Forced unfolding of proteins within cells.
To identify cytoskeletal proteins that change conformation or assembly within stressed cells, in situ labeling of sterically shielded cysteines with fluorophores was analyzed by fluorescence imaging, quantitative mass spectrometry, and sequential two-dye labeling. Within red blood cells, shotgun labeling showed that shielded cysteines in the two isoforms of the cytoskeletal protein spectrin were increasingly labeled as a function of shear stress and time, indicative of forced unfolding of specific domains. Within mesenchymal stem cells-as a prototypical adherent cell-nonmuscle 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 colocalize signaling events such as phosphorylation with forced unfolding.
Johnson
Colin P
CP
Biophysical Engineering Lab, University of Pennsylvania, Philadelphia, PA 19104, USA.
Tang
Hsin-Yao
HY
Carag
Christine
C
Speicher
David W
DW
Discher
Dennis E
DE
D002853:Chromatography, Liquid; D003545:Cysteine / Q000737:chemistry; D003598:Cytoskeletal Proteins / Q000737:chemistry*; D004912:Erythrocytes / Q000737:chemistry*; D005453:Fluorescence; D005455:Fluorescent Antibody Technique; D005456:Fluorescent Dyes; D006576:Heterocyclic Compounds, 4 or More Rings / Q000494:pharmacology; D006801:Humans; D059630:Mesenchymal Stem Cells / Q000737:chemistry*; D009282:Naphthalenesulfonates; D024507:Nonmuscle Myosin Type IIA / Q000737:chemistry; D011487:Protein Conformation; D017510:Protein Folding; D020836:Protein Structure, Quaternary; D017434:Protein Structure, Tertiary; D013049:Spectrin / Q000737:chemistry; D013314:Stress, Mechanical; D053719:Tandem Mass Spectrometry; D013696:Temperature; D014746:Vimentin / Q000737:chemistry
D003598:Cytoskeletal Proteins; D005456:Fluorescent Dyes; D006576:Heterocyclic Compounds, 4 or More Rings; D009282:Naphthalenesulfonates; D014746:Vimentin; D013049:Spectrin; C472645:blebbistatin; C006422:1,8-I-AEDANS; D024507:Nonmuscle Myosin Type IIA; D003545:Cysteine