At the heart of the cell’s ability to maintain its mechanical integrity while dynamically altering its shape is an intimate interaction between plasma membrane and the underlying actin cortex. There is mounting evidence that regulation of actin–membrane anchoring by lipids and dedicated anchoring proteins is critical for the formation of dynamic actin structures needed for essential functions such as cell division, migration, and mechanosensing. The underlying mechanisms are difficult to probe, however, due to the complexity and redundancy of biochemical processes in cells. Here I propose to elucidate how actin–membrane anchoring is involved in cytoskeletal organization and mechanics using a biomimetic model of contractile cells with well-defined composition. I will develop new microfluidic methods to encapsulate active cortical networks of actin and myosin motors in cell-sized liposomes, and I will use an important physiological protein, moesin, to implement actin–membrane anchoring. By combining fluorescence microscopy, micromanipulation techniques, and fluctuation analysis, I will investigate the interplay between actin–membrane anchoring and motor activity in cortical organization and rigidity in a systematic and quantitative manner. Ultimately, I will take the biomimetic approach a step further and investigate the molecular basis of cell adhesion and mechanosensing by incorporating focal adhesion proteins and seeding the biomimetic cells on extracellular matrix substrates. The project will elucidate the basic physical principles behind cell mechanics, which is vital for the development of predictive theoretical models and for understanding cellular shape changes in living organisms. Thus, I expect to obtain valuable molecular insights into diseases associated with malfunction of cytoskeleton–membrane anchoring, most notably cancer. Moreover, the experimental assays will be directly applicable to related fields, such as synthetic biology and membrane technology.