Fluorogenic Pt complexes distinguish the quantity and folding behavior of RNA G-quadruplexes

Fluorogenic Pt complexes

Two Pt complexes with high quantum yields and photostability, and low cytotoxicity, were developed to track RNA G-quadruplexes (GQs) in live cells. Higher number and intensity, and longer lifetime of fluorescent foci in cancer cells than those in healthy cells suggest that the quantity and folding dynamics of RNA GQs could not only correlate to their biological functions, but be two novel biomarkers to characterize cancerous cells.

Functional Characterization of the N-Acetylmuramyl-l-Alanine Amidase, Ami1, from Mycobacterium

Peptidoglycan (PG) is made of a polymer of disaccharides organized as a three-dimensional mesh-like network connected together by peptidic cross-links. PG is a dynamic structure that is essential for resistance to environmental stressors. Remodeling of PG occurs throughout the bacterial life cycle, particularly during bacterial division and separation into daughter cells. Numerous autolysins with various substrate specificities participate in PG remodeling. Expression of these enzymes must be tightly regulated, as an excess of hydrolytic activity can be detrimental for the bacteria. In non-tuberculous mycobacteria such as Mycobacterium abscessus, the function of PG-modifying enzymes has been poorly investigated. In this study, we characterized the function of the PG amidase, Ami1 from M. abscessus. An ami1 deletion mutant was generated and the phenotypes of the mutant were evaluated with respect to susceptibility to antibiotics and virulence in human macrophages and zebrafish. The capacity of purified Ami1 to hydrolyze muramyl-dipeptide was demonstrated in vitro. In addition, the screening of a 9200 compounds library led to the selection of three compounds inhibiting Ami1 in vitro. We also report the structural characterization of Ami1 which, combined with in silico docking studies, allows us to propose a mode of action for these inhibitors.

Charles-Henri Buffet

JUNIOR GROUP LEADER POSITION IMMUNOLOGY AND/OR IMMUNOTHERAPY

bandeau logos IC labex inserm

JUNIOR GROUP LEADER POSITION
IMMUNOLOGY AND/OR IMMUNOTHERAPY
INSTITUT CURIE, PARIS – FRANCE

Call JPI Position INSTITUT CURIE

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Institut Curie (https://institut-curie.org) is constituted of a hospital group and a world-class multidisciplinary research center combining research in cell biology, genetics, epigenetics, immunology, soft matter physics, organic and medicinal chemistry. It includes over 3,300 researchers, physicians, clinicians, technicians and administrative staff working on three sites: Paris, Orsay and Saint-Cloud. The institute facilities include an advanced imaging platform with a wide variety of top-of-the line microscopes from super-resolution to cell and small animal live imaging. Other facilities include a small molecule collection platform, next generation sequencing, bioinformatics, reverse phase protein array, proteomics and mass spectrometry, antibody technologies and protein purification, nano-SIMS, cytometry, and animal housing (https://science.institut-curie.org/platforms/). In addition, the hospital proximity allows access to large clinical databases and sample collections.

Institut Curie will recruit a Junior Group Leader in the fields of Immunology and/or Immunotherapy. Research programs focused on both basic science and/or translational approaches are welcomed. Relevance to cancer will be positively considered.

The successful applicant will join the U932 INSERM “Immunity and Cancer ” Unit (https://u932.curie.fr/en). It currently includes 7 senior and 2 junior research teams. All have broad interests in the fields of innate and adaptive immunology, from the most fundamental aspects of immune cell biology and biophysics, to mechanistic aspects of immune responses in animal models, and to translational and clinical immunology in the context of infection and cancer. A joint appointment with the Curie hospital can be envisioned, where the recently created “Cancer Immunotherapy Center” facilitates discovery transfer from the bench to the bedside.

The newly recruited group leader will benefit from the highly competitive scientific environment of the unit, the LabEx DCBIOL and the institute, and from state-of-the-art research equipment and platforms. Laboratory space for 6-7 people will be available as well as a starting package. The successful candidate should meet the criteria to compete for national and international funding, and for French institutional research positions (University or INSERM).

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Send full CV, motivation letter, 3-4 pages research plan and three reference letters (with contacts of references) to elodie.mieville@curie.fr
For information please contact:
Sebastian Amigorena (sebastian.amigorena@curie.fr) and/or
Ana-Maria Lennon-Duménil (ana-maria.lennon@curie.fr).

Deadline for applications: January 31, 2021.
Interviews will be scheduled at mid-February 2021.

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Institut Curie is an inclusive, equal opportunities employer and is dedicated to the highest standards of research integrity.

Prognostic impact of cancer stem cell markers and in colorectal cancer.

Colon cancer develops according to a defined temporal sequence of genetic and epigenetic molecular events that may primarily affect cancer stem cells. In an attempt to identify new markers of such cells that would help predict patient outcome, we performed a comparative transcriptome analysis of colon cancer stem cells and normal colon stem cells. We identified 162 mRNAs, either over- or under-expressed. According to Cox multivariate regression with our set of 83 colorectal cancers, low expression of , tumor size and the presence of distant metastases were predictive factors for overall survival. Combined expression of and was a significant predictor for overall survival in our cohort, which was confirmed by external validation in 221 colorectal cancers from the Cancer Genome Atlas (TCGA) portal. Tumor size, lymph node involvement and expression were also independently correlated with disease-free survival. Taken together, our results suggest that molecular markers of colorectal cancers and are prognostic factors in colorectal cancer patients. It can be proposed that surveying expression of these marker genes should help better characterizing CRC prognosis, and help selecting the best therapeutic options.

The nucleus acts as a ruler tailoring cell responses to spatial constraints.

The microscopic environment inside a metazoan organism is highly crowded. Whether individual cells can tailor their behavior to the limited space remains unclear. In this study, we found that cells measure the degree of spatial confinement by using their largest and stiffest organelle, the nucleus. Cell confinement below a resting nucleus size deforms the nucleus, which expands and stretches its envelope. This activates signaling to the actomyosin cortex via nuclear envelope stretch-sensitive proteins, up-regulating cell contractility. We established that the tailored contractile response constitutes a nuclear ruler-based signaling pathway involved in migratory cell behaviors. Cells rely on the nuclear ruler to modulate the motive force that enables their passage through restrictive pores in complex three-dimensional environments, a process relevant to cancer cell invasion, immune responses, and embryonic development.

From evolution to function: Two sides of the same CENP-B coin?

The centromere is the nucleoproteic chromosomal structure necessary for accurate chromosome segregation during cell division. One of the earliest centromeric proteins to be discovered was CENP-B, the only one capable of recognizing a specific centromeric DNA binding motif. The phylogenetic history of this protein and of its DNA binding site shows independent events of function acquisition across different species and raises questions on the evolutionary dynamics of CENP-B, including what may be the selective advantage provided by its role at the centromere. Recent results have provided insight into potential functions of CENP-B in chromosome dynamics, however, its function is still object of debate. The recurrent appearance of CENP-B centromeric activity along phylogenesis, together with its dispensability, represent strictly intertwined facets of this controversy. This chapter focuses on the evolution, function and homeostasis of CENP-B and its importance in centromere biology.

Centromere strength: just a sense of proportion.

The overall structure and composition of human centromeres have been well reported, but how these elements vary between individual chromosomes and influence the chromosome-specific behavior during mitosis remains untested. In our study, we discover the existence of heterogeneity of centromeric DNA features that dictates the chromosome segregation fidelity during mitosis.

Centromeres: genetic input to calibrate an epigenetic feedback loop.

Centromeres are chromatin domains maintained by a self-templating feedback loop based on nucleosomes bearing the histone H3 variant CENP-A. The underlying centromeric DNA sequence is largely dispensable, yet paradoxically, it has highly conserved features. Hoffmann et al (2020) now uncover that when the epigenetic chromatin cycle falters, a genetically hardwired mechanism offers robustness to a dynamic epigenetic feedback loop ensuring long-term centromere inheritance.

A genetic memory initiates the epigenetic loop necessary to preserve centromere position.

Centromeres are built on repetitive DNA sequences (CenDNA) and a specific chromatin enriched with the histone H3 variant CENP-A, the epigenetic mark that identifies centromere position. Here, we interrogate the importance of CenDNA in centromere specification by developing a system to rapidly remove and reactivate CENP-A (CENP-A ). Using this system, we define the temporal cascade of events necessary to maintain centromere position. We unveil that CENP-B bound to CenDNA provides memory for maintenance on human centromeres by promoting de novo CENP-A deposition. Indeed, lack of CENP-B favors neocentromere formation under selective pressure. Occasionally, CENP-B triggers centromere re-activation initiated by CENP-C, but not CENP-A, recruitment at both ectopic and native centromeres. This is then sufficient to initiate the CENP-A-based epigenetic loop. Finally, we identify a population of CENP-A-negative, CENP-B/C-positive resting CD4 T cells capable to re-express and reassembles CENP-A upon cell cycle entry, demonstrating the physiological importance of the genetic memory.

Artificial chromosomes.

Camellia Chakraborty

Mary Saad, MD

Low-frequency variation near common germline susceptibility loci are associated with risk of Ewing

Ewing sarcoma (EwS) is a rare, aggressive solid tumor of childhood, adolescence and young adulthood associated with pathognomonic EWSR1-ETS fusion oncoproteins altering transcriptional regulation. Genome-wide association studies (GWAS) have identified 6 common germline susceptibility loci but have not investigated low-frequency inherited variants with minor allele frequencies below 5% due to limited genotyped cases of this rare tumor.

Neuroblastomas. Report From the SIOPEN Biology Group on the LNESG Trials and a COG Validation Group.

For localized, resectable neuroblastoma without amplification, surgery only is recommended even if incomplete. However, it is not known whether the genomic background of these tumors may influence outcome.

Frédérique Quignon

Phenotypic profiling with a living biobank of primary rhabdomyosarcoma unravels disease

Cancer therapy is currently shifting from broadly used cytotoxic drugs to patient-specific precision therapies. Druggable driver oncogenes, identified by molecular analyses, are present in only a subset of patients. Functional profiling of primary tumor cells could circumvent these limitations, but suitable platforms are unavailable for most cancer entities. Here, we describe an in vitro drug profiling platform for rhabdomyosarcoma (RMS), using a living biobank composed of twenty RMS patient-derived xenografts (PDX) for high-throughput drug testing. Optimized in vitro conditions preserve phenotypic and molecular characteristics of primary PDX cells and are compatible with propagation of cells directly isolated from patient tumors. Besides a heterogeneous spectrum of responses of largely patient-specific vulnerabilities, profiling with a large drug library reveals a strong sensitivity towards AKT inhibitors in a subgroup of RMS. Overall, our study highlights the feasibility of in vitro drug profiling of primary RMS for patient-specific treatment selection in a co-clinical setting.

Cell-sized liposomes reveal how actin-myosin cortical tension drives shape change

Abstract

Animal cells actively generate contractile stress in the actin cortex, a thin actin network beneath the cell membrane, to facilitate shape changes during processes like cytokinesis and motility. On the microscopic scale, this stress is generated by myosin molecular motors, which bind to actin cytoskeletal filaments and use chemical energy to exert pulling forces. To decipher the physical basis for the regulation of cell shape changes, here, we use a cell-like system with a cortex anchored to the outside or inside of a liposome membrane. This system enables us to dissect the interplay between motor pulling forces, cortex–membrane anchoring, and network connectivity. We show that cortices on the outside of liposomes either spontaneously rupture and relax built-up mechanical stress by peeling away around the liposome or actively compress and crush the liposome. The decision between peeling and crushing depends on the cortical tension determined by the amount of motors and also on the connectivity of the cortex and its attachment to the membrane. Membrane anchoring strongly affects the morphology of cortex contraction inside liposomes: cortices contract inward when weakly attached, whereas they contract toward the membrane when strongly attached. We propose a physical model based on a balance of active tension and mechanical resistance to rupture. Our findings show how membrane attachment and network connectivity are able to regulate actin cortex remodeling and membrane-shape changes for cell polarization.

Shape control of lipid bilayer membranes by confined actin bundles

Abstract

In living cells, lipid membranes and biopolymers determine each other’s conformation in a delicate force balance. Cellular polymers such as actin filaments are strongly confined by the plasma membrane in cell protrusions such as lamellipodia and filopodia. Conversely, protrusion formation is facilitated by actin-driven membrane deformation and these protrusions are maintained by dense actin networks or bundles of actin filaments. Here we investigate the mechanical interplay between actin bundles and lipid bilayer membranes by reconstituting a minimal model system based on cell-sized liposomes with encapsulated actin filaments bundled by fascin. To address the competition between the deformability of the membrane and the enclosed actin bundles, we tune the bundle stiffness (through the fascin-to-actin molar ratio) and the membrane rigidity (through protein decoration). Using confocal microscopy and quantitative image analysis, we show that actin bundles deform the liposomes into a rich set of morphologies. For liposomes having a small membrane bending rigidity, the actin bundles tend to generate finger-like membrane protrusions that resemble cellular filopodia. Stiffer bundles formed at high crosslink density stay straight in the liposome body, whereas softer bundles formed at low crosslink density are bent and kinked. When the membrane has a large bending rigidity, membrane protrusions are suppressed. In this case, membrane enclosure forces the actin bundles to organize into cortical rings, to minimize the energy cost associated with filament bending. Our results highlight the importance of taking into account mechanical interactions between the actin cytoskeleton and the membrane to understand cell shape control.