Final Report Summary - SURFACE ENDOCYTOSIS (Effect of nano-patterned substrate properties on cell attachment and endocytosis)
The principal objective of this project was to study the effect of biomaterial properties on cell function and in particular endocytosis. The results and conclusion of this project are summarized below.
An artificial, polymer-based hydrogel with tunable elasticity was designed and developed during the project. Hydrogels were characterized mechanically and chemically and appropriate setups were designed to allow easy handling, sterilization and long-term cell culture conditions. The elastic modulus of the hydrogels could be controlled in the range of 1 to 100 kPa. Protocols for biofunctionalization of hydrogels using small peptides or full-length extracellular matrix (ECM) proteins were established.
The above constitute a successful implementation of the 1st specific aim of the proposal. The aspect of hydrogel nanopatterning was deemed to be of lesser importance for the questions addressed and it was developed in the group by another researcher with whom discussion and collaborations were initiated.
The interactions of cell lines with hydrogels were then examined. Several cell lines were initially tested and rat embryonic fibroblasts (REF) were selected. Preliminary experiments established the requirements of cell-adhesive ligand concentrations for sustained cell growth and the elasticity range that cells respond to.
Substrate rigidity had a profound effect on cell morphology and cytoskeletal organization on hydrogels functionalized with RGD peptides. REF cells spread to a lower extent on softer hydrogels where they failed to develop large focal adhesions and stress fibers. Immunofluorescence data revealed lower amounts of signaling proteins and kinases as elasticity decreased. These differences were manifested as a reduction of cell proliferation rates on the softer hydrogels.
Cell spreading experiments showed that cells sense differences in rigidity within minutes from plating with cells spreading slower on softer hydrogels, which is a novel finding. On the other hand, migration was enhanced at lower substrate elasticities.
An important question examined was whether substrate sensing was mediated by specific integrins. We modulated integrin engagement by coating hydrogels with either fibronectin (FN) or vitronectin (VN), ECM proteins that are known to engage differentially integrins. REF cells exhibited the same trends in spread area, growth rates and focal adhesion formation and characteristics on both coatings, albeit with a quantitative difference between them. Cell spreading increased with substrate stiffness and was always lower on VN. At the same time, focal adhesion area and aspect ratio increased with substrate stiffness, with adhesions being larger on VN. A difference in migration persistence was also evident, with cells showing persistent migration on stiff FN hydrogels. These findings contribute to the understanding of how specific integrins regulate cell behavior.
The third specific aim of the project was to determine whether different forms of endocytosis were affected by substrate elasticity. Results obtained using markers for 1) non-specific fluid-phase endocytosis 2) clathrin-mediated endocytosis and 3) lipid-raft mediated endocytosis showed that there were only minor differences of their uptake on substrates of varying rigidity. These small differences were attributed to differences in cell surface area and not mechanical feedback to the cells. There was additionally no dependence noted on transfection efficiency of cells on the different substrates. These conclusions discouraged us to test internalization of cell penetrating peptides and peptide amphiphiles (as initially proposed) and led us to focus our attention towards other aspects of the project, including adaptation of our hydrogels for 3-dimensional cell culture.
The results and conclusions of the funded work are in the process of being written as two manuscripts. The researcher has been offered to stay in the laboratory for an additional year in order to better prepare the work to be published. The publication of our findings will contribute in the growing body of knowledge in the field of cell mechanotransduction with implications in understanding processes in development, wound healing and cancer progression. In these biological events, cells integrate mechanical input from their microenvironment and make decisions.
The funded project also accomplished the goal of integrating the researcher in the European scientific community. The researcher participated in 3 international conferences, was invited at 3 laboratories in other countries to present seminars and initiated collaborations. The laboratory benefited from his presence, interactions and work. The hydrogels developed will be an important tool for extending the current work to answer new fundamental questions and should provide a good platform to initiate future collaborations.
An artificial, polymer-based hydrogel with tunable elasticity was designed and developed during the project. Hydrogels were characterized mechanically and chemically and appropriate setups were designed to allow easy handling, sterilization and long-term cell culture conditions. The elastic modulus of the hydrogels could be controlled in the range of 1 to 100 kPa. Protocols for biofunctionalization of hydrogels using small peptides or full-length extracellular matrix (ECM) proteins were established.
The above constitute a successful implementation of the 1st specific aim of the proposal. The aspect of hydrogel nanopatterning was deemed to be of lesser importance for the questions addressed and it was developed in the group by another researcher with whom discussion and collaborations were initiated.
The interactions of cell lines with hydrogels were then examined. Several cell lines were initially tested and rat embryonic fibroblasts (REF) were selected. Preliminary experiments established the requirements of cell-adhesive ligand concentrations for sustained cell growth and the elasticity range that cells respond to.
Substrate rigidity had a profound effect on cell morphology and cytoskeletal organization on hydrogels functionalized with RGD peptides. REF cells spread to a lower extent on softer hydrogels where they failed to develop large focal adhesions and stress fibers. Immunofluorescence data revealed lower amounts of signaling proteins and kinases as elasticity decreased. These differences were manifested as a reduction of cell proliferation rates on the softer hydrogels.
Cell spreading experiments showed that cells sense differences in rigidity within minutes from plating with cells spreading slower on softer hydrogels, which is a novel finding. On the other hand, migration was enhanced at lower substrate elasticities.
An important question examined was whether substrate sensing was mediated by specific integrins. We modulated integrin engagement by coating hydrogels with either fibronectin (FN) or vitronectin (VN), ECM proteins that are known to engage differentially integrins. REF cells exhibited the same trends in spread area, growth rates and focal adhesion formation and characteristics on both coatings, albeit with a quantitative difference between them. Cell spreading increased with substrate stiffness and was always lower on VN. At the same time, focal adhesion area and aspect ratio increased with substrate stiffness, with adhesions being larger on VN. A difference in migration persistence was also evident, with cells showing persistent migration on stiff FN hydrogels. These findings contribute to the understanding of how specific integrins regulate cell behavior.
The third specific aim of the project was to determine whether different forms of endocytosis were affected by substrate elasticity. Results obtained using markers for 1) non-specific fluid-phase endocytosis 2) clathrin-mediated endocytosis and 3) lipid-raft mediated endocytosis showed that there were only minor differences of their uptake on substrates of varying rigidity. These small differences were attributed to differences in cell surface area and not mechanical feedback to the cells. There was additionally no dependence noted on transfection efficiency of cells on the different substrates. These conclusions discouraged us to test internalization of cell penetrating peptides and peptide amphiphiles (as initially proposed) and led us to focus our attention towards other aspects of the project, including adaptation of our hydrogels for 3-dimensional cell culture.
The results and conclusions of the funded work are in the process of being written as two manuscripts. The researcher has been offered to stay in the laboratory for an additional year in order to better prepare the work to be published. The publication of our findings will contribute in the growing body of knowledge in the field of cell mechanotransduction with implications in understanding processes in development, wound healing and cancer progression. In these biological events, cells integrate mechanical input from their microenvironment and make decisions.
The funded project also accomplished the goal of integrating the researcher in the European scientific community. The researcher participated in 3 international conferences, was invited at 3 laboratories in other countries to present seminars and initiated collaborations. The laboratory benefited from his presence, interactions and work. The hydrogels developed will be an important tool for extending the current work to answer new fundamental questions and should provide a good platform to initiate future collaborations.