Cysteine-Shotgun Mass Spectrometry (CS-MS) for Probing Nuclear Lamin Conformation during Mechanical Stress

Abstract

Nuclear envelope proteins including the various lamins are expressed in all adult tissues, but lamins that are implicated in aging, cancer, and various dystrophies often affect specific tissues for unknown reasons. We have shown that the level of lamin-A,C and additional components of the nuclear envelope proteome that link the nucleus to the cytoskeleton scale systematically with tissue elasticity, while physical manipulation has demonstrated that nuclear stiffness scales with lamin-A,C. For example, brain tissue, which is relatively unaffected in the lamin-A,C-based aging disease progeria, has an elasticity about ten-fold softer than striated muscle with proportionately less lamin-A,C and a dominant amount of constitutive B-type lamins. Evidence suggests a mechano-sensitive regulation of the lamina, but the process of mechanical stimulation of molecular processes is poorly understood. Cysteine-shotgun mass spectrometry (CS-MS), a method capable of mapping the exposure of cysteine residues as proteins are stressed in complex biological systems such as isolated nuclear or whole cells, allowed us to identify stress sensitive proteins. We discovered a number of stress-sensitive proteins in isolated nuclei - including lamin-A,C - consistent with cell and tissue evidence that the nucleus transduces physical stress. Further work enquires whether substrate stiffnesses representative of soft and stiff tissue are reflected in changes to protein conformation.