Assays to Measure Nuclear Mechanics in Interphase Cells

Philipp Isermann1, Patricia M. Davidson1, Josiah D. Sliz1, Jan Lammerding2

1 These authors contributed equally to this work., 2 Cornell University, Ithaca, New York
Publication Name:  Current Protocols in Cell Biology
Unit Number:  Unit 22.16
DOI:  10.1002/0471143030.cb2216s56
Online Posting Date:  September, 2012
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Abstract

The nucleus is the characteristic hallmark of all eukaryotic cells. The physical properties of the nucleus reflect important biological characteristics, such as chromatin organization or nuclear envelope composition; they can also directly affect cellular function, e.g., when cells pass through narrow constrictions, where the stiff nucleus may present a limiting factor. We present two complementary techniques to probe the mechanical properties of the nucleus. In the first, nuclear stiffness relative to the surrounding cytoskeleton is inferred from induced nuclear deformations during strain application to cells on an elastic substrate. In the second approach, nuclear deformability is deduced from the transit time through a perfusion‐based microfabricated device with constrictions smaller than the size of the nucleus. These complementary methods, which can be applied to measure nuclear stiffness in large numbers of living adherent or suspended cells, can help identify important changes in nuclear mechanics associated with disease or development. Curr. Protoc. Cell Biol. 56:22.16.1‐22.16.21. © 2012 by John Wiley & Sons, Inc.

Keywords: nucleus; microfluidics; cell/nuclear mechanics; deformation

     
 
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Table of Contents

  • Introduction
  • Strategic Planning
  • Basic Protocol 1: Nuclear Deformation During Applied Substrate Strain
  • Basic Protocol 2: Assessing Nuclear Deformability with a Microfluidic Perfusion Device
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
     
 
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Materials

Basic Protocol 1: Nuclear Deformation During Applied Substrate Strain

  Materials
  • Deionized water to rinse assembled strain dishes
  • 70% ethanol
  • Phosphate‐buffered saline (PBS) or Hank's Balanced Salt Solution (HBSS), both Ca/Mg‐ion‐free
  • Fibronectin or other suitable extracellular matrix proteins to coat silicone membrane
  • Appropriate cell culture medium, e.g., Dulbecco's modified Eagle medium (DMEM) supplemented with fetal bovine serum (FBS) and penicillin/streptomycin
  • Trypsin/EDTA
  • Hoechst 33342
  • HEPES buffered, phenol red‐free imaging medium: e.g., Dulbecco's modified Eagle medium (DMEM) without phenol red and with HEPES
  • Chemically inert, silicone‐impermeable grease (e.g., Braycote804; Castrol)
  • Custom‐built strain device consisting of microscope‐mounted base plate, dish holding plate, and components for strain dish (Fig. ); alternatively, one can use commercially available systems (e.g., from Flexcell International Corporation, the Cell Stretcher by Electron Microscopy Sciences, or the STREX instruments from B‐Bridge International)
  • Silicone membrane: 0.005‐in.‐thick silicone sheeting in 12 × 12‐in. sheets (Gloss/Gloss nonreinforced silicone sheeting; Specialty Manufacturing)
  • Scissors
  • Kimwipes or other tissues
  • Ethanol prep pad: 1.1 × 2.6‐in.
  • Autoclavable bags
  • Autoclave
  • 10‐cm disposable plastic dishes, sterile
  • Fine‐tip black permanent marker suitable to mark silicone membrane
  • Scotch tape (19‐mm width)
  • 4°C incubator
  • Centrifuge
  • Hemacytometer
  • 37°C cell culture incubator
  • Microscope
  • Inverted epifluorescence microscope equipped for fluorescence and phase‐contrast image acquisition with light sensitive CCD camera
  • Filter sets for DAPI/Hoechst 33342
  • 60× non‐immersion phase‐contrast objective with long working distance
  • Weight plate (∼5 lb) to place on strain dish for strain application (Fig. )
  • Image acquisition software
  • Linear‐encoded motorized stage (optional)
  • Image analysis software: e.g., ImageJ, Adobe Photoshop or MATLAB (Mathworks)
NOTE: DIC objectives can be used as an alternative for phase contrast; however, in our experience, DIC illumination is more sensitive to deviations from the ideal Koehler illumination that can occur with custom‐build strain devices, resulting in reduced image quality that can make it difficult to analyze the transmitted light images.NOTE: We have also successfully used (water) immersion objectives for the strain experiments; however, care must be taken that the silicone membrane is not accidentally stretched over the objective, particularly when applying strain, as this could cause additional local strain in the substrate. When using immersion objectives, it is best to lower the objective when applying the weight to the strain dish and the refocusing once full membrane strain is reached.

Basic Protocol 2: Assessing Nuclear Deformability with a Microfluidic Perfusion Device

  Materials
  • SU‐8 2010 photoresist (Microchem)
  • SU‐8 Developer (1‐Methoxy‐2‐propyl acetate, Microchem)
  • Isopropanol
  • Nitrogen
  • Anti‐stiction coating: e.g., (tridecafluoro‐1,1,2,2‐tetrahydrooctyl)trichlorosilane, a fluorinated silane (also known as FOTS)
  • Sylgard 184 silicone elastomer base
  • Sylgard 184 silicone elastomer curing agent
  • 0.2 M HCl
  • 70% ethanol
  • Deionized water
  • Bovine serum albumin (BSA; see recipe)
  • Phosphate‐buffered saline (PBS; see recipe)
  • Pluronic I‐127 (optional)
  • Cells for analysis (1 ml cell suspension in PBS with BSA at a density of 3 × 106 cells/ml)
  • Trypsin
  • Cell culture medium appropriate for the cell type studied [e.g., Dulbecco's modified Eagle medium (DMEM) with 10% fetal bovine serum (FBS)]
  • Cytochalasin D (optional)
  • 10% bleach in water (optional)
  • Microfabrication facility with the following tools: mask generator, contact aligner, molecular vapor deposition and spin coater
  • CAD software (e.g., AutoCAD, L‐edit)
  • 4‐in. silicon wafers
  • Oven
  • Hot plate
  • 150‐mm (diameter) plastic petri dishes
  • Premium vacuum pump, 115 V (Harrick Plasma)
  • Desiccator
  • Packing tape
  • Utility knife
  • Cutting mat
  • 0.75‐mm (diameter) Uni‐Core hole‐puncher (Harris)
  • Glass coverslips (22 × 50 mm, thickness 1.5)
  • Glass slides (27 × 75 × 1.0–mm)
  • Compressed air tank with 5% CO 2
  • Expanded plasma cleaner, 115 V (Harrick Plasma)
  • 0.2‐µm (pore size) syringe filter
  • 5‐ml syringes
  • 0.015‐in. i.d., 1/32‐in. o. d. pre‐cut natural PEEK tubing for microfluidics (Ing)
  • Forceps (rounded tip)
  • Cell culture incubator
  • Hemacytometer
  • Centrifuge
  • 40‐µm (pore size) cell strainer
  • High‐speed microscope camera, 60 frames per second or higher: e.g., PIKE F‐032 1/3” CCD FireWire.B Monochrome Camera with up to 208 frames per second (Edmund Optics)
  • Inverted microscope with camera mount
  • Image acquisition software
  • Image Analysis software (ImageJ)
  • 15‐ and 50‐ml Falcon tubes
  • Pressurized cap for 15‐ml Falcon tubes for microfluidics (World Precision Instruments, FLUIWELL‐1C‐15ML)
  • Two stage brass analytical pressure regulator, 0 to 25 psi (Airgas)
  • 5/16‐in. (i.d.) PVC tubing, 40’ length
  • 0.05‐in. (i.d.) PVC tubing, 2’ length
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Figures

Literature Cited

   Caille, N., Tardy, Y., and Meister, J.J. 1998. Assessment of strain field in endothelial cells subjected to uniaxial deformation of their substrate. Ann. Biomed. Eng. 26:409‐416.
   Dahl, K.N., Kahn, S.M., Wilson, K.L., and Discher, D.E. 2004. The nuclear envelope lamina network has elasticity and a compressibility limit suggestive of a molecular shock absorber. J. Cell Sci. 117:4779‐4786.
   Friedl, P., Wolf, K., and Lammerding, J. 2010. Nuclear mechanics during cell migration. Curr. Opin. Cell Biol. 23:55‐64.
   Holaska, J.M. 2008. Emerin and the nuclear lamina in muscle and cardiac disease. Circ. Res. 103:16‐23.
   Lammerding, J., Schulze, P.C., Takahashi, T., Kozlov, S., Sullivan, T., Kamm, R.D., Stewart, C.L., and Lee, R.T. 2004. Lamin A/C deficiency causes defective nuclear mechanics and mechanotransduction. J. Clin. Invest. 113:370‐378.
   Lammerding, J., Dahl, K.N., Discher, D.E., and Kamm, R.D. 2007. Nuclear mechanics and methods. Methods Cell Biol. 83:269‐294.
   Lombardi, M.L. and Lammerding, J. 2011. Keeping the LINC: The importance of nucleocytoskeletal coupling in intracellular force transmission and cellular function. Biochem. Soc. Trans. 39:1729‐1734.
   Lombardi, M.L., Zwerger, M., and Lammerding, J. 2011. Biophysical assays to probe the mechanical properties of the interphase cell nucleus: Substrate strain application and microneedle manipulation. J. Vis. Exp. 55:e3087.
   Pajerowski, J.D., Dahl, K.N., Zhong, F.L., Sammak, P.J., and Discher, D.E. 2007. Physical plasticity of the nucleus in stem cell differentiation. Proc. Natl. Acad. Sci. U.S.A. 104:15619‐15624.
   Rosenbluth, M.J., Lam, W.A., and Fletcher, D.A. 2008. Analyzing cell mechanics in hematologic diseases with microfluidic biophysical flow cytometry. Lab. Chip 8:1062‐1070.
   Rowat, A.C., Lammerding, J., Herrmann, H., and Aebi, U. 2008. Towards an integrated understanding of the structure and mechanics of the cell nucleus. BioEssays 30:226‐236.
   Simon, D.N. and Wilson, K.L. 2011. The nucleoskeleton as a genome‐associated dynamic “network of networks”. Nat. Rev. Mol. Cell Biol. 12:695‐708.
   Zwerger, M., Ho, C.Y., and Lammerding, J. 2011. Nuclear mechanics in disease. Annu. Rev. Biomed. Eng. 13:397‐428.
Key Reference
   Caille et al., 1998. See above.
  
   Lammerding et al., 2004. See above.
  This article characterizes the effect of substrate strain application on nuclear and cytoskeletal deformations.
   Huang, Y., Agrawal, B., Sun, D., Kuo, J.S., and Williams, J.C. 2011. Microfluidics‐based devices: New tools for studying cancer and cancer stem cell migration. Biomicrofluidics 5:13412.
  This article provides an example of the application of the nuclear strain assay to compare the mechanical stiffness of nucleus in lamin A/C‐deficient and wild‐type fibroblasts.
   Rosenbluth et al., 2008. See above.
  
   Whitesides, G.M., Ostuni, E., Takayama, S., Jiang, X., and Ingber, D.E. 2001. Soft lithography in biology and biochemistry. Annu. Rev. Biomed. Eng. 3:335‐373.
  This review provides an overview of the microfabrication process involved in the fabrication of microfluidic devices and the current applications of microfluidics.
Internet Resources
  http://www.jove.com/details.php?id=3087
  This is a JoVE article: Biophysical Assays to Probe the Mechanical Properties of the Interphase Cell Nucleus: Substrate Strain Application and Microneedle Manipulation (Lombardi et al., ).
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