Final Report Summary - GELVESICLE (Biophysical principles of cellular morphogenesis)
Cells are the smallest living building blocks of an organism. Enclosed by a soft plasma membrane and spanning the cell interior, the composite protein filament network of the cytoskeleton provides structure and mechanical stability to animal cells. The dynamic nature of the cytoskeleton allows a cell to fulfill physiological functions, such as division, migration, and sensing and adaptation to the mechanical environment. Functional failure is associated with abnormal development and disease, including fibrosis, cardiovascular disorders, and cancer. It is hence of biological and medical importance to understand the emergence of the cytoskeletal structure in the soft confinement of the plasma membrane as well as resulting mechanical and dynamical properties of the cell material.
Because the inherent complexity of cells often hampers identifying physical processes on the nanoscale, an attractive alternative to investigating actual cells is using simplified mechanical analogs thereof. The development and structure-mechanical characterisation of such biomimetic cell models were the Milestones of this project.
In the first year of the project we (the post-doctoral fellow B. Stuhrmann funded under this grant agreement and the PhD student F.-C. Tsai) developed a method for incorporating actin-myosin networks in cell-sized liposomes, hence successfully creating mechanical cell analogs. Existing methods'problems of vanishing yield under physiological (high) salt concentrations and/or in the presence of proteins could be overcome with an adaptation of the classical "Gentle Swelling" technique; we hydrate pre-formed lipid bilayer stacks from spin-coated agarose gels (see publication [1]) instead of from traditional solid substrates. In collaboration with Prof. C. Sykes (Curie Institute, Paris) we developed yet another liposome fabrication technique which allows faster protein inclusion and thereby better biological activity of proteins in the final assay. The supported fellow set up new collaborations with B. Baum (University College London) and A. Cambi (Radboud University Nijmegen, The Netherlands) and started developing a network-membrane anchoring scheme which exceeds existing model systems in terms of physiological relevance.
B. Stuhrmann developed an optical platform which allows combined active and passive laser microrheology to characterise mechanics of and stress generation in the biomimetic cell construct. A respective publication is in preparation. This unique setup allows structural investigation using confocal or phase contrast microscopy truly simultaneously to mechanical probing. The PhD student F.-C. Tsai developed a complementary flicker spectroscopy approach which allows extraction of membrane mechanical properties from fluctuations of liposome contours (see publication [1]).
Together with the (then) PhD student M. Soares e Silva, B. Stuhrmann investigated structural evolution of motor-driven actin networks in bulk (see publication [2]). This work suggests physical principles for active network coarsening into contractile spots reminiscent of features observed in living cells, and serves as a basis for an understanding of the respective processes in liposomal confinement.
The developed methods pioneer the incorporation of actin networks together with physiological motor proteins in cell-sized liposomes and pave the way for systematic investigation of cytoskeletal self-organisation in realistically small cellular volumes. The developed biomimetic cells give the community at hand an experimental system which allows for the first time the systematic investigation of structural, mechanical, and dynamic properties of living cells. Understanding these cellular properties is essential for an understanding of development and disease, and highly relevant for the key technology of tissue engineering. The supported fellow will continue building his own research line based on his experience in the direction of active soft matter
Because the inherent complexity of cells often hampers identifying physical processes on the nanoscale, an attractive alternative to investigating actual cells is using simplified mechanical analogs thereof. The development and structure-mechanical characterisation of such biomimetic cell models were the Milestones of this project.
In the first year of the project we (the post-doctoral fellow B. Stuhrmann funded under this grant agreement and the PhD student F.-C. Tsai) developed a method for incorporating actin-myosin networks in cell-sized liposomes, hence successfully creating mechanical cell analogs. Existing methods'problems of vanishing yield under physiological (high) salt concentrations and/or in the presence of proteins could be overcome with an adaptation of the classical "Gentle Swelling" technique; we hydrate pre-formed lipid bilayer stacks from spin-coated agarose gels (see publication [1]) instead of from traditional solid substrates. In collaboration with Prof. C. Sykes (Curie Institute, Paris) we developed yet another liposome fabrication technique which allows faster protein inclusion and thereby better biological activity of proteins in the final assay. The supported fellow set up new collaborations with B. Baum (University College London) and A. Cambi (Radboud University Nijmegen, The Netherlands) and started developing a network-membrane anchoring scheme which exceeds existing model systems in terms of physiological relevance.
B. Stuhrmann developed an optical platform which allows combined active and passive laser microrheology to characterise mechanics of and stress generation in the biomimetic cell construct. A respective publication is in preparation. This unique setup allows structural investigation using confocal or phase contrast microscopy truly simultaneously to mechanical probing. The PhD student F.-C. Tsai developed a complementary flicker spectroscopy approach which allows extraction of membrane mechanical properties from fluctuations of liposome contours (see publication [1]).
Together with the (then) PhD student M. Soares e Silva, B. Stuhrmann investigated structural evolution of motor-driven actin networks in bulk (see publication [2]). This work suggests physical principles for active network coarsening into contractile spots reminiscent of features observed in living cells, and serves as a basis for an understanding of the respective processes in liposomal confinement.
The developed methods pioneer the incorporation of actin networks together with physiological motor proteins in cell-sized liposomes and pave the way for systematic investigation of cytoskeletal self-organisation in realistically small cellular volumes. The developed biomimetic cells give the community at hand an experimental system which allows for the first time the systematic investigation of structural, mechanical, and dynamic properties of living cells. Understanding these cellular properties is essential for an understanding of development and disease, and highly relevant for the key technology of tissue engineering. The supported fellow will continue building his own research line based on his experience in the direction of active soft matter