Genetically Engineered Myoblasts For Measuring Nuclear Lamina Stress

The nuclear lamina is a protective shell that controls nuclear stress, and is composed primarily of the protein lamin. Grafting of optical force probes into lamins would provide a real-time readout of nuclear stress, but the expression levels of the chimeric reporter proteins need to be regulated throughout myogenesis. We achieved this by using CRISPR-Cas9 to genetically insert the optical force sensing probe cpstFRET into the nuclear lamins A/C and B1 genes of C2C12 myoblasts, where the cells own genetic regulatory elements control expression. Cell lines, heterozygous for the WT and cpstFRET chimeras of lamin were identified, that formed spontaneously contracting multinucleated myotubes having expression levels of 3:1 of WT:chimeric protein. Isolated nuclei were stretched by swelling via cation depletion, showing an increase in FRET with a force sensitivity of 0.023 and 0.013 FRET units per 1% change in surface area for lamin A/C and B1 respectively. Static and dynamic stresses in vivo caused the greatest magnitude, and most rapid changes, in nuclear stress in myoblasts bound to fibronectin coated surfaces. Substrate stiffness dictates the nuclear stress in flattened myoblasts where the nucleus is in close proximity to the surface. During myogenesis, the nuclear stress is more strongly influenced by the developing contractile machinery. During cell cycle, both lamin subtypes displayed an expected stress decrease during nuclear disassembly at mitosis, but smaller transient stress changes were observed during interphase that differed in timing between type A and B lamins. A surprising property of the angularly sensitive cpstFRET probe was that FRET increased as stress increased. This suggests a length mismatch between heterotypic WT:chimeric lamin pairs causes compression at the cpstFRET insertion site that relaxes with stress. These studies demonstrate the ability to target the insertion of optical force probes into structural proteins.