3.6 Data Processing
3.6.4 Correction of Force Offset
In general, the force calibration and therefore the measurements were very stable and very eas-ily comparable. Probably due to dirt on the objective, one set of measurements at the C-Trap in Amsterdam had very different values for the force calibration (spring constant) than all other measurements. To still gain comparable data, the data with the calibration outliner was cor-rected by dividing the force values by the spring constant of the calibration that was performed for this measurements. The new values were multiplied by the mean spring constant of the cal-ibrations of the same experiments, performed during the other weeks of measurement. Data sets, corrected by this procedure, fitted very well to the other experimental data. No other cor-rection procedures were performed for any of the optical trap data sets.
Table 3.1:List of Chemicals and Enzymes
Chemical/Protein Full Name, Company
AMP ampicillin sodium salt, Sigma-Aldrich, Munich, Germany
ATTO 647N-maleimide ATTO-TEC GmbH, Siegen, Germany Bio-Gel P medium BioRad, Hercules, CA, USA
biotin-maleimide Jena Bioscience GmbH, Jena, Germany
BSA bovine serum albumin, Sigma-Aldrich, Munich,
Germany, A7906-10G, Lot: SLBB476V
CM sepharose column material, GE Healthcare, Munich, Ger-many
L-cysteine Carl-Roth GmbH, Karlsruhe, Germany
DEAE sepharose column material, GE Healthcare, Munich, Ger-many
DMSO ≥99,8 %, p.A., dimethyl sulfoxide, Carl-Roth GmbH, Karlsruhe, Germany
DNAse1 Sigma-Aldrich, Munich, Germany
C2H5OH ethanol, Carl-Roth GmbH, Karlsruhe, Germany
EDC
glutaraldehyde Polysciences Europe GmbH, Hirschberg an der Bergstrasse, Germany
glycerol Carl-Roth GmbH, Karlsruhe, Germany
HCl hydrochloric acid, Carl-Roth GmbH, Karlsruhe, Germany
DOC 3α-12α,dihydroxy-5β-cholanic acid sodium salt, Sigma-Aldrich, Munich, Germany
3.6. Data Processing 53 Chemical/Protein Full Name, Company
InstantBlueT M BIOZOL Diagnostica Vertrieb GmbH, #EXP-ISBO1L, Eiching, Germany
LB agar plate lysogeny broth agar plate with ampicillin, Sigma-Aldrich, Munich, Germany, L5667
lysozyme Roche Diagnostics, Mannheim, Germany MgCl2 magnesium dichloride, Sigma-Aldrich, Munich,
Germany
Maleimide-PEG-NH2 MW 5000, PG2-AMM2-5k, Nanocs, New York, NY, USA
Pefabloc SC serine protease inhibitor, Carl-Roth GmbH, Karlsruhe, Germany
PMSF phenylmethylsulfonyl fluoride, Serva, Heidel-berg, Germany
protein marker VWR, A5418.0250, Darmstadt, Germany PBS phosphate buffer saline, Invitrogen AG,
Carls-bad, CA, USA, LOT: 73604799A
KCl potassium chloride, Carl-Roth GmbH, Karlsruhe, Germany
RNAse Roche Diagnostics, Mannheim, Germany
saccharose D-sucrose,≥99,5 %, p.A., Carl-Roth GmbH, Karlsruhe, Germany
sample buffer for SDS-gel VWR/CBS, #FB31010, Darmstadt, Germany NaCl sodium chloride, Carl-Roth GmbH, Karlsruhe,
Germany
NaH2PO4 sodium dihydrogen phosphate dihydrate, Carl-Roth GmbH, Karlsruhe, Germany
NH2-PEG-OH Iris-Biotech, Marktredwitz, Germany
Chemical/Protein Full Name, Company
SDS sodium dodecyl sulphate, Carl-Roth GmbH,
Karlsruhe, Germany
SDS-gel VWR/CBS, #FK00812-10, Darmstadt, Germany Na2HPO4 disodium hydrogen phosphate dihydrate,
Carl-Roth GmbH, Karlsruhe, Germany
NaHSO3 sodium hydrogensulfit, Carl-Roth GmbH, Karls-ruhe, Germany
TB terrific broth, Sigma-Aldrich, Munich, Germany,
#T0918
DTT 1,4-di-thiothreitol, Carl-Roth GmbH, Karlsruhe, Germany
tricine N-[tris(hydroxymethyl)methyl]glycine, Carl-Roth GmbH, Karlsruhe, Germany
TRIS-HCl Tris(hydroxymethyl) aminomethane, Carl-Roth GmbH, Karlsruhe, Germany
Triton X100
4-(1,1,3,3-tetramethylbutyl)phenyl-polyethylene glycol, Carl-Roth GmbH, Karlsruhe, Germany
CH4N2O urea, Carl-Roth GmbH, Karlsruhe, Germany
References 55
References
1. Block, J.et al.Nonlinear Loading-Rate-Dependent Force Response of Individual Vimentin Intermediate Filaments to Applied Strain.Phys. Rev. Lett.118,048101 (2017).
2. Block, J.et al.Viscoelastic properties of vimentin originate from nonequilibrium confor-mational changes.Sci. Adv.4,eaat1161 (2018).
3. Herrmann, H. & Aebi, U. Intermediate Filaments: Molecular Structure, Assembly Mech-anism, and Integration into Functionally Distinct Intracellular Scaffolds. Annu. Rev.
Biochem.73,749–789 (2004).
4. Winheim, S.et al.Deconstructing the Late Phase of Vimentin Assembly by Total Internal Reflection Fluorescence Microscopy (TIRFM).PLOS One6,e19202 (2011).
5. Nöding, B., Herrmann, H. & Köster, S. Direct Observation of Subunit Exchange along Ma-ture Vimentin Intermediate Filaments.Biophys J107,2923–2931 (2014).
6. Janissen, R.et al. Invincible DNA Tethers: Covalent DNA Anchoring for Enhanced Tem-poral and Force Stability in Magnetic Tweezers Experiments.Nucleic Acids Res.42,e137 (2014).
7. Gross, P., Farge, G., Peterman, Erwin J. G. & Wuite, Gijs J. L. Combining Opticalt Tweezers, Single-Molecule Fluorescence Microscopy, and Microfluidics for Studies of DNA-Protein Interactions.Method. Enzymol.475,427–453 (2010).
8. Candelli, A., Wuite, Gijs J L & Peterman, Erwin J G. Combining Optical Trapping, Fluores-cence Microscopy and Micro-Fluidics for Single Molecule Studies of DNA-Protein Interac-tions.Phys. Chem. Chem. Phys.13,7263–7272 (2011).
9. Savitzky, A. & Golay, M. J. E. Smoothing and Differentiation of Data by Simplified Least Squares Procedures.Anal. Chem.36,1627–1639 (1964).
10. Barak, P. Smoothing and Differentiation by an Adaptive-Degree Polynomial Filter. Anal.
Chem.67,2758–2762 (1995).
Chapter 4
Nonlinear Loading-Rate- Dependent Force Response of Individual Vimentin
Intermediate Filaments to Applied Strain
This chapter was published as "Nonlinear Loading-Rate-Dependent Force Response of Indi-vidual Vimentin Intermediate Filaments to Applied Strain" (J. Block et al., Physical Review Letters 118, 048101, ISSN : 0031-9007 (4 2017), DOI: 10.1103/PhysRevLett.118.048101), and reproduced in this dissertation with the permission of the American Physical Society under the terms of the Creative Commons Attribution 3.0 License.
Johanna Block1, Hannes Witt2, Andrea Candelli3,4, Erwin J. G. Peterman3, Gijs J. L. Wuite3, Andreas Janshoff2, Sarah Köster1
1Institute for X-Ray Physics, University of Goettingen, 37077 Göttingen, Germany.
2Institute of Physical Chemistry, University of Goettingen, 37077 Göttingen, Germany.
3Department of Physics and Astronomy and LaserLab, Vrije Universiteit Amsterdam, 1081 HV Amsterdam, Netherlands.
4LUMICKS B.V., 1081 HV Amsterdam, Netherlands.
Contribution to the article: Vimentin has been produced and isolated by Susanne Bauch. I have prepared all buffers and the vimentin filaments for all experiments. I have also performed and optimized the experiments and analyzed the data of the optical tweezers experiments.
57
Hannes Witt performed the AFM experiments and analyzed the AFM data. I produced all fig-ures for the manuscript. The article was written and then iteratively optimized by Hannes Witt, Sarah Köster, Andreas Janshoff and myself.
4.1. Abstract 59
4.1 Abstract
The mechanical properties of eukaryotic cells are to a great extent determined by the cytoskele-ton, a composite network of different filamentous proteins. Among these, intermediate fila-ments (IFs) are exceptional in their molecular architecture and mechanical properties. Here we directly record stress-strain curves of individual vimentin IFs using optical traps and atomic force microscopy. We find a strong loading rate dependence of the mechanical response, sup-porting the hypothesis that IFs could serve to protect eukaryotic cells from fast, large deforma-tions. Our experimental results show different unfolding regimes, which we can quantitatively reproduce by an elastically coupled system of multiple two-state elements.