Mechanical Stress in Actinin and Actin in Stem Cells

Abstract

cpstFRET is a FRET based force sensor designed to be modulated primarily by the angle between the donor and acceptor. It is physically smaller, less invasive, and has greater dynamic range and better signal-to-noise behavior than the linear probes. Using this sensor with FRET polarization imaging we measured the gradients of constitutional stresses in time and space for actinin and actin in HEK and MDCK cells. We created eight stable cell lines with these probes. Then we derived stem cells from the cell lines and measured stress changes during differentiation and dedifferentiation. We calculated FRET with the parallel/perpendicular polarization images of FRET. Because the FRET signal is always more depolarized, high ratios represent low FRET and high stress, while low ratios represent high FRET and low stress. We induced HEK and MDCK cells into embryoid bodies (EB) which is the characteristic morphology of induced pluripotent stem cells and cancer stem cells. We verified the stem cell in the EBs with alkaline phosphatase (AP) staining. Stem cells in EBs derived from HEKs showed higher AP activities than those derived from MDCK cells. All stem cells showed escalated stress in both actinin and actin relative to the parent. Stress was higher in stem cells of HEK origination. After we removed the EB-induction factors, these stresses declined as the stem cells differentiated into HEK or epithelial cells. We also induced the stem cells differentiate into neurons. The cell body showed low stress while axon extrusions showed increased stress in actin and actinin. The significant stress changes in stem cells and differentiated descendants hint at the potential of inducing pluripotent stem cells through changing cell mechanics. The data also shows that stem cell differentiation involves changes in internal stresses as well as changes in biochemistry.