INTELLECTUAL MERIT: The central paradigm of mammalian cell migration states that cell motility, which is an important aspect of cellular functionality, is the result of cytoskeleton-induced protrusion and contraction forces that are transduced to the cell environment through specific cell linkages, such as integrin-based focal adhesions. However, in contrast to most mammalian cells which are well characterized by this paradigm, the underlying migration processes of weakly adhering cells with less prominent cell substrate linkages, such as leukocytes and certain cancer cells, remain elusive. This type of migration is known as amoeboid migration and is characterized by relatively rapid cell body translocation, enhanced plasticity, and pronounced cellular shape fluctuations. The current proposal seeks to explore underlying processes of amoeboid migration by the use of a novel biomembrane-mimicking cell substrate that allows the systematic variation of viscous drag of cell linkers without permitting formation of tensile force-carrying focal adhesions. By varying the viscous drag of cell-substrate linkages, as well as by selective modification of protrusion and contraction forces using pharmacological agents, this experimental system will enable the PI to determine the interplay between adhesion, contraction, and protrusion forces during amoeboid migration. Tuning of the viscous drag of cell linkers will be accomplished using a stack of multiple polymer-tethered lipid bilayers of adjustable bilayer number on a solid support. Specifically, the PI proposes migration studies in the presence of two types of cell-substrate linkages: (1) cell-extracellular matrix (ECM) mimicking integrin-laminin linkages (Specific Aim 1) and cell-cell mimicking cadherin based linkages (Specific Aim 2). Cell morphologies, cell migration velocities, cellular shape fluctuations, and cytoskeletal organization will be monitored using complementary optical microscopy methods.
BROADER IMPACTS: The proposed biomembrane-mimicking cell substrates system represents a powerful tool to explore poorly understood properties of weakly adhering cells under well-controlled conditions. These substrates also have substantial translational potential in biomedical cell assays, including drug screening, as they may mimic native tissue environments more realistically than currently existing substrates. Biosensor applications are also envisioned because the enhanced distance between top bilayer and underlying solid in multi-bilayer stacks will likely improve the functional reconstitution of membrane proteins. `The interdisciplinary character of the project will provide excellent training for graduate and undergraduate students. The PI will remain committed to the training of a broad pool of students with diverse social, racial, and ethnic backgrounds. The PI will also expand previous outreach activities at the high-school level and through the IUPUI Nanoscale Imaging Center. In addition, research results from this project will be developed into undergraduate and graduate courses in physical chemistry and biomimetic chemistry. Research will also be disseminated via scientific meetings, peer-reviewed journals, as well as via the internet.
A major outcome of the grant is that linker-functionalized, polymer-tethered single- and multi-bilayers were found to be well-suited as biomembrane-mimicking cell substrates for the analysis of cellular mechano-sensing. As confirmed experimentally, an attractive feature of these cell substrate architectures is that traction stress against adhesion points can be rationalized on the basis of the viscous drag of individual cell linkers and a rather elastic response with respect to linker clusters. Unlike on traditional polymeric cell substrates with immobilized linkers, this feature allows cellular adhesions to run through the characteristic stages of activation, assembly and maturation under well controlled conditions. Polymer-tethered single- and multi-bilayers were found to have remarkable materials properties. For example, magnetic tweezer experiments on bilayer-bound magnetic beads demonstrated that substrate stiffness can be adjusted by the number of bilayers in the polymer-tethered multi-bilayer stack. Alternatively, elastic properties of membranes can be modified by altering the concentration of polymer-tethered lipids (lipopolymers) in a single polymer-tethered lipid bilayer. A set of complementary experiments confirmed the integrity of these polymer-tethered membrane systems in the presence of plated cells. Analysis of plated cells (fibroblasts and myoblasts) showed that different degrees of bilayer stacking are associated with changes in cellular phenotype, cytoskeletal organization, and motility. Furthermore, a traction force microscopy assay was developed, which identified a clear correlation between cellular traction forces and substrate stiffness of polymer-tethered membranes. Comparing analysis of fibroblasts and myoblasts on bilayer substrates with laminin and N-cadherin-linkers, respectively, illustrated the ability to design biomembrane-mimicking cell substrates, which mimic either cell-extracellular matrix (ECM) or cell-cell interfaces. These experiments also revealed that plated fibroblasts and myoblasts respond to changes in bilayer stacking, but not to variations of linker type. The grant supported the interdisciplinary training of five graduate students and four undergraduate students. In addition, research training was provided to two high-school students through Project SEED.
