Integrins are a family of conserved cell surface receptors found throughout the animal kingdom. They comprise 24 dimers in mammals, and regulate a number of processes including cell survival, differentiation, and migration. These complex cellular responses involve processes such as cell attachment, spreading, and various signaling pathways, which in turn depend on the composition of the extracellular environment, on its mechanical properties, and involved integrin types. This thesis focuses on identifying molecules that signal downstream of integrins and how integrin-induced signals may differ dependent on the type of mechanical stimulus that is given. In Paper I, we show that cell spreading and the activation of AKT is regulated by the catalytic PI3K isoform p110α. An intact β1 integrin cytoplasmic tail and actin polymerization was needed for spreading, whereas the presence of FAK or SRC, or the interaction between p110α and RAS was dispensable. Paper II reports that the RICTOR-mTOR complex (TORC2) acts as the kinase downstream of β1 integrins in order to phosphorylate AKT on Ser473, which was functionally linked to cell survival. β1 integrins activated both AKT1 and AKT2, but seemed to prefer AKT2. The investigation of several receptor types with regard to their requirement of TORC2, PAK, and ILK for AKT Ser473 phosphorylation revealed that different kinds of receptors engage specific enzyme combinations depending on cell type and context. In the third paper, we demonstrate that adhesion- and mechanical stretch-induced integrin signaling lead to divergent protein phosphorylation patterns, and that most signals from cell adhesion were not dependent on intracellular contractility. This indicates that integrin ligand binding and mechanical stretch induce signaling via distinct mechanisms. Reactive oxygen species (ROS) derived from different cellular sources modulated these responses. Stretching primarily induced phosphorylation of ERK1/2, and this signal was markedly increased by a derivative of the antioxidant ascorbate and extracellularly administered catalase. The robust AKT phosphorylation in response to adhesion was almost completely abolished with an inhibitor targeting mitochondrial ROS, whereas phosphorylation levels were only marginally affected in stretch assays. Similar results were obtained with siRNA knock-down of a critical subunit of ROS-producing NADPH oxidases.

Editorial on the Research Topic. Editorial: Editor’s Pick 2021: Highlights in Cell Adhesion and Migration.

Electrospun nanofibers have been researched extensively in the culturing of stem cells to understand their behavior since electrospun fibers mimic the native extracellular matrix (ECM) in many types of mammalian tissues. Here, electrospun nanofibers with defined structure (orientation/alignment) and pore size could significantly modulate human mesenchymal stem cell (hMSC) behavior. Controlling the fiber membrane pore size was predominantly influenced by the duration of electrospinning, while the alignment of the fiber membrane was determined by parallel electrode collector design. Electric field simulation data provided information on the electrostatic interactions in this electrospinning apparatus.hMSCs on small-sized pores (~3-10 µm²) tended to promote the cytoplasmic retention of Yes-associated protein (YAP), while larger pores (~30-45 µm²) promoted the nuclear activation of YAP. hMSCs also displayed architecture-mediated behavior, as the cells aligned along with the fiber membranes orientation. Additionally, fiber membranes affected nuclear size and shape, indicating changes in cytoskeletal tension, which coincided with YAP activity. The mechanistic understanding of hMSC behavior on defined nanofiber structures seeks to advance their translation into more clinical settings and increase biomanufacturing efficiencies.