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Fabian Anderegg

The characterization of surface features that control the assembly of the resorption apparatus in cultured osteoclasts

This approach will be primarily based on micro- and nanofabrication of biomimetic adhesive surfaces. One of the used technologies is photolithography, which is a widely applied technology in the semiconductor industry to create micro patterned surfaces with a resolution down to few hundred nanometers. Using a combined photolithographic and molecular assembly technique called Molecular Assembly Patterning by Lift-off (MAPL) we create polymer and protein derived micro patterns, which allow us to tailor both the chemical and the physical composition of the substrate. By this we hope to gain insight into the role substrate properties such as topography or adhesion have in sealing zone assembly and dynamics.

 

Kristyna Bicanova

Focal degradation of the extracellular matrix by invasive tumour cells. The central role of caveolin 1 in the regulation of lipid composition and protein phosphorylation at invadopodia.

I will be working towards revealing the morpho-functional basis of the highly organized structure and function of invadopodia in tumour cells. We have previously shown that invadopodia exhibit features of raft-like microdomains and that their integrity and functionality rely tightly on cholesterol levels at the plasma membrane.  In my current research, I will study the role of cholesterol-interacting protein caveolin 1 and membrane lipids in cytoskeletal organization and ECM degradation at invadopodia taking advantage of available caveolin 1 mutants and manipulation of membrane lipid composition. In addition, I plan to extend the observed effects in other invasion-related cell behaviours, such as motility, and in 3D settings.
Furthermore, in collaboration with RUMC, studies will be performed to directly assess the membrane order state at forming and fully formed invadopodia by multiphoton microscopy.

 

Pasquale Cervero

Analysis of podosome enriched cell fractions from macrophages

The aim of the project  is the proteomic analysis of podosome enriched cell fractions from macrophages using SILAC (Stable Isotope Labelling with Aminoacids in Cell culture) and subsequent Mass Spectrometry in order to identify and study new podosome components.
Moreover, I will employ micro-patterned substrates to elucidate the minimal/maximal inter-podosome distances supporting the formation of a podosome network in macrophages, as well as the associated changes in local contractility.

 

Paolo Ciufici

Characterization of the post-translational modifications and novel protein partner affecting the Cdc42-specific guanine nucleotide exchange factor Fgd1 in cell invasion

The Cdc42 guanine exchange factor Fgd1 is a novel invadosome component. It is required for invadosome formation and function in endothelial cells and carcinoma cells. How Fgd1 is regulated is still unknown. My aim is to characterize the post-translational modifications and novel protein partners affecting Fgd1 in the process of invadosome formation in these two models. We expect to reveal common regulatory mechanisms operating on Fgd1 but also regulatory mechanisms specifically involved in invadosome formation. Altogether, the expected results should help to understand the initial steps of invadosome formation in tumor angiogenesis and cancer metastasis.

 

Filipa Dos Santos Curado

Molecular mechanisms of TGFβ-induced endothelial podosome formation

Cultured aortic endothelial cells form podosomes upon exposure to the inflammatory cytokine TGFβ in vitro. Experiments will be performed in this model to identify the TGFβ receptor(s) and co-receptor(s) involved, and dissect out the molecular links to downstream effectors in the process of endothelial podosome assembly and subsequent matrix degradation. We will also analyse the phenotypic and behavioural characteristics of these cells. These studies will continue by testing arterial endothelial cells isolated from genetically engineered mice with TGFβ signalling disorders in order to determine the consequences of defective endothelial podosome formation at the vessel level.

 

Isabel Egaña

Podosomes in the living endothelium and their role in vascular remodelling

Work in our lab has now provided evidence that the process of endothelial formation in cultured aortic endothelial cells also occurs in native tissues. Rosettes of podosomes are detected in situ, in the endothelium of living aortic vessel segments from mice, when exogenous TGFβ is applied. Our aim is now to demonstrate that endothelial podosomes are formed upon endogenous TGFβ release. To this end, several models based on the use of genetically engineered mice presenting increased TGFβ signalling will be employed. The architectural and functional characteristics of endothelial podosomes will then be examined in detail to establish the role of these structures and the fate of podosome forming cells in vivo.

 

Karim El-Azzouzi

Characterization of proteinases involved in ECM degradation by podosomes

The aim of my project is the identification of specific proteinases involved in podosome-localised matrix degradation and their mode of transport. We will also focus on intracellular transport processes in podosome regulation. By labelling the matrix (e. g. gelatin or fibronectin) and using fluorescently tagged isoforms of proteinases in live cell imaging, we expect to visualise their trafficking through several subcellular compartments, and monitor the release of proteinases at the podosome level before the matrix degradation process takes place.  

 

Dan Georgess

Molecular mechanisms implicated in bone resorption

Osteoclasts (OCs) are multinucleated bone resorbing cells that result from monocyte/macrophage fusion in the presence of two cytokines: M-CSF (Macrophage Colony Stimulating Factor) and RANKL (Receptor Activator on NF-kB Ligand). Besides this canonical path of differentiation, my host laboratory has shown that OCs can also be transdifferentiated from Dendritic Cells (DCs) when treated with M-CSF and RANKL.
Recently, our collaborators have described a new population of multinucleated giant cells (MGCs) resulting from DC fusion in the presence of Interferon
g and IL-17 (Interleukin 17) (Coury et al, Nat Med, 2008). These MGCs present most of the OC characteristics such as TRAP (Tartrate-Resistant Acid Phosphatase) and cathepsin-K but are, however, unable to resorb bone. A transcriptomic comparison between bone resorbing monocyte- or DC-derived OCs and non-resorbing DC-derived MGCs has aimed to identify candidate genes specifically implicated in bone resorption.
The statistical analysis of the transcriptomic data has identified 98 annotated genes that are expressed in monocyte- and DC-derived OCs but not in MGCs, monocytes nor DCs. These genes have been categorized on a functional basis using Gene Ontology Molecular Function software. Briefly, 35 genes code for proteins implicated in cellular metabolism among which proteases, 22 genes code for transporters, 16 genes code for cytoskeleton-related proteins, 7 genes code for transcription regulators and 7 genes code for secreted proteins.
Based on the results of the transcriptomic study, my PhD project aims to investigate the function of these genes and to find those that characterize bone resorption.

 

Olga Ilina

Using 3D collagen matrix cultures, lytic actin-rich structures towards collagen fibers are being visualized and inhibited using small compounds and RNAi approaches. The outcome of migration mode and efficiency after protease inhibition is being monitored by live-cell microscopy.

 

 
 

Vinoth K.M. Khandelwal

Study of the molecular machinery regulating focal degradation of the extracellular matrix by cancer cells. Role of Dynamin 2 in extracellular matrix degradation and motility.

Human Dynamin 2 is a member of the large GTP-binding protein (98 kDa) family. Dynamin 2 (Dyn2) is encoded by the DNM2 gene located on the short arm of chromosome 19. Dyn2 has been intensely studied in the recent years, especially with respect to its prominent role on the regulation of endocytosis through the control of membrane fissioning events. Latest studies, however, have indicated direct or indirect roles of Dyn2 in focal adhesion turnover, degradation of the extra cellular matrix (ECM) at invadopodia and podosomes, and actin reorganization. Furthermore, Dyn2 mutations have been shown to cause human hereditary diseases such as Charcot–Marie-Tooth disease and centronuclear myopathy. Our aim is to extend our previous studies demonstrating the role of Dyn2 in ECM degradation, invadopodia formation and cell motility, especially with regard to its regulation by phosphorylation and interaction with other pivotal regulatory proteins such as cortactin and caveolin 1.

 

Franco Klingberg

The influence of cell stress and extracellular matrix stiffness on the bio-availability of transforming growth factor beta

Epithelial tumors activate resident stroma cells (fibroblasts) to generate a chemical and mechanical environment that stimulates tumor progression and that has many aspects of fibrosis. Responsible for these detrimental conditions are myofibroblasts that are characterized by excessive production of collagenous extracellular matrix (ECM) and neo-formation of contractile bundles of actin and myosin (stress fibers). Myofibroblast differentiation depends on the presence of mechanical stress and transforming growth factor beta1 (TGFβ1). Using novel polymer culture substrates with tunable stiffness that are the core expertise of Excellness Biotech SA and the laboratory of Dr. Boris Hinz, we previously demonstrated that myofibroblast contraction directly activates TGFβ1 from self-generated latent stores in the ECM. This requires binding of latent TGFβ1 to a stiff ECM and transmission of cell forces to latent TGFβ1 via yet unidentified integrins. The activation potential of TGFβ1 is influenced by the way it is presented in the ECM to the activation machineries of cells. We propose that myofibroblasts mechanically prime TGFβ1 for activation by pre-organizing the latent complex during its secretion into the ECM.
It is our hypothesis that the mechanical state of the microenvironment influences activation of TGFβ1 by myofibroblasts and progression of fibrosis. We will determine the molecular mechanisms of how myofibroblast contraction and ECM stress influence the availability and activation of latent TGFβ1 in the ECM.

 

Marina Krause

 

Ground-state depletion microscopy (GSD-IM) is a super-resolution imaging approach to visualize structures far below the diffraction limit. As such resolutions below 50 nm can be achieved. Small complexes can be resolved in a single-protein-manner. Thus proteins could be much more precisely located and maturation of specific structures could be studied. This project aims to visualize proteolytic adhesion complexes in invading cancer cells at unprecedendent resolution using GSD-IM. Using 3D collagen matrices for detection of adhesion complexes and their locations to individual collagen fibers, we aim to detect specific signals for beta-1 integrins and surface-localized membrane-type matrix metalloproteinases (MT-MMPs). This will provide a map of adhesion and proteolytic cell surface subregions and zones of ECM remodeling.

 

Nilesh Talele

Fibrogenesis of mesenchymal stem cells:
Roles of extracellular matrix stiffness

Mesenchymal stem cells (MSCs) are pluripotent cells with the potential to regenerate organs. MSC have gained recent attention as delivery agents of anti-cancer drugs, encompasses their engraftment into fibrotic tissue that surrounds epithelial tumors. This hostile environment can drive engrafted MSCs to develop fibrogenic rather than regenerative or tumor-resolving characteristics. MSCs were shown to differentiate into myofibroblasts when delivered into post-infarct myocardial scar, fibrotic kidney, liver and lung. The progression of fibrosis at the time of MSC engraftment is critical. MSCs engrafted into early stages of fibrosis appear to improve organ healing; MSCs delivered to mature scar are more prone to fibrogenesis. The molecular mechanisms underlying this discrepancy are virtually unknown. It is known, however, that fibrotic tissue becomes significantly stiffer with progression of the disease and that stiff extracellular matrix (ECM) is one of the main factors driving myofibroblast fibrogenesis. Myofibroblasts substantially contribute to this stiffening by their high remodeling and ECM secreting activities.
We hypothesize that the stiff fibrotic microenvironment drives the differentiation of MSCs into myofibroblasts and thereby modulates their contractile and ECM remodeling behavior. We want to determine the role of ECM stiffness in MSC-to-myofibroblast fibrogenesis, using the unique soft culture technologies from Excellness Biotech SA and MCSs from different sources.

 

Vineetha Vijayakumar

The Role of WIP/WASP in regulating podosome turnover

Podosomes are actin rich structures present in cells of monocytic lineage that are involved in cell migration and invasion. Our group's focus is on understanding the molecular mechanism involved in regulating the formation and dissociation of podosomes.  As a member of Professor Jones laboratory, my project consists on studying the mechanism by which phosphorylation of WIP and its interacting partners such as WASP mediate podosome turnover in migration. To begin with different microscopic and biochemical approaches will be exploited to examine both the phosphorylation status and the spatial kinetics of WIP and selected domain-deleted variants. Early experiments will be conducted in THP-1 human monocytic cell lines and, once validated, I shall move on to human DC and macrophages.

 

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