On chip cellular dielectric spectroscopy

In the recent years, the development of automated systems for cellular monitoring has been highly promoted in basic biological research as well as in industries and medical diagnostic. Several methods for transduction of cellular signals have been investigated and it has been demonstrated that impedance-based techniques have great potential for real-time, non invasive cell-based assays for both high throughput screening and research laboratory environments. This thesis presents the fabrication and design of a microelectrodes-based dielectric sensor for cellular analysis, and its application to monitor in-vitro physiological functions of different cell types. A dual array of planar interdigitated microelectrodes has been patterned on the bottom of two microchambers used for cells culture. The system allows real-time dynamic cellular monitoring with high temporal resolution and high sensitivity. The device enables multifrequency characterization (frequency between 100Hz and 30MHz), so that the integral cellular response is obtained and several independent effects can be monitored in parallel at different frequencies. Analytical models are used to evaluate the evolution of various cell parameters, such as cell membrane capacitance and cytoplasm conductivity, and to obtain information about cell morphology and adhesion. To interface the microelectrode array to the external instrumentation, a printed circuit board has been developed, which includes the circuitry controlling the switch between reference/sensing chamber and calibration/measurement mode. In addition, the device is controlled by a computer interface, to automatize the acquisition of the impedance spectra and select long or short term response monitoring. Two innovative biological applications are proposed for the presented device. The effect of D-glucose on biconcave erythrocytes has been investigated. The variation of glucose concentration in plasma is an important biological process and it has been extensively studied because of its implication in diseases like diabetes. Measurements have been performed on erythrocyte suspensions exposed to several D-glucose concentrations. The results have shown that glucose induces variations in the dielectric properties of erythrocytes and affects mainly the plasma membrane capacitance. A better understanding of this mechanism is fundamental for a future development of impedance glucose sensors based on the evaluation of the dielectric properties of the red blood cells. Furthermore, a functional assay of the anthrax toxin receptor on endothelial cells has been performed using the developed device. This transmembrane protein is encoded by the Capillary Morphogenesis Gene 2 (CMG2), which was identified as a gene overexpressed in endothelial cells during capillary morphogenesis. This protein is known as an anthrax toxin receptor and it has been discovered that its mutations lead to a rare human disease (Hyaline Fibromatosis Symdrome). On the contrary, its physiological function has still to be revealed. To investigate a potential physiological ligand of CMG2, vascular endothelial cells (HUVEC) have been cultured on the microelectrodes array coated with different extracellular matrix protein. The adhesion kinetic of wild-type HUVEC has been recorded for several hours and compared with the adhesion of vascular cells in which the receptor CMG2 is inhibited. The results obtained in this work provide new knowledge about the physiological role of this receptor associated with different ECM proteins.

Unraveling the Physiological Role of Capillary Morphogenesis Gene 2 in Health and Disease

Jérôme Bürgi

Capillary Morphogenesis Gene 2 (CMG2) is a 55kDa single-pass transmembrane protein best described as the main Anthrax Toxin Receptor. However, CMG2 physiological role remains elusive and understanding better its in vivo function was the main objective of my thesis. My first aim was to investigate the prevalence of intrinsically disordered domains in the human transmembrane proteome. Using prediction tools, I found that 50% of transmembrane proteins had at least one domain of minimum 30 amino-acids predicted as intrinsically disordered (IDD), these proteins being mainly involved in cell signaling and adhesion. The majority of the IDDs were localized in the cytoplasm, and the intrinsic disorder containing proteins were significantly more phosphorylated and interacted on average with more proteins than fully ordered proteins, corroborating their role in signaling. CMG2 loss-of-function mutations is the known cause of a rare inherited genetic disorder called Hyaline Fibromatosis Syndrome (HFS). HFS patients develop subcutaneous nodules, gingival hypertrophy, contracture of the large joints, and in the most severe cases diarrhea and premature death. In a second part of my thesis, I was involved in understanding the molecular defects underlying missense mutations localized in the ectodomain and a highly conserved cytoplasmic domain of CMG2. We found that CMG2 was able to bind the actin cytoskeleton through Talin and Vinculin when free of its extracellular ligand. Interestingly, the HFS mutation p.C218R, altered the cytoskeleton release and led to a CMG2 protein able to bind both the extracellular ligand and the actin cytoskeleton. Strikingly, we observed that all but one cytoplasmic missense mutation hindered actin binding, a defect potentially relevant to HFS pathogenesis. Furthermore, we showed that the Src-like kinase family are involved in the release of Talin upon ligand binding and that CMG2 binding to its extracellular ligand is critical for the correct orientation of the mitotic spindle of zebrafish epiblast cells, which allows cell division along the Animal/Vegetal axis. Through the analysis of cmg2 knock-out mice, along with the study of HFS patient samples, we aimed at better defining CMG2 function. We showed that type VI Collagen was strongly accumulating in the cmg2 KO mice uterus tissue leading to failure of parturition, an accumulation that was also observed in HFS patient nodules. In addition, we observed that CMG2 was able to interact and potentiate MT1-MMP, and that type VI Collagen is a bona fide ligand for CMG2, its binding leading to the phosphorylation of the receptor. With the scope to understand the HFS patient subcutaneous nodules pathogenesis, we investigated the gene expression profile of fibroblasts derived from non-affected and affected tissues by RNA sequencing. Fibroblasts derived from affected tissues appeared to differentially express genes involved in the TGFBeta pathway and cytoskeleton, such as alpha smooth muscle actin, a marker of myofibroblast activation. We observed the presence of myofibroblasts in the patient nodules, and showed that CMG2 was limiting their activation process, loss-of-function mutations in CMG2 leading to an exacerbated differentiation. As we observed CMG2 interaction with the TGFBeta receptor 2, we postulated that CMG2 influence the TGFBeta pathway, a process that is altered in HFS patient and potentially lead to the formation of nodules.

EPFL2015

Investigating molecular interactions of G protein coupled receptors by fluorescence techniques

Jean-Baptiste Perez

G Protein coupled receptors (GPCRs) constitute an abundant family of membrane proteins which play a central role in cellular signaling and are important targets for modern medicine. Despite intensive research, central questions of the molecular mechanism of GPCR mediated signal transduction remain unresolved. This thesis concerns α1b-adrenergic receptor (α1b-AR) as a prototypic GPCR. The question whether ARs form homo- or hetero-oligomers was investigated using fluorescence resonance energy transfer (FRET). An important finding was that both α1a and α1b-AR subtypes form homo-oligomers. Also hetero-oligomers have been observed between the α1b- and the α1a-AR subtypes, but not with less related receptors. Another interesting finding concerns α1-AR co-internalization which correlates with the ability of the receptor to hetero-oligomerize. Investigating particular processes in living cells is often complicated by the complex cellular environment. Here we have developed a rather simple approach to study transmembrane signaling by using supported cell membrane sheets. They were prepared by pressing a glass coverslip on the apical part of cultured cells, ripping off large regions of the native plasma membrane. For the α1b-AR we still could observe ligand binding to such sheets, indicating that GPCR functionality was at least partly conserved. Due to the absence of cytosolic autofluorescence of the cells, supported membrane sheets were also found to be suited for single-molecule microscopy. Because both leaflets of the supported membranes remained fluid, it was possible to follow the diffusion of certain membrane proteins. For example using fluorescence recovery after photobleaching (FRAP) and single-molecule microscopy, we investigated how G proteins diffused along the membrane and compartmentalized. The predominant role of lipid anchors in G protein localization was highlighted by measuring the diffusion of two Gαq proteins fused with citrine at two different positions and citrine based constructs designed to mimic the monomeric and the trimeric form of a G protein. Finally, first results were obtained showing the feasibility to form supported cell membrane sheets on microstructured devices consisting of a planar silicon support perforated with arrays of holes. This geometry provides a full accessibility to both intra- and extracellular sides of the bilayer. This concept could potentially be applied for the development of chip-based screening assays.

EPFL2005

Molecular Mechanisms Underlying Hyaline Fibromatosis Syndrome

Julie Deuquet Ariosa

Hyaline Fibromatosis Syndrome (HFS) is a rare inherited disease that is characterized by an accumulation of an unidentified hyaline material, largely affecting connective tissues. Patients afflicted with HFS present a wide range of clinical symptoms such as nodules, papules, hyperplasia of the gingiva, joint contractions, thickness and hyperpigmentation of the skin, and osteopenia. Depending of the severity and the delay of appearance of these symptoms, the life expectancy varies between 2 years and > 30 years. Death earlier in life is due to recurrent diarrhea and chest infections of unknown origin. In 2003, the gene responsible for HFS was identified to be Capillary Morphoqenesis Gene 2 (CMG2), a gene upregulated during capillary morphogenesis in three-dimensional collagen matrices. This gene encodes a type I transmembrane glycoprotein composed of a von Willebrand (vWA) domain followed by an immunoglobulin (Ig)-like domain in its extracellular part, a single α-helix spanning transmembrane domain, and a 148 amino acids cytoplasmic tail predicted as unstructured. Even though the physiological function of CMG2 remains to be elucidated, it has been shown that this protein induces endothelial proliferation, and dictates cell polarization during embryogenesis in a zebrafish model. Besides its physiological function, CMG2 was found to be an anthrax toxin receptor, thus its alternative name ANTXR2, and has been extensively studied in this context. Genetic analysis has led to the identification of 34 CMG2 mutations in HFS patients. The molecular mechanisms underlying the disease have however not yet been identified. During the five years of my thesis, we were interested in understanding the cellular consequences of these mutations and in getting a better understanding of the physiological function of CMG2. I first showed that most of the HFS mutations in the vWA domain lead to alterations in folding of the domain, either in terms of kinetics or of structure, leading to recognition of the protein by the ER quality control (ERQC) and subsequent degradation by the ERAD pathway. A direct consequence of this retention is the loss of function at the cell surface or other cellular sites. In a second study, I studied the effect of novel HFS mutations that our collaborators and we identified in the CMG2 Ig-like domain. Since the structure of this domain was uncharacterized, we first generated a model of the domain and found that it has an Ig-like fold, despite the very low sequence homology, and harbors two disulfide bonds essential for folding and stabilization of the domain. HFS mutations localized to this domain led to aberrant intermolecular disulfide binding, ER retention and enhanced ERAD when compared to the vWA domain HFS mutations as shown in patients fibroblasts. The consequential loss of CMG2 could be partly alleviated by treating cells with proteasome inhibitor, such as Bortezomib, illustrating that ERAD components are potential therapeutic targets for HFS. As most of the HFS mutations in the ectodomain of CMG2 trigger this ER retention during the CMG2 biosynthesis, we then addressed this process by studying one of the most described post-translational modifications occurring in the ER for protein folding, namely the N-glycosylation. We found that the two potential CMG2 N-glycosylation sites, N250 and N260, are both occupied. Neither of the two sites is essential for ER exit. Glycosylation of CMG2 on N260 is however important since the expression levels of the N260A mutant were significant lower that for the WT protein, despite equivalent or higher synthesis, suggesting that glycosylation at this site is important for efficient folding. Finally, in order to better understand the CMG2 function, we investigated what the physiological ligands could be, assuming it is a receptor as suggested by its role in anthrax infection. We identified type VI collagen as a potential ligand in-vitro that activates CMG2 in-vivo, by phosphorylation of CMG2 cytoplasmic tail. This CMG2 activation upon type VI collagen binding could be a signal for ligand-triggered endocytosis, as is has been described with the protective antigen (PA) of anthrax toxin. The results of this work allow us to gain insights in the molecular mechanisms underlying HFS. Our studies indicate that the identification of the genotype-phenotype correlation of this disease is important for proper diagnosis and clinical approaches, and also that a therapeutic strategy based on proteasome inhibitors, such as Bortezomib, could be envisioned to treat some HFS symptoms in order to improve the life conditions of the patients.

EPFL2011