model of breast cancer. Interestingly, the knock-down of Cxcr2 in PyMT animals led to an increased growth of the primary tumor and lung metastasis. The analysis of tumor content of PyMT-Cxcr2-/- animals highlighted an increased infiltration of tumor associated neutrophils (TANs), mirrored by a decreased recruitment of tumor associated macrophages (TAMs) compared to PyMT animals. Analysis of PyMT-Cxcr2-/- TANs revealed that they lost their killing ability compared to PyMT-Cxcr2+/+ TANs. The transcriptomic analysis of PyMT-Cxcr2-/- TANs showed that they had a more pronounced pro-tumor TAN2 profile compared to PyMT TANs. In particular, PyMT-Cxcr2-/- TANs displayed an up-regulation of the pathways involved in reactive oxygen species (ROS) production and angiogenesis and factors favoring metastasis, but reduced apoptosis. In summary, our data reveal that a lack of Cxcr2 provides TANs with pro-tumor effects.

Imaging endocytic vesicle formation at high spatial and temporal resolutions with the pulsed-pH protocol.

Endocytosis is a fundamental process occurring in all eukaryotic cells. Live cell imaging of endocytosis has helped to decipher many of its mechanisms and regulations. With the pulsed-pH (ppH) protocol, one can detect the formation of individual endocytic vesicles (EVs) with an unmatched temporal resolution of 2 s. The ppH protocol makes use of cargo protein (e.g., the transferrin receptor) coupled to a pH-sensitive fluorescent protein, such as superecliptic pHluorin (SEP), which is brightly fluorescent at pH 7.4 but not fluorescent at pH <6.0. If the SEP moiety is at the surface, its fluorescence will decrease when cells are exposed to a low pH (5.5) buffer. If the SEP moiety has been internalized, SEP will remain fluorescent even during application of the low pH buffer. Fast perfusion enables the complete exchange of low and high pH extracellular solutions every 2 s, defining the temporal resolution of the technique. Unlike other imaging-based endocytosis assays, the ppH protocol detects EVs without a priori hypotheses on the dynamics of vesicle formation. Here, we explain how the ppH protocol quantifies the endocytic activity of living cells and the recruitment of associated proteins in real time. We provide a step-by-step procedure for expression of the reporter proteins with transient transfection, live cell image acquisition with synchronized pH changes and automated analysis. The whole protocol can be performed in 2 d to provide quantitative information on the endocytic process being studied.

Deciphering the internalization mechanism of WRAP:siRNA nanoparticles.

Gene silencing mediated by double-stranded small interfering RNA (siRNA) has been widely investigated as a potential therapeutic approach for a variety of diseases and, indeed, the first therapeutic siRNA was approved by the FDA in 2018. As an alternative to the traditional delivery systems for nucleic acids, peptide-based nanoparticles (PBNs) have been applied successfully for siRNA delivery. Recently, we have developed amphipathic cell-penetrating peptides (CPPs), called WRAP allowing a rapid and efficient siRNA delivery into several cell lines at low doses (20 to 50 nM). In this study, using a highly specific gene silencing system, we aimed to elucidate the cellular uptake mechanism of WRAP:siRNA nanoparticles by combining biophysical, biological, confocal and electron microscopy approaches. We demonstrated that WRAP:siRNA complexes remain fully active in the presence of chemical inhibitors of different endosomal pathways suggesting a direct cell membrane translocation mechanism. Leakage studies on lipid vesicles indicated membrane destabilization properties of the nanoparticles and this was supported by the measurement of WRAP:siRNA internalization in dynamin triple-KO cells. However, we also observed some evidences for an endocytosis-dependent cellular internalization. Indeed, nanoparticles co-localized with transferrin, siRNA silencing was inhibited by the scavenger receptor A inhibitor Poly I and nanoparticles encapsulated in vesicles were observed by electron microscopy in U87 cells. In conclusion, we demonstrate here that the efficiency of WRAP:siRNA nanoparticles is mainly based on the use of multiple internalization mechanisms including direct translocation as well as endocytosis-dependent pathways.

Functional recruitment of dynamin requires multimeric interactions for efficient endocytosis.

During clathrin mediated endocytosis (CME), the concerted action of dynamin and its interacting partners drives membrane scission. Essential interactions occur between the proline/arginine-rich domain of dynamin (dynPRD) and the Src-homology domain 3 (SH3) of various proteins including amphiphysins. Here we show that multiple SH3 domains must bind simultaneously to dynPRD through three adjacent motifs for dynamin's efficient recruitment and function. First, we show that mutant dynamins modified in a single motif, including the central amphiphysin SH3 (amphSH3) binding motif, partially rescue CME in dynamin triple knock-out cells. However, mutating two motifs largely prevents that ability. Furthermore, we designed divalent dynPRD-derived peptides. These ligands bind multimers of amphSH3 with >100-fold higher affinity than monovalent ones in vitro. Accordingly, dialyzing living cells with these divalent peptides through a patch-clamp pipette blocks CME much more effectively than with monovalent ones. We conclude that dynamin drives vesicle scission via multivalent interactions in cells.

Simple, sensitive and robust chicken specific sexing assays, compliant with large scale analysis.

Chicken meat and eggs are important sources of food for the world population. The significant increase in food demand has pushed the food industry toward a rapid non-expensive production which in turn raises ethical issues. How chicken are cultivated and processed in food industry is no longer acceptable. Ethical and economical concerns emerging from chicken culling need to be solved in the near future. Indeed, in egg production industry, male chicken are killed at the age of 1-day post-hatching since they are not egg producers. A number of laboratory all over the world are looking for innovative non-invasive sexing methods to determine the sex of chicken in the early stages of the development before hatching. It will allow males' chicken elimination before the pain-feeling stages. In order to evaluate the efficiency of these methods, the scientific community need a reliable, easy to use and cost-effective in-ovo invasive sexing method. In this report, we developed two new invasive assays based on PCR and Q-PCR techniques respectively, which fulfil the above mentioned requirements. In the same line with other groups, we exploited the differences betweed males (ZZ) and females (ZW) chicken sexual chromosomes. We identified two genes, SWIM and Xho-I, on chromosome W and DMRT gene on chromosome Z allowing a clear discrimination between the two sexes using PCR and qPCR respectively. These two new genomic markers and their corresponding methods not only increase the accuracy but also reduce time and cost of the test compared to previously developed sexing methods. Depending on the technology available in the lab, one can choose between the two techniques requiring different machines and expertise.

Fluorescent biosensors for drug discovery new tools for old targets--screening for inhibitors of cyclin-dependent kinases.

Cyclin-dependent kinases play central roles in regulation of cell cycle progression, transcriptional regulation and other major biological processes such as neuronal differentiation and metabolism. These kinases are hyperactivated in most human cancers and constitute attractive pharmacological targets. A large number of ATP-competitive inhibitors of CDKs have been identified from natural substances, in high throughput screening assays, or through structure-guided approaches. Alternative strategies have been explored to target essential protein/protein interfaces and screen for allosteric inhibitors that trap inactive intermediates or prevent conformational activation. However this remains a major challenge given the highly conserved structural features of these kinases, and calls for new and alternative screening technologies. Fluorescent biosensors constitute powerful tools for the detection of biomolecules in complex biological samples, and are well suited to study dynamic processes and highlight molecular alterations associated with pathological disorders. They further constitute sensitive and selective tools which can be readily implemented to high throughput and high content screens in drug discovery programmes. Our group has developed fluorescent biosensors to probe cyclin-dependent kinases and gain insight into their molecular behaviour in vitro and in living cells. These tools provide a means of monitoring subtle alterations in the abundance and activity of CDK/Cyclins and can respond to compounds that interfere with the conformational dynamics of these kinases. In this review we discuss the different strategies which have been devised to target CDK/Cyclins, and describe the implementation of our CDK/Cyclin biosensors to develop HTS/HCS assays in view of identifying new classes of inhibitors for cancer therapeutics.

Fluorescent protein biosensor for probing CDK/cyclin activity in vitro and in living cells.

Cyclin-dependent kinases (CDKs) play an essential role in the coordination of cell cycle progression and transcriptional regulation; hyperactivation is associated with cancer. However there are few means of measuring their activity in a physiological context or their inhibition in response to therapeutics. To this aim we engineered a modular fluorescent protein biosensor that reports on phosphorylation by CDK/cyclins through real-time changes in fluorescence intensity. This allowed a comparison of enzymatic activity of recombinant kinases, monitoring inhibition by small molecules, and probing endogenous activities in lysates from healthy and cancer cell lines in a sensitive and quantitative fashion. This versatile tool was further implemented to probe the oscillatory activity of these kinases throughout the cell cycle by time-lapse imaging and ratiometric fluorescence quantification, following delivery of a red fluorescent protein fusion mediated by cell-penetrating peptides.

Fluorescent biosensors for high throughput screening of protein kinase inhibitors.

High throughput screening assays aim to identify small molecules that interfere with protein function, activity, or conformation, which can serve as effective tools for chemical biology studies of targets involved in physiological processes or pathways of interest or disease models, as well as templates for development of therapeutics in medicinal chemistry. Fluorescent biosensors constitute attractive and powerful tools for drug discovery programs, from high throughput screening assays, to postscreen characterization of hits, optimization of lead compounds, and preclinical evaluation of candidate drugs. They provide a means of screening for inhibitors that selectively target enzymatic activity, conformation, and/or function in vitro. Moreover, fluorescent biosensors constitute useful tools for cell- and image-based, multiplex and multiparametric, high-content screening. Application of fluorescence-based sensors to screen large and complex libraries of compounds in vitro, in cell-based formats or whole organisms requires several levels of optimization to establish robust and reproducible assays. In this review, we describe the different fluorescent biosensor technologies which have been applied to high throughput screens, and discuss the prerequisite criteria underlying their successful application. Special emphasis is placed on protein kinase biosensors, since these enzymes constitute one of the most important classes of therapeutic targets in drug discovery.