Subproject: Elastomeric substrate development, cell mechanics of myocytes.
Heart muscle cells (myocytes) are the main component of the heart myocard and the mechanically most active cells in mammalian organisms. Essential for blood transport through the organism, it is easy to believe that even small changes in the mechanical properties and functions of myocytes can lead to devastating cardiomyopathies, one of the most frequent diseases in humans. On the other hand, environmental changes often barely affect the mechanical functions of myocytes which is essential for heart function upon aging or even after mild heart strokes. Although myocytes are of immense importance for life, the knowledge about their mechanics is still highly limited due to the lack of appropriate analysis systems and methods. This is due to the fact that mechanically active cells cannot be analyzed on classical cell culture surfaces that are additionally approximately 105 to 106 times stiffer than natural tissue conditions and therefore cause artificial cell behavior. For this reason, our aim is split into two topics. The first topic deals with the development of elastomeric substrates mimicking natural elasticity conditions. As material cross-linkable silicone oils are used (PDMS, poly-dimethylsiloxanes) that can be ideally tuned in their elasticity. These substrates become deformed by mechanically active cells and deformations can be detected due to substrate incorporated microstructures (Fig. 1).
Fig. 1: Cells (red) adhere to the substrate via adhesion structures (green). Upon use of elastomeric substrates (PDMS) cell forces become applied at sites of adhesion and deform the substrate. Deformations can be detected through micropatterns at the top layers of the substrate and subsequently recalculated into cell forces.
Since the cell types, myocytes and podocytes, used within this program are naturally embedded in different environmental topographies, elastomeric substrates are formed as flat substrates, with various curvatures as well as 3D-substrates. Elastomeric substrates therefore allow for the first time the analysis of cellular mechanics under close to nature conditions. In the second topic those substrates are used for a careful analysis of cell mechanical processes of myocytes (Fig. 2). Special focus is given on the characterization on the force generation apparatus of myocytes. Questions about generated force intensities, force transmission sites, involved protein compositions and protein dynamics are getting analyzed and tested whether those parameters depend on physical environmental parameters by modifying elasticity and topography. We furthermore analyze how mutations that cause cardiomyopathies affect the mechanical functions of myocytes. For this purpose temporal siRNA mutants of myocyte cells are getting analyzed. Acquisitions of multidimensional data sets on single cells analyze force levels, morphology, adhesion sites, protein composition as well as bead frequency and amplitude. Those data sets will allow a deep understanding of the mechanical mechanisms responsible for myocyte function and are at the same time the basis for comparative systems biological approaches and theoretical models/simulations as further important aspect within the MechanoSys program.
Subproject: Cell mechanics of podocytes.
Podocyte foot processes cover the outer aspect of the capillaries in the renal glomerulus. They are important determinants of the permeability of the glomerular filtration barrier. In addition, podocytes stabilize the capillary wall with a contractile actin cytoskeleton counteracting the high hydrostatic capillary pressure. Chronic kidney disease (CKD) is predominantly caused by damage and failure of podocytes, e.g. due to increased capillary pressure. The aim of the project is to elucidate the molecular mechanisms of podocyte cell mechanics by developing and using novel methods of systems biology. In the project, force vectors and parameters of cell mechanics will be measured by high resolution microscopy in living podocytes on elastomeres expressing fluorescent fusion proteins. Cell mechanics will be correlated with protein localization and will be simulated by mathematical modeling. The effect of actin polymerization, of contractility and of actin-associated proteins involved in CKD on cell mechanics will be studied in detail. Proteins of the cell adhesome will be investigated by RNAi with respect to their function in cell mechanics of podocytes. Moreover, we will determine the effect of the geometry of non-planar substrates on podocyte differentiation. It is expected that the results of the project will contribute to a deeper understanding of podocyte cell mechanics, leading to novel therapeutic approaches for CKD. At the same time, novel methods and products for studying cell mechanics by systems biology will be developed.
Subproject: Theoretical and simulation approaches to cell mechanics.
Adhesion and mechanics of animal cells are tightly coupled and together form a highly complex and dynamic system whose biological function can be understood only on a systems level. This statement is particularly true for rigidity sensing, which is of central importance for the fate and differentiation decisions of tissue cells. At the heart of this process is intracellular force production by non-muscle myosin II motors tensing the actin cytoskeleton. This mechanical tension is propagated to integrin-based focal adhesions anchored in the extracellular matrix. As tension builds at the focal adhesions, mechanotransduction processes in their cytoplasmic plaques lead to biochemical signals propagating into the cytosol, most prominently those controlled by the small GTPases from the Rho-family. These signals lead to strengthening of the actin cytoskeleton and increased force generation, thus closing a positive feedback loop including both mechanical and biochemical elements. The dynamics of this feedback loop is modulated by extracellular stiffness and thus allow different cell types to compare it with their specific set points (for example, myocyte differentiation has been shown to work best at 12 kPa). It is obvious that the mechanochemical feedback loop of rigidity sensing cannot be understood in terms of its single components. It is the interplay of these components in a system which leads to biological function (compare cartoon).
Due to the complex nature of cell mechanical systems and the vast array of different data sets available, modeling and simulation are important tools to decipher the way these systems function. Systems biology approaches allow us to develop and test different hypotheses which then can be compared with experimental data in a systematic manner. In cell adhesion and mechanics, a particular challenge exists in the requirement to couple biochemical and mechanical approaches, for example reaction-diffusion systems with models for force generation in filament bundles. In the framework of MechanoSys, the Schwarz group will follow a two-fold strategy. On the one hand, it supports experiments and data analysis by developing new tools for measuring cellular traction on non-planar elastic substrates (mimicking natural conditions for myocytes and podocytes) and for correlating them with biochemical data, for example protein localization. On the other hand, the group develops a general modeling framework for the coupling of biochemistry and mechanics, which couples biochemical data (network motifs, binding affinities) with spatial data (protein localization and transport, traction force patterns).
Schematics of the mechanochemical feedback loop of rigidity sensing: myosin motors create tension in the actin cytoskeleton. This leads to activation of soluble factors at focal adhesions, which in turn regulate cytoskeleton remodeling and force generation. Different theoretical modeling approaches have to be coupled to achieve a systems level description.
Subproject: Exploration and design of elastomer-based cell-biochips.
ibidi work packages focus on the exploration and design of relevant
cell-biochips that will be used for systems biological as well as
industrial approaches. This includes the Definition of the specifications
for the cell-biochip. All specifications for cultivation, function and
behaviour of podocytes and myocytes will be continuously collected from
the partners. Special focus is given on multi-functional approaches as
cell force analysis and fluorescence microscopy on elastic substrates
under flow. Additionally, all optical parameters (e.g. fluorescence
techniques, high resolution, DIC, index of refraction) as well as
material properties of various types of PDMS, integrated particles
like fluorescent beads or microtubes will be collected. The influence
of different bonding technologies on the measured system parameters will
be explored. Moreover, the different possible glues will be analyzed with
respect to e.g. biocompatibility or processability.