The Foldome in cellular force transduction.
The Human Genome is essentially complete, and yet the impact on how we understand physiological processes such as cellular force transduction has been minimal in part because of our inability to work from known sequence to structure, i.e. the Foldome. In order to specifically identify cytoskeletal proteins that change conformation or assembly in stressed versus static cells, in situ labeling of sterically-shielded or 'cryptic' cysteines with fluorophores is analyzed by quantitative mass spectrometry, sequential two-dye labeling, and fluorescence imaging. Within red blood cells, shotgun labeling shows that shielded cysteines in the two isoforms of the cytoskeletal protein spectrin are increasingly labeled as a function of shear stress and time, indicative of forced unfolding of specific domains. Within mesenchymal stem cells - as a prototypical nucleated cell - non-muscle myosin IIA and vimentin are just two of the cytoskeletal proteins identified that show differential labeling in tensed versus drug-relaxed cells. Cysteine labeling of proteins within live cells can thus be used to fluorescently map out sites of molecular-scale deformation, and the results also suggest means to co-localize signaling events such as phosphorylation with forced unfolding.
Discher
Dennis E
DE
University of Pennsylvania, USA. discher@seas.upenn.edu
D000205:Actomyosin / Q000737:chemistry; D001703:Biophysics / Q000379:methods*; D003545:Cysteine / Q000737:chemistry; D003599:Cytoskeleton / Q000737:chemistry; D004912:Erythrocytes / Q000378:metabolism; D005456:Fluorescent Dyes / Q000494:pharmacology; D015894:Genome, Human; D006801:Humans; D013058:Mass Spectrometry / Q000379:methods; D008968:Molecular Conformation; D024507:Nonmuscle Myosin Type IIA / Q000737:chemistry; D010766:Phosphorylation; D011994:Recombinant Proteins / Q000737:chemistry; D013314:Stress, Mechanical; D014746:Vimentin / Q000737:chemistry
D005456:Fluorescent Dyes; D011994:Recombinant Proteins; D014746:Vimentin; D000205:Actomyosin; D024507:Nonmuscle Myosin Type IIA; D003545:Cysteine