Tissue homeostasis depends on proper adhesion of cells to their environment. Specialized protein complexes mediate adhesion to the extracellular matrix (ECM) and to neighbouring cells. These adhesion complexes serve structural roles, but also function as (mechano-) sensors of the extracellular environment. During embryonic development, tissue regeneration and malignant transformation of tumor cells, adhesion complexes are actively regulated to induce cell migration and reduce intercellular interactions. This process is referred to as EMT (Epithelial-mesenchymal transition). We aim to elucidate the signaling pathways that regulate adhesive activity during this process and to resolve the molecular structure of the adhesion complexes that allows their regulation.
The regulation of E-cadherin by acto-myosin controlled tension
Our main focus is the E-cadherin complex and its regulation by acto-myosin activity, downstream of the transforming hormone HGF (De Rooij et al. JCB 2005). We investigate the complex of proteins that forms the link between E-cadherin and actin and we investigate the changes in this complex that mediate the response to increased or decreased cytoskeletal tension. This response can be strengthening or loss of cell-cell adhesion dependent on the magnitude and context of the cytoskeletal force. Because of the specific localization of adhesion structures and the transient nature of adhesion activity, we use high-resolution live-cell microscopy to monitor the localization of adhesion proteins. Furthermore, we use FRET (fluorescence resonance energy transfer) to study the interaction between proteins and the activation of proteins in cell-cell adhesions.
Automated image analysis software to investigate metastatic cell behavior
Using HGF-induced MDCK cell-scattering as a model for malignant cell transformation, we developed automated image analysis software that tracks cells throughout time-lapse recordings of this process. From these tracks, the software derives several mathematical descriptors to quantitate migration vs cell-cell adhesion. Using this assay to screen a library of chemical inhibitors identified several specific inhibitors of the disruption of cell-cell adhesion or of the induction of cell-migration (Loerke et al. submitted). We are currently expanding such screens and developing image analysis software for the automated analysis of subcellular structures such as cell-matrix adhesions.
The regulation of epithelial cell-scattering by Epac/Rap signaling pathways
Epac is a cAMP-regulated guanine-nucleotide exchange factor (GEF) that activates the small GTPases of the Rap family (De Rooij et al. nature 1998). Rap GTPases regulate cell-cell as well as cell-matrix adhesions and as a consequence inhibit HGF-induced MDCK-cell scattering as well as other types of EMT. In collaboration with the lab of Hans Bos, we have shown that inhibition of these transformations is at the level of cell-migration, which is strongly impaired. Direct, allosteric activation of ECM-adhesion receptors, called integrins, (as proposed earlier) is probably not involved. Instead our results indicate that the dynamic coupling of integrins to the actin cytoskeleton is impaired (Lyle et al Cell. Signal. 2008). We are investigating which proteins are involved downstream of Rap1, to further elucidate this mechanism of adhesion-regulation.
Regulation of VE-cadherin during angiogenesis
Angiogenesis, the formation of new blood vessels, is very important for processes such as development, wound healing, inflammatory response and tumor progression. Angiogenesis involves multiple steps and is initiated by the disruption of cell-cell adhesions in the endothelial monolayer of existing vessels, followed by the formation of sprouts, and it ends with the strong sealing of cell-cell contacts at the encountering tips of sprouting vessels. Many angiogenic factors including VEGF, angiopoietin, and thrombin are known to stimulate the formation and/or disruption of cell-cell contacts of endothelial cells. However, the details of how cell-cell adhesion are regulated during sprouting angiogenesis remain poorly understood.
To investigate endothelial cell-cell adhesions, we are using high resolution imaging methods to visualize the localization of fluorescently labeled junctional proteins, such as VE-cadherin and associated proteins. We follow these adhesion molecules in time by live-cell microscopy, which gives fascinating insights into the molecular dynamics that are associated with the formation, or the disruption of cell-cell junctions. Currently, we are investigating the role of novel interactors of VE-cadherin in regulating cell-cell junctions and validate their function in 3D sprouting angiogenesis assays.
What regulates VE-cadherin mediated cell-cell adhesion?
Immunofluorescent image of HUVECs staied for F-actin (green) and VE-cadherin (red). The right panels show that the organization of the actin cytoskleton influences the organization of VE-cadherin at cell-cell junctions.
We would welcome motivated undergraduate students to participate in one of these projects. Besides learning many cell biology techniques (tissue culture, Westerns, generating DNA constructs, transfections, viral transductions etc.), students can get familiar with imaging techniques depending on their own interests and experience.
If you are interested please contact Johan de Rooij.
About the group leader