Extracting Weak Signal with the Help of Data Clustering: Application to Motoneuron Adhesion

International audienceIt is known that the external forces exerted on cells can have a strong influence through the mechano-transduction processes. They may drive the cell differentiation into osteocytes or neuron, for example. In this work, the cell adhesion of motoneuron cells have been investigated by picosecond acoustic experiment coupled to microscope objectives. Acoustic echoes bouncing on a metal/cell interface have been imaged at the micron scale. The echo?s temporal shape can reveal the spatial and dynamical properties of the cell adhesion. Due to the data acquisition time, a trade-off has to be found between signal to noise ratio and spatio-temporal resolution. In order to improve the data analysis, the signals recorded at different location are clustered according to their temporal variation which may be partially masked by the noise. It is based on a spectral clustering method where the similarity matrix is given by the cross-correlation of the time traces. Actin layers, thinner than the acoustic wavelength, have been located and their viscosity have been extracted. Complementary AFM images show good coorelation with the acoustic images. Since the cell viscosity may affect cell adhesion measurement as performed in picosecond acoustics experiments, the problem arising from the standard massless Hook spring used to model the adhesion layer will be discussed

Correlative imaging of motoneuronal cell elasticity by pump and probe spectroscopy

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Picosecond ultrasounds as elasticity probes in neuron-like cells models

International audienceWe report on elasticity measurements in neuron-like cells using picosecond acoustics pump and probe spectroscopy. The stimulated Brillouin oscillations were mapped in PC12 cells to reveal their internal elastic structure. Thanks to a Pearson correlation coefficient mapping, different areas could be distinguished. The nucleus material shows a bulk modulus equal to 12.9 GPa in the case of dry cell. Attenuation of the Brillouin signature gives access to dynamical longitudinal viscosity equal to 10.6 mPa · s, one order magnitude higher than water. The modulus considerably drops to 2.6 GPa in the most physiologically relevant case of a hydrated cell. Keywords: Neuronal cell, elasticity, pump and probe spectroscopy. Cells respond to mechanical signals perceived from the nearest extracellular world 1-4. For instance, it has been suggested that mechanical constraints prevail over biochemical signaling in the early stage of embryogenesis 5. Substrate stiffness has also been identified as a key factor driving cell proliferation and differentiation 6. Both endothelial and smooth muscle cells were shown to proliferate in response to stretching ; however, in the case of endothelial cells this response depends on cell-cell adhesion 7. In the mechanotransduction process, external forces exerted on the cells transit inside them through microscale adhesions domains that serve as anchoring points for the structuration of the cellular cytoskeletal network. This phenomenon allows the cell to sense its surrounding environment and is followed by the activation of fundamental cellular processes involving motility or changes in cell shape 8. Obviously, how this regulation occurs will depend on the cell type and function. In the case of tumors, the increase in rigidity could be related to various factors, including an increase in the modulus of elasticity of transformed cells due to cellular disturbances. This leads to tumors being generally stiffer than normal tissues 9,10. Perturbation of tissue rigidity is associated with different types of pathology. However, it is sometimes difficult to conclude if this change in stiffness of cells or tissue is the effect or the source of the pathologies 11. This is why the characterization of the mechanical properties of cells is essential to understand their behavior during mitosis, apoptosis, adhesion, a) Electronic mail: [email protected] mobility and disease development 12-14. However, the complexity of the inner cell composition and the intricate mesh-work formed by molecular mediators of the transmembrane cell-substrate interactions requires non-invasive techniques to probe and quantify local mechanical properties of cells, including modulus of elasticity, viscoelastic properties, adhesion , and forces created at the single-cell scale. Several recent reviews describe tools used to study cell mechanics 15,16 and to apply forces on them 17. The vast majority of conventional methods of measuring the local mechanical properties of cells are based on the use of solid probes, such as AFM, and as a result the measured mechanical properties can strongly depend on the contact/adhesion between the probe and the cell. In contrast, acoustic waves generated by lasers provide a very adequate tool for probing the mechanical properties of biological cells or tissues in a non-contact, non-invasive configuration. In the optical pump probe technique usually called picosecond acoustics (PA), high frequency acoustic pulses (in the 1 − 1000 GHz range) can be generated by the pump beam and detected using a delayed probe beam. Since acoustic waves travel several microns per nanosecond, it is possible to study material on a submicron scale with acoustic waves of 10 GHz or more. Such time resolved measurements are known to achieve sound velocity characterization with an accuracy less than < 5 %, parameter directly related to the elasticity behavior. In addition, by combining the time and space aspects, it is possible to perform 3D elastic investigations with sub micrometer resolution. To finish, the all-optical approach allows to consider complex environments to address issues related to relevant biological conditions, aqueous media, controlled temperature. For more than 30 years, properties of matter, mainly solid thin metallic films and transparent me-This is the author's peer reviewed, accepted manuscript. However, the online version of record will be different from this version once it has been copyedited and typeset. PLEASE CITE THIS ARTICLE A

Molecular Analysis of a Congenital Myasthenic Syndrome Due to a Pathogenic Variant Affecting the C-Terminus of ColQ

International audienceCongenital Myasthenic Syndromes (CMSs) are rare inherited diseases of the neuromuscular junction characterized by muscle weakness. CMSs with acetylcholinesterase deficiency are due to pathogenic variants in COLQ, a collagen that anchors the enzyme at the synapse. The two COLQ N-terminal domains have been characterized as being biochemical and functional. They are responsible for the structure of the protein in the triple helix and the association of COLQ with acetylcholinesterase. To deepen the analysis of the distal C-terminal peptide properties and understand the CMSs associated to pathogenic variants in this domain, we have analyzed the case of a 32 year old male patient bearing a homozygote splice site variant c.1281 C > T that changes the sequence of the last 28 aa in COLQ. Using COS cell and mouse muscle cell expression, we show that the COLQ variant does not impair the formation of the collagen triple helix in these cells, nor its association with acetylcholinesterase, and that the hetero-oligomers are secreted. However, the interaction of COLQ variant with LRP4, a signaling hub at the neuromuscular junction, is decreased by 44% as demonstrated by in vitro biochemical methods. In addition, an increase in all acetylcholine receptor subunit mRNA levels is observed in muscle cells derived from the patient iPSC. All these approaches point to pathophysiological mechanisms essentially characterized by a decrease in signaling and the presence of immature acetylcholine receptors

Overexpression of Nucleolin and Associated Genes in Prostate Cancer

International audienceProstate cancer (PCa) is the second most frequent cancer and the fifth leading cause of cancer death in men worldwide. If local PCa presents a favorable prognosis, available treatments for advanced PCa display limiting benefits due to therapeutic resistances. Nucleolin (NCL) is a ubiquitous protein involved in numerous cell processes, such as ribosome biogenesis, cell cycles, or angiogenesis. NCL is overexpressed in several tumor types in which it has been proposed as a diagnostic and prognostic biomarker. In PCa, NCL has mainly been studied as a target for new therapeutic agents. Nevertheless, little data are available concerning its expression in patient tissues. Here, we investigated the expression of NCL using a new cohort from Mondor Hospital and data from published cohorts. Results were then compared with NCL expression using in vitro models. NCL was overexpressed in PCa tissues compared to the normal tissues, but no prognostic values were demonstrated. Nine genes were highly co-expressed with NCL in patient tissues and tumor prostate cell lines. Our data demonstrate that NCL is an interesting diagnostic biomarker and propose a signature of genes co-expressed with NCL

Implication of NPM1 phosphorylation and preclinical evaluation of the nucleoprotein antagonist N6L in prostate cancer

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CRIPTO overexpression promotes mesenchymal differentiation in prostate carcinoma cells through parallel regulation of AKT and FGFR activities

Members of the EGF-CFC (Cripto, FRL-1, Cryptic) protein family are increasingly recognized as key mediators of cell movement and cell differentiation during vertebrate embryogenesis. The founding member of this protein family, CRIPTO, is overexpressed in various human carcinomas. Yet, the biological role of CRIPTO in this setting remains unclear. Here, we find CRIPTO expression as especially high in a subgroup of primary prostate carcinomas with poorer outcome, wherein resides cancer cell clones with mesenchymal traits. Experimental studies in PCa models showed that one notable function of CRIPTO expression in prostate carcinoma cells may be to augment PI3K/AKT and FGFR1 signaling, which promotes epithelial-mesenchymal transition and sustains a mesenchymal state. In the observed signaling events, FGFR1 appears to function parallel to AKT, and the two pathways act cooperatively to enhance migratory, invasive and transformation properties specifically in the CRIPTO overexpressing cells. Collectively, these findings suggest a novel molecular network, involving CRIPTO, AKT, and FGFR signaling, in favor of the emergence of mesenchymal-like cancer cells during the development of aggressive prostate tumors

The Neuropilin-1/PKC axis promotes neuroendocrine differentiation and drug resistance of prostate cancer

International audienceAbstract Background Neuroendocrine prostate cancer (NEPC) is a multi-resistant variant of prostate cancer (PCa) that has become a major challenge in clinics. Understanding the neuroendocrine differentiation (NED) process at the molecular level is therefore critical to define therapeutic strategies that can prevent multi-drug resistance. Methods Using RNA expression profiling and immunohistochemistry, we have identified and characterised a gene expression signature associated with the emergence of NED in a large PCa cohort, including 169 hormone-naïve PCa (HNPC) and 48 castration-resistance PCa (CRPC) patients. In vitro and preclinical in vivo NED models were used to explore the cellular mechanism and to characterise the effects of castration on PCa progression. Results We show for the first time that Neuropilin-1 (NRP1) is a key component of NED in PCa cells. NRP1 is upregulated in response to androgen deprivation therapies (ADT) and elicits cell survival through induction of the PKC pathway. Downmodulation of either NRP1 protein expression or PKC activation suppresses NED, prevents tumour evolution toward castration resistance and increases the efficacy of docetaxel-based chemotherapy in preclinical models in vivo. Conclusions This study reveals the NRP1/PKC axis as a promising therapeutic target for the prevention of neuroendocrine castration-resistant variants of PCa and indicates NRP1 as an early transitional biomarker