Probing the compressibility of tumor cell nuclei by combined atomic force–confocal microscopy

The cell nucleus is the largest and stiffest organelle rendering it the limiting compartment during migration of invasive tumor cells through dense connective tissue. We here describe a combined atomic force microscopy (AFM)-confocal microscopy approach for measurement of bulk nuclear stiffness together with simultaneous visualization of the cantilever-nucleus contact and the fate of the cell. Using cantilevers functionalized with either tips or beads and spring constants ranging from 0.06-10 N m(-1), force-deformation curves were generated from nuclear positions of adherent HT1080 fibrosarcoma cell populations at unchallenged integrity, and a nuclear stiffness range of 0.2 to 2.5 kPa was identified depending on cantilever type and the use of extended fitting models. Chromatin-decondensating agent trichostatin A (TSA) induced nuclear softening of up to 50%, demonstrating the feasibility of our approach. Finally, using a stiff bead-functionalized cantilever pushing at maximal system-intrinsic force, the nucleus was deformed to 20% of its original height which after TSA treatment reduced further to 5% remaining height confirming chromatin organization as an important determinant of nuclear stiffness. Thus, combined AFM-confocal microscopy is a feasible approach to study nuclear compressibility to complement concepts of limiting nuclear deformation in cancer cell invasion and other biological processes.

[1]  P. Friedl,et al.  Extracellular matrix determinants of proteolytic and non-proteolytic cell migration. , 2011, Trends in cell biology.

[2]  Richard T. Lee,et al.  Lamins A and C but Not Lamin B1 Regulate Nuclear Mechanics* , 2006, Journal of Biological Chemistry.

[3]  P. Janmey,et al.  Tissue Cells Feel and Respond to the Stiffness of Their Substrate , 2005, Science.

[4]  Cwj Cees Oomens,et al.  Monitoring the biomechanical response of individual cells under compression: A new compression device , 2003, Medical and Biological Engineering and Computing.

[5]  Ferenc Horkay,et al.  Determination of elastic moduli of thin layers of soft material using the atomic force microscope. , 2002, Biophysical journal.

[6]  Anthony W. Parker,et al.  Measuring protein dynamics with ultrafast two-dimensional infrared spectroscopy , 2012 .

[7]  Robert M. Hoffman,et al.  Physical limits of cell migration: Control by ECM space and nuclear deformation and tuning by proteolysis and traction force , 2013, The Journal of cell biology.

[8]  K. Stroka,et al.  Physical confinement alters tumor cell adhesion and migration phenotypes , 2012, FASEB journal : official publication of the Federation of American Societies for Experimental Biology.

[9]  Wenna Badjo,et al.  Micropipette aspiration of a living cell , 2014 .

[10]  Gilbert C. Walker,et al.  Finite Sample Thickness Effects on Elasticity Determination Using Atomic Force Microscopy , 1999 .

[11]  Alain Richert,et al.  Real-time single-cell response to stiffness , 2010, Proceedings of the National Academy of Sciences.

[12]  G. Gerlitz,et al.  The role of chromatin structure in cell migration. , 2011, Trends in cell biology.

[13]  C. Oomens,et al.  Decreased mechanical stiffness in LMNA-/- cells is caused by defective nucleo-cytoskeletal integrity: implications for the development of laminopathies. , 2004, Human molecular genetics.

[14]  F. Byfield,et al.  Micropipette aspiration of substrate-attached cells to estimate cell stiffness. , 2012, Journal of visualized experiments : JoVE.

[15]  G. Charras,et al.  The cytoplasm of living cells behaves as a poroelastic material , 2013, Nature materials.

[16]  L. Lamberti,et al.  Whole-Depth Change in Bovine Zona Pellucida Biomechanics after Fertilization: How Relevant in Hindering Polyspermy? , 2012, PloS one.

[17]  Denis Wirtz,et al.  A perinuclear actin cap regulates nuclear shape , 2009, Proceedings of the National Academy of Sciences.

[18]  H. Gaub,et al.  Interlaboratory round robin on cantilever calibration for AFM force spectroscopy. , 2011, Ultramicroscopy.

[19]  James K. Gimzewski,et al.  Applicability of AFM in cancer detection , 2009 .

[20]  Kheya Sengupta,et al.  Fibroblast adaptation and stiffness matching to soft elastic substrates. , 2007, Biophysical journal.

[21]  R. Mahaffy,et al.  Quantitative analysis of the viscoelastic properties of thin regions of fibroblasts using atomic force microscopy. , 2004, Biophysical journal.

[22]  Gang-Yu Liu,et al.  Cell mechanics using atomic force microscopy-based single-cell compression. , 2006, Langmuir : the ACS journal of surfaces and colloids.

[23]  Dennis E. Discher,et al.  Nuclear Lamin-A Scales with Tissue Stiffness and Enhances Matrix-Directed Differentiation , 2013, Science.

[24]  Dennis E Discher,et al.  Matrix elasticity, cytoskeletal forces and physics of the nucleus: how deeply do cells ‘feel’ outside and in? , 2010, Journal of Cell Science.

[25]  Christopher Beadle,et al.  The role of myosin II in glioma invasion of the brain. , 2008, Molecular biology of the cell.

[26]  P. Libby,et al.  Mechanical strain tightly controls fibroblast growth factor-2 release from cultured human vascular smooth muscle cells. , 1997, Circulation research.

[27]  Valerie M. Weaver,et al.  A tense situation: forcing tumour progression , 2009, Nature Reviews Cancer.

[28]  T. Shirai,et al.  FE‐SEM observation of swelled seaweed using hydrophilic ionic liquid; 1‐butyl‐3‐methylimidazolium tetrafluoroborate , 2013, Microscopy research and technique.

[29]  I. N. Sneddon The relation between load and penetration in the axisymmetric boussinesq problem for a punch of arbitrary profile , 1965 .

[30]  David C Lin,et al.  Robust strategies for automated AFM force curve analysis--I. Non-adhesive indentation of soft, inhomogeneous materials. , 2007, Journal of biomechanical engineering.

[31]  F. MacKintosh,et al.  Scanning probe-based frequency-dependent microrheology of polymer gels and biological cells. , 2000, Physical review letters.

[32]  Erin C. Vintinner,et al.  Linear Arrays of Nuclear Envelope Proteins Harness Retrograde Actin Flow for Nuclear Movement , 2010, Science.

[33]  G. Shivashankar,et al.  Dynamics of chromatin decondensation reveals the structural integrity of a mechanically prestressed nucleus. , 2008, Biophysical journal.

[34]  C. Shih,et al.  Cell motility and local viscoelasticity of fibroblasts. , 2005, Biophysical journal.

[35]  Jan Lammerding,et al.  Nuclear mechanics during cell migration. , 2011, Current opinion in cell biology.

[36]  Cynthia A. Reinhart-King,et al.  Tensional homeostasis and the malignant phenotype. , 2005, Cancer cell.

[37]  Rachel E. Factor,et al.  The nuclear envelope environment and its cancer connections , 2012, Nature Reviews Cancer.

[38]  H. Gong,et al.  The effect of the endothelial cell cortex on atomic force microscopy measurements. , 2013, Biophysical journal.

[39]  Shamik Sen,et al.  Indentation and adhesive probing of a cell membrane with AFM: theoretical model and experiments. , 2005, Biophysical journal.

[40]  Dennis E. Discher,et al.  Physical plasticity of the nucleus in stem cell differentiation , 2007, Proceedings of the National Academy of Sciences.

[41]  Mikala Egeblad,et al.  Matrix Crosslinking Forces Tumor Progression by Enhancing Integrin Signaling , 2009, Cell.

[42]  J. Käs,et al.  The optical stretcher: a novel laser tool to micromanipulate cells. , 2001, Biophysical journal.

[43]  J. Aubertin,et al.  Fine control of nuclear confinement identifies a threshold deformation leading to lamina rupture and induction of specific genes. , 2012, Integrative biology : quantitative biosciences from nano to macro.

[44]  A. Martinez-Arias,et al.  Chromatin decondensation and nuclear softening accompany Nanog downregulation in embryonic stem cells. , 2012, Biophysical journal.

[45]  Jan Lammerding,et al.  Nuclear Envelope Composition Determines the Ability of Neutrophil-type Cells to Passage through Micron-scale Constrictions* , 2013, The Journal of Biological Chemistry.

[46]  P. Friedl,et al.  Intravital third harmonic generation microscopy of collective melanoma cell invasion , 2012, Intravital.

[47]  Andreea Trache,et al.  Atomic force-multi-optical imaging integrated microscope for monitoring molecular dynamics in live cells. , 2005, Journal of biomedical optics.

[48]  B. Geiger,et al.  Environmental sensing through focal adhesions , 2009, Nature Reviews Molecular Cell Biology.

[49]  Josef A. Käs,et al.  Cell migration through small gaps , 2006, European Biophysics Journal.

[50]  G. Charras,et al.  Experimental validation of atomic force microscopy-based cell elasticity measurements , 2011, Nanotechnology.

[51]  Sanjay Kumar,et al.  The mechanical rigidity of the extracellular matrix regulates the structure, motility, and proliferation of glioma cells. , 2009, Cancer research.

[52]  M. Goldberg,et al.  A new model for nuclear lamina organization. , 2008, Biochemical Society transactions.

[53]  Sean X. Sun,et al.  Actin cap associated focal adhesions and their distinct role in cellular mechanosensing , 2012, Scientific Reports.

[54]  E. Kumacheva,et al.  Characterization of the mechanical properties of microgels acting as cellular microenvironments , 2013 .

[55]  R. King,et al.  Elastic analysis of some punch problems for a layered medium , 1987 .

[56]  M. Radmacher,et al.  Amphibian oocyte nuclei expressing lamin A with the progeria mutation E145K exhibit an increased elastic modulus , 2011, Nucleus.

[57]  Atef Asnacios,et al.  Microplates-based rheometer for a single living cell , 2006 .

[58]  Hermann Schillers,et al.  Elasticity measurement of living cells with an atomic force microscope: data acquisition and processing , 2008, Pflügers Archiv - European Journal of Physiology.

[59]  G. Gerlitz,et al.  Efficient cell migration requires global chromatin condensation , 2010, Journal of Cell Science.

[60]  G. Fröschl,et al.  Chromatin condensation during apoptosis is accompanied by degradation of lamin A+B, without enhanced activation of cdc2 kinase , 1994, The Journal of cell biology.

[61]  M. Radmacher,et al.  Influence of lamin A on the mechanical properties of amphibian oocyte nuclei measured by atomic force microscopy. , 2009, Biophysical journal.

[62]  D. Discher,et al.  Power-law rheology of isolated nuclei with deformation mapping of nuclear substructures. , 2005, Biophysical journal.