Cellular function is greatly influenced by biochemical and physical properties of the extracellular matrix (ECM) environment. Cells not only receive and process external signals but also actively interact with their environment by exerting mechanical forces and secreting signaling molecules. The magnitude of the cell-generated traction forces affects intracellular processes and cell function. Traction force microscopy (TFM) is an established technique to measure and quantify cell traction forces in 2D and 3D culture conditions. Standard 3D TFM analysis involves extensive investigation and modelling of the material properties and deformation to estimate cell-exerted forces. In this project, we want to investigate cellular traction forces using intracellular traction biosensors that measure forces across specific proteins in cells with pico-Newton (pN) precision. The fluorescence-resonance energy transfer (FRET) donor and acceptor protein fluorophores are genetically encoded into vinculin, a protein that connects integrins to actin filaments and whose recruitment to focal adhesions (FAs) is force-dependent. Vinculin has been shown to represent the mechanotransduction of the focal adhesion, which relates directly to the forces applied by the cells onto the ECM. To assess tension across vinculin traction forces are estimated via measuring FRET efficiency between donor and acceptor fluorophores. (Grashoff 2010) This method removes the underlying dependence on finite element models previously used for TFM and can help identifying local force distributions within the cells.
3D traction force estimations are routinely performed in bulk culture conditions which offer only limited control over cell environment. Existing 3D culture methods do not provide control over geometrical properties of the cell niche such as size and geometry. These properties influence cell morphology and cytoskeleton alignment and therefore are determinant for cell generated traction forces. In our lab, we have developed a cell culture platform with highly defined single cell microenvironments which allow testing of the ECM properties involved in mechanotransduction with high precision. In particular, we want to investigate how the niche stiffness and geometry regulate cell morphology and cell traction forces and influence cell function. Our previous findings demonstrate that in confined 3D niches, cell viability is reduced at low stiffness and small volume niches relative to 2D. The cell proliferation rate in confined 3D environments is also significantly lower. Localization studies of mechanosensing proteins (e.g., YAP) suggest that the influence of 3D confinement on stem cell fate is coordinated through canonical mechanosensitive signaling pathways. In this project we will investigate how cell morphology and traction forces influence the underlying intracellular biological pathways, transcription, and expression of the proteins.
Cellular function is greatly influenced by biochemical and physical properties of the extracellular matrix (ECM) environment. Cells not only receive and process external signals but also actively interact with their environment by exerting mechanical forces and secreting signaling molecules. The magnitude of the cell-generated traction forces affects intracellular processes and cell function. Traction force microscopy (TFM) is an established technique to measure and quantify cell traction forces in 2D and 3D culture conditions. Standard 3D TFM analysis involves extensive investigation and modelling of the material properties and deformation to estimate cell-exerted forces. In this project, we want to investigate cellular traction forces using intracellular traction biosensors that measure forces across specific proteins in cells with pico-Newton (pN) precision. The fluorescence-resonance energy transfer (FRET) donor and acceptor protein fluorophores are genetically encoded into vinculin, a protein that connects integrins to actin filaments and whose recruitment to focal adhesions (FAs) is force-dependent. Vinculin has been shown to represent the mechanotransduction of the focal adhesion, which relates directly to the forces applied by the cells onto the ECM. To assess tension across vinculin traction forces are estimated via measuring FRET efficiency between donor and acceptor fluorophores. (Grashoff 2010) This method removes the underlying dependence on finite element models previously used for TFM and can help identifying local force distributions within the cells. 3D traction force estimations are routinely performed in bulk culture conditions which offer only limited control over cell environment. Existing 3D culture methods do not provide control over geometrical properties of the cell niche such as size and geometry. These properties influence cell morphology and cytoskeleton alignment and therefore are determinant for cell generated traction forces. In our lab, we have developed a cell culture platform with highly defined single cell microenvironments which allow testing of the ECM properties involved in mechanotransduction with high precision. In particular, we want to investigate how the niche stiffness and geometry regulate cell morphology and cell traction forces and influence cell function. Our previous findings demonstrate that in confined 3D niches, cell viability is reduced at low stiffness and small volume niches relative to 2D. The cell proliferation rate in confined 3D environments is also significantly lower. Localization studies of mechanosensing proteins (e.g., YAP) suggest that the influence of 3D confinement on stem cell fate is coordinated through canonical mechanosensitive signaling pathways. In this project we will investigate how cell morphology and traction forces influence the underlying intracellular biological pathways, transcription, and expression of the proteins.
1. Literature review intracellular traction sensors
2. Cell culture and transfection with intracellular FRET tension sensors
3. Imaging of cells within hydrogel niches
4. Studying cell traction forces within 3D niches of different geometry
5. Relating the force profiles to localization of mechanosensing transcription factors such as YAP and MRTF
6. Investigate the link between cell shape and cell function
1. Literature review intracellular traction sensors 2. Cell culture and transfection with intracellular FRET tension sensors 3. Imaging of cells within hydrogel niches 4. Studying cell traction forces within 3D niches of different geometry 5. Relating the force profiles to localization of mechanosensing transcription factors such as YAP and MRTF 6. Investigate the link between cell shape and cell function