Research within T3Net is conducted in several work packages, which all focus on different cellular and molecular aspects of invasive cell migration.
1) Cancer invasion and dissemination
1.1 Morpho-functional basis of invadopodia structure and function
This task aims to unveil the morpho-functional basis of the highly organized structure and function of invadopodia in tumour cells. The role of membrane lipids, particularly cholesterol and caveolin 1, will be studied through the manipulation of membrane lipid composition.
1.2 Role of Fgd1 and podoplanin in linking ECM-cell interactions and formation of invadopodia
This task will analyse the role of two key proteins, Fgd1 and podoplanin, mediating the signalling events linking extracellular matrix degrading (ECM)-cell interactions and the formation of invadopodia in tumor cells and podosomes in dendritic cells (DCs) and endothelial cells.
2) Immunity, inflammation and vascular remodelling
2.1 TGFβ receptors involved in podosome formation in endothelial cells
Experiments will be performed aimed at identifying the TGFβ receptor and co-receptor involved in podosome formation in endothelial cells and dissecting the molecular links to downstreameffectors in the process of endothelial podosome formation and ECM degradation.
2.2 Regulation of WASP, a critical player in podosome formation in dendritic cells
Regulation of WASP (Wiskott-Aldrich Syndrome Protein) by WASP-Interacting Protein (WIP) will be investigated, including the effect of WIP phosphorylation, regulation of actin filament assembly and control of WASP degradation.
2.3 Intracellular transport processes in podosome regulation
This project will investigate intracellular transport processes in podosome regulation, with particular emphasis on the role of vesicle-regulating proteins.
2.4 Characterisation of proteinases involved in ECM degradation by podosomes
This project aims to identify the specific proteinases involved in podosome-localized matrix degradation, their modes of transport to podosomes, and the mechanisms of their subcellular activation.
2.5 Role of integrin receptors and mechanical stress in release of TGFb by fibroblasts
This task investigates the cell-ECM integrin receptors and the forces involved in the physical activation of TGFb by contracting fibroblasts . These investigations will also tie in synergistically with WP2.1, which aims at identifying events downstream of TGFb receptors during podosome formation.
3) Interactions with the ECM
3.1 Mechanisms of myofibroblast contraction
Myofibroblasts will be grown in 3D collagen gels to correlate spontaneously occurring intracellular Ca2+ oscillations with contractile events on the subcellular level. These processes will be further analyzed as a function of ECM rigidity, using 2D and 3D culture substrates with tuneable stiffness.
3.2 Factors influencing matrix degradation by macrophages
This project will employ micro-patterned substrates to elucidate the minimal/maximal inter-podosome distances supporting matrix degradation by macrophages, as well as the associated changes in ocal contractility.
3.3 Structural characteristics of podosomes formed in vitro and in vivo
The differing structural characteristics of podosomes formed under in vitro and in vivo conditions will be explored in detail, using immunofluorescence and confocal microscopy. 3D reconstruction of serial images will allow visualization of the architectural organisation of the structures.
3.4 Assembly of the sealing zone by osteoclasts
In this project, the mechanisms will be addressed whereby osteoclasts interact with bone surfaces, “sense” chemical and physical features of the bone and assemble the resorptive “sealing zone” which can degrade bone
4) Unravelling the basic molecular machinery- the search for new components
4.1 Genes involved in regulation of podosome formation
In this task, the expression of specific adhesion-associated and signalling molecules that might be involved in the regulation of podosome and invadopodia formation, will be suppressed using a siRNA approach.
4.2 Comparative analysis of invadopodia and podosome-enriched cell fractions
This two-pronged approach will allow for an in-depth comparative analysis between podosomes and invadopodia, which are similar yet unique structures in different cell models.
4.3 Transcriptomic analysis of DC-derived osteoclasts
This taskwill identify genes specifically implicated in bone resorption. Selected proteins will also be validated in a 3D environment
5) The third dimension
5.1 Role of lytic protrusions in 3D tissue invasion
These studies will show precisely how invadopodia contribute to 3D cell invasion and tissue structure remodelling.
5.2 Matrix-degrading structures in additional models
The approach in 5.1 will be extended to relevant 3D models of endothelial cell, macrophage and osteoclast podosomes
5.3 Investigation in human tumour samples
A direct effort to detect lytic protrusive structures will be carried out in human tumour samples, using high-end microscopy and ECM degradation assays.