Amandine Pitaval

Cell shape and contractility regulate ciliogenesis in cell cycle–arrested cells

Adhesive micropatterns show the effect of spatial confinement and actin network architecture on basal body positioning and primary cilium formation.

Prion protein prevents human breast carcinoma cell line from tumor necrosis factor α-induced cell death

To define genetic determinants of tumor cell resistance to the cytotoxic action of tumor necrosis factor α (TNF), we have applied cDNA microarrays to a human breast carcinoma TNF-sensitive MCF7 cell line and its established TNF-resistant clone. Of a total of 5760 samples of cDNA examined, 3.6% were found to be differentially expressed in TNF-resistant 1001 cells as compared with TNF-sensitive MCF7 cells. On the basis of available literature data, the striking finding is the association of some differentially expressed genes involved in the phosphatidylinositol-3-kinase/Akt signaling pathway. More notably, we found that the PRNP gene coding for the cellular prion protein (PrPc), was 17-fold overexpressed in the 1001 cell line as compared with the MCF7 cell line. This differential expression was confirmed at the cell surface by immunostaining that indicated that PrPc is overexpressed at both mRNA and protein levels in the TNF-resistant derivative. Using recombinant adenoviruses expressing the human PrPc, our data demonstrate that PrPc overexpression converted TNF-sensitive MCF7 cells into TNF-resistant cells, at least in part, by a mechanism involving alteration of cytochrome c release from mitochondria and nuclear condensation.

Expression profiling of genes and proteins in HaCaT keratinocytes: proliferating versus differentiated state.

The knowledge of the mechanism of keratinocyte differentiation in culture is still uncompleted. The emergence of new technologies, such as cDNA microarrays or 2D electrophoresis followed by mass spectrometry analysis, has allowed the identification of genes and proteins expressed in biological processes in keratinocytes. Here, we report a genome wide analysis of proliferating versus differentiated human HaCaT keratinocytes. We found that genes and proteins which take part in the cell cycle control, carbohydrate metabolism, cell auto‐immunity, adhesion and cytokine signal transduction pathways were regulated in differentiated HaCaT keratinocytes. In addition, we identified seven proteins and 33 transcripts that had not been previously described as differentially expressed in proliferating versus differentiated HaCaT cells. Furthermore, some of these transcripts or proteins were similarly regulated in human primary keratinocytes and in human epidermis. The present study opens new areas of investigation in the comprehension of keratinocyte differentiation.

A statistically inferred microRNA network identifies breast cancer target miR-940 as an actin cytoskeleton regulator

MiRNAs are key regulators of gene expression. By binding to many genes, they create a complex network of gene co-regulation. Here, using a network-based approach, we identified miRNA hub groups by their close connections and common targets. In one cluster containing three miRNAs, miR-612, miR-661 and miR-940, the annotated functions of the co-regulated genes suggested a role in small GTPase signalling. Although the three members of this cluster targeted the same subset of predicted genes, we showed that their overexpression impacted cell fates differently. miR-661 demonstrated enhanced phosphorylation of myosin II and an increase in cell invasion, indicating a possible oncogenic miRNA. On the contrary, miR-612 and miR-940 inhibit phosphorylation of myosin II and cell invasion. Finally, expression profiling in human breast tissues showed that miR-940 was consistently downregulated in breast cancer tissues

Cell Microarray for Functional Exploration of Genomes

As more genomes are sequenced, challenge of rapidly unraveling the functions of genes was faced. To that end, cell microarrays have been recently described that permit transfection of thousands of nucleic acids in parallel and enable the analysis of phenotypic consequences of such perturbations. As many parameters can influence the efficiency of transfection and consequently protein expression or extinction, some important features in manufacturing cell microarrays for functional exploration of genomes were described.

A kinome-targeted RNAi-based screen links FGF signaling to H2AX phosphorylation in response to radiation

AbstractA general radioprotective effect by fibroblast growth factor (FGF) has been extensively described since the early 1990s; however, the molecular mechanisms involved remain largely unknown. Radiation-induced DNA double-strand breaks (DSBs) lead to a complex set of responses in eukaryotic cells. One of the earliest consequences is phosphorylation of histone H2AX to form nuclear foci of the phosphorylated form of H2AX (γH2AX) in the chromatin adjacent to sites of DSBs and to initiate the recruitment of DNA-repair molecules. Upon a DSB event, a rapid signaling network is activated to coordinate DNA repair with the induction of cell-cycle checkpoints. To date, three kinases (ATM, ATR, and DNA-PK) have been shown to phosphorylate histone H2AX in response to irradiation. Here, we report a kinome-targeted small interfering RNA (siRNA) screen to characterize human kinases involved in H2AX phosphorylation. By analyzing γH2AX foci at a single-nucleus level, we identified 46 kinases involved either directly or indirectly in H2AX phosphorylation in response to irradiation in human keratinocytes. Furthermore, we demonstrate that in response to irradiation, the FGFR4 signaling cascade promotes JNK1 activation and direct H2AX phosphorylation leading, in turn, to more efficient DNA repair. This can explain, at least partially, the radioprotective effect of FGF.

Probing Ciliogenesis Using Micropatterned Substrates

The primary cilium is a biomechanical sensor plugged in at the cell surface. It is implicated in the processing of extracellular signals and its absence or misfunctioning lead to a broad variety of serious defects known as ciliopathies. Unfortunately, the precise mechanisms underlying primary cilium assembly and operation are still poorly understood. Molecular dynamics and intracellular morphogenesis are easier to study in cell culture than in tissues. However, cultured cells are usually nonciliated and the empirical methods that are used to induce ciliogenesis in these cells have variable efficiencies. In addition, these methods require cells to be cultured at high density, which is not convenient for further automated image analysis. Here, we describe a method to induce and modulate ciliogenesis in mammalian cells in culture that is compatible with high-throughput imaging and analysis. Surface micropatterning is used to normalize cell shape and actin network architecture. In these conditions, the deprivation of growth factor induces ciliogenesis in individual single cells. The manipulation of cell-spreading area is used to modulate the proportion of ciliated cells. The manipulation of cell adhesion geometry is used to orient the position of the primary cilium. The spatial disposition of cells on a regular array offers a simple way to perform automated image acquisition. In addition, the regular cell shape is convenient to perform robust and automated image analysis to quantify the presence and location of primary cilia. This method offers a new way to study ciliogenesis in automated and high-throughput assays.