Note to users. If you're seeing this message, it means that your browser cannot find this page's style/presentation instructions -- or possibly that you are using a browser that does not support current Web standards. Find out more about why this message is appearing, and what you can do to make your experience of our site the best it can be.


Logo for

Science 317 (5838): 663-666

Copyright © 2007 by the American Association for the Advancement of Science

Forced Unfolding of Proteins Within Cells

Colin P. Johnson,1*{dagger}{ddagger} Hsin-Yao Tang,2* Christine Carag,1{dagger}{ddagger} David W. Speicher,2{dagger} Dennis E. Discher1,2{dagger}{ddagger}

Abstract: 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.

1 Biophysical Engineering Lab, University of Pennsylvania, Philadelphia, PA 19104, USA.
2 Systems Biology Division—The Wistar Institute, Philadelphia, PA 19104, USA.

* These authors conducted experiments.

{dagger} These authors designed, refined, and analyzed experiments.

{ddagger} These authors modeled the data and wrote the paper.

To whom correspondence should be addressed. E-mail: discher{at}

Nuclear lamin stiffness is a barrier to 3D migration, but softness can limit survival.
T. Harada, J. Swift, J. Irianto, J.-W. Shin, K. R. Spinler, A. Athirasala, R. Diegmiller, P. C. D. P. Dingal, I. L. Ivanovska, and D. E. Discher (2014)
J. Cell Biol. 204, 669-682
   Abstract »    Full Text »    PDF »
Low-force transitions in single titin molecules reflect a memory of contractile history.
Z. Martonfalvi, P. Bianco, M. Linari, M. Caremani, A. Nagy, V. Lombardi, and M. Kellermayer (2014)
J. Cell Sci. 127, 858-870
   Abstract »    Full Text »    PDF »
Nuclear Lamin-A Scales with Tissue Stiffness and Enhances Matrix-Directed Differentiation.
J. Swift, I. L. Ivanovska, A. Buxboim, T. Harada, P. C. D. P. Dingal, J. Pinter, J. D. Pajerowski, K. R. Spinler, J.-W. Shin, M. Tewari, et al. (2013)
Science 341, 1240104
   Abstract »    Full Text »    PDF »
Crawling from soft to stiff matrix polarizes the cytoskeleton and phosphoregulates myosin-II heavy chain.
M. Raab, J. Swift, P. C. D. P. Dingal, P. Shah, J.-W. Shin, and D. E. Discher (2012)
J. Cell Biol. 199, 669-683
   Abstract »    Full Text »    PDF »
Finding the weakest link - exploring integrin-mediated mechanical molecular pathways.
P. Roca-Cusachs, T. Iskratsch, and M. P. Sheetz (2012)
J. Cell Sci. 125, 3025-3038
   Abstract »    Full Text »    PDF »
Marker-of-self becomes marker-of-senescence.
N. Sosale and D. E. Discher (2012)
Blood 119, 5343-5344
   Full Text »    PDF »
Mutation of Conserved Histidines Alters Tertiary Structure and Nanomechanics of Consensus Ankyrin Repeats.
W. Lee, J. Strumpfer, V. Bennett, K. Schulten, and P. E. Marszalek (2012)
J. Biol. Chem. 287, 19115-19121
   Abstract »    Full Text »    PDF »
Phage-based molecular probes that discriminate force-induced structural states of fibronectin in vivo.
L. Cao, M. K. Zeller, V. F. Fiore, P. Strane, H. Bermudez, and T. H. Barker (2012)
PNAS 109, 7251-7256
   Abstract »    Full Text »    PDF »
Actin stress fibers - assembly, dynamics and biological roles.
S. Tojkander, G. Gateva, and P. Lappalainen (2012)
J. Cell Sci. 125, 1855-1864
   Abstract »    Full Text »    PDF »
Orientation-based FRET sensor for real-time imaging of cellular forces.
F. Meng and F. Sachs (2012)
J. Cell Sci. 125, 743-750
   Abstract »    Full Text »    PDF »
Single-molecule Force Spectroscopy Reveals the Individual Mechanical Unfolding Pathways of a Surface Layer Protein.
C. Horejs, R. Ristl, R. Tscheliessnig, U. B. Sleytr, and D. Pum (2011)
J. Biol. Chem. 286, 27416-27424
   Abstract »    Full Text »    PDF »
Probing the Folded State of Fibronectin Type III Domains in Stretched Fibrils by Measuring Buried Cysteine Accessibility.
C. A. Lemmon, T. Ohashi, and H. P. Erickson (2011)
J. Biol. Chem. 286, 26375-26382
   Abstract »    Full Text »    PDF »
Structural and kinetic mapping of side-chain exposure onto the protein energy landscape.
R. Bernstein, K. L. Schmidt, P. B. Harbury, and S. Marqusee (2011)
PNAS 108, 10532-10537
   Abstract »    Full Text »    PDF »
Cysteine shotgun-mass spectrometry (CS-MS) reveals dynamic sequence of protein structure changes within mutant and stressed cells.
C. C. Krieger, X. An, H.-Y. Tang, N. Mohandas, D. W. Speicher, and D. E. Discher (2011)
PNAS 108, 8269-8274
   Abstract »    Full Text »    PDF »
Visualizing dynamic cytoplasmic forces with a compliance-matched FRET sensor.
F. Meng and F. Sachs (2011)
J. Cell Sci. 124, 261-269
   Abstract »    Full Text »    PDF »
ATP-dependent Mechanism Protects Spectrin against Glycation in Human Erythrocytes.
S. Manno, N. Mohandas, and Y. Takakuwa (2010)
J. Biol. Chem. 285, 33923-33929
   Abstract »    Full Text »    PDF »
Free Edges in Epithelial Cell Sheets Stimulate Epidermal Growth Factor Receptor Signaling.
E. R. Block, M. A. Tolino, J. S. Lozano, K. L. Lathrop, R. S. Sullenberger, A. R. Mazie, and J. K. Klarlund (2010)
Mol. Biol. Cell 21, 2172-2181
   Abstract »    Full Text »    PDF »
Structural basis for spectrin recognition by ankyrin.
J. J. Ipsaro and A. Mondragon (2010)
Blood 115, 4093-4101
   Abstract »    Full Text »    PDF »
Withaferin A Targets Intermediate Filaments Glial Fibrillary Acidic Protein and Vimentin in a Model of Retinal Gliosis.
P. Bargagna-Mohan, R. R. Paranthan, A. Hamza, N. Dimova, B. Trucchi, C. Srinivasan, G. I. Elliott, C.-G. Zhan, D. L. Lau, H. Zhu, et al. (2010)
J. Biol. Chem. 285, 7657-7669
   Abstract »    Full Text »    PDF »
PR65, the HEAT-repeat scaffold of phosphatase PP2A, is an elastic connector that links force and catalysis.
A. Grinthal, I. Adamovic, B. Weiner, M. Karplus, and N. Kleckner (2010)
PNAS 107, 2467-2472
   Abstract »    Full Text »    PDF »
Matrix elasticity, cytoskeletal forces and physics of the nucleus: how deeply do cells 'feel' outside and in?.
A. Buxboim, I. L. Ivanovska, and D. E. Discher (2010)
J. Cell Sci. 123, 297-308
   Abstract »    Full Text »    PDF »
Fibronectin forms the most extensible biological fibers displaying switchable force-exposed cryptic binding sites.
E. Klotzsch, M. L. Smith, K. E. Kubow, S. Muntwyler, W. C. Little, F. Beyeler, D. Gourdon, B. J. Nelson, and V. Vogel (2009)
PNAS 106, 18267-18272
   Abstract »    Full Text »    PDF »
Amino acid sequence dependence of nanoscale deformation mechanisms in alpha-helical protein filaments.
J. Bertaud, Z. Qin, and M. J. Buehler (2009)
The Journal of Strain Analysis for Engineering Design 44, 517-531
   Abstract »    PDF »
Atomic Structures of Two Novel Immunoglobulin-like Domain Pairs in the Actin Cross-linking Protein Filamin.
O. K. Heikkinen, S. Ruskamo, P. V. Konarev, D. I. Svergun, T. Iivanainen, S. M. Heikkinen, P. Permi, H. Koskela, I. Kilpelainen, and J. Ylanne (2009)
J. Biol. Chem. 284, 25450-25458
   Abstract »    Full Text »    PDF »
ATP-dependent mechanics of red blood cells.
T. Betz, M. Lenz, J.-F. Joanny, and C. Sykes (2009)
PNAS 106, 15320-15325
   Abstract »    Full Text »    PDF »
Multiscale Mechanics of Fibrin Polymer: Gel Stretching with Protein Unfolding and Loss of Water.
A. E. X. Brown, R. I. Litvinov, D. E. Discher, P. K. Purohit, and J. W. Weisel (2009)
Science 325, 741-744
   Abstract »    Full Text »    PDF »
Growth Factors, Matrices, and Forces Combine and Control Stem Cells.
D. E. Discher, D. J. Mooney, and P. W. Zandstra (2009)
Science 324, 1673-1677
   Abstract »    Full Text »    PDF »
Force Signaling in Biology.
J. Christof, M. Gebhardt, and M. Rief (2009)
Science 324, 1278-1280
   Abstract »    Full Text »    PDF »
The structure of the ankyrin-binding site of {beta}-spectrin reveals how tandem spectrin-repeats generate unique ligand-binding properties.
P. R. Stabach, I. Simonovic, M. A. Ranieri, M. S. Aboodi, T. A. Steitz, M. Simonovic, and J. S. Morrow (2009)
Blood 113, 5377-5384
   Abstract »    Full Text »    PDF »
Multiscale Modeling of Form and Function.
A. J. Engler, P. O. Humbert, B. Wehrle-Haller, and V. M. Weaver (2009)
Science 324, 208-212
   Abstract »    Full Text »    PDF »
Plectin contributes to mechanical properties of living cells.
S. Na, F. Chowdhury, B. Tay, M. Ouyang, M. Gregor, Y. Wang, G. Wiche, and N. Wang (2009)
Am J Physiol Cell Physiol 296, C868-C877
   Abstract »    Full Text »    PDF »
Localization and Structure of the Ankyrin-binding Site on {beta}2-Spectrin.
L. Davis, K. Abdi, M. Machius, C. Brautigam, D. R. Tomchick, V. Bennett, and P. Michaely (2009)
J. Biol. Chem. 284, 6982-6987
   Abstract »    Full Text »    PDF »
Embryonic cardiomyocytes beat best on a matrix with heart-like elasticity: scar-like rigidity inhibits beating.
A. J. Engler, C. Carag-Krieger, C. P. Johnson, M. Raab, H.-Y. Tang, D. W. Speicher, J. W. Sanger, J. M. Sanger, and D. E. Discher (2008)
J. Cell Sci. 121, 3794-3802
   Abstract »    Full Text »    PDF »
Red cell membrane: past, present, and future.
N. Mohandas and P. G. Gallagher (2008)
Blood 112, 3939-3948
   Abstract »    Full Text »    PDF »
Visualizing myosin-actin interaction with a genetically-encoded fluorescent strain sensor.
S. Iwai and T. Q. P. Uyeda (2008)
PNAS 105, 16882-16887
   Abstract »    Full Text »    PDF »
In vitro and in vivo evidence for shear-induced activation of latent transforming growth factor-{beta}1.
J. Ahamed, N. Burg, K. Yoshinaga, C. A. Janczak, D. B. Rifkin, and B. S. Coller (2008)
Blood 112, 3650-3660
   Abstract »    Full Text »    PDF »
Mechanotransduction - a field pulling together?.
C. S. Chen (2008)
J. Cell Sci. 121, 3285-3292
   Abstract »    Full Text »    PDF »
Force-Clamp Spectroscopy Detects Residue Co-evolution in Enzyme Catalysis.
R. Perez-Jimenez, A. P. Wiita, D. Rodriguez-Larrea, P. Kosuri, J. A. Gavira, J. M. Sanchez-Ruiz, and J. M. Fernandez (2008)
J. Biol. Chem. 283, 27121-27129
   Abstract »    Full Text »    PDF »
Application of Fluorescence Resonance Energy Transfer and Magnetic Twisting Cytometry to Quantify Mechanochemical Signaling Activities in a Living Cell.
S. Na and N. Wang (2008)
Science Signaling 1, pl1
   Abstract »    Full Text »    PDF »
Structural and functional effects of hereditary hemolytic anemia-associated point mutations in the alpha spectrin tetramer site.
M. Gaetani, S. Mootien, S. Harper, P. G. Gallagher, and D. W. Speicher (2008)
Blood 111, 5712-5720
   Abstract »    Full Text »    PDF »
Nuclear Shape, Mechanics, and Mechanotransduction.
K. N. Dahl, A. J.S. Ribeiro, and J. Lammerding (2008)
Circ. Res. 102, 1307-1318
   Abstract »    Full Text »    PDF »
Rapid signal transduction in living cells is a unique feature of mechanotransduction.
S. Na, O. Collin, F. Chowdhury, B. Tay, M. Ouyang, Y. Wang, and N. Wang (2008)
PNAS 105, 6626-6631
   Abstract »    Full Text »    PDF »
Mechanical Biochemistry of Proteins One Molecule at a Time.
A. F. Oberhauser and M. Carrion-Vazquez (2008)
J. Biol. Chem. 283, 6617-6621
   Abstract »    Full Text »    PDF »
Matrix Elasticity, Cytoskeletal Tension, and TGF-{beta}: The Insoluble and Soluble Meet.
R. G. Wells and D. E. Discher (2008)
Science Signaling 1, pe13
   Abstract »    Full Text »    PDF »

To Advertise     Find Products

Science Signaling. ISSN 1937-9145 (online), 1945-0877 (print). Pre-2008: Science's STKE. ISSN 1525-8882