We report a technique for generating controllable, time-varying and localizable forces on arrays of cells in a massively parallel fashion. To achieve this, we grow magnetic nanoparticle–dosed cells in defined patterns on micromagnetic substrates. By manipulating and coalescing nanoparticles within cells, we apply localized nanoparticle-mediated forces approaching cellular yield tensions on the cortex of HeLa cells. We observed highly coordinated responses in cellular behavior, including the p21-activated kinase–dependent generation of active, leading edge–type filopodia and biasing of the metaphase plate during mitosis. The large sample size and rapid sample generation inherent to this approach allow the analysis of cells at an unprecedented rate: in a single experiment, potentially tens of thousands of cells can be stimulated for high statistical accuracy in measurements. This technique shows promise as a tool for both cell analysis and control.
Subscribe to Journal
Get full journal access for 1 year
only $4.92 per issue
All prices are NET prices.
VAT will be added later in the checkout.
Tax calculation will be finalised during checkout.
Rent or Buy article
Get time limited or full article access on ReadCube.
All prices are NET prices.
Ingber, D.E. Tensegrity: the architectural basis of cellular mechanotransduction. Annu. Rev. Physiol. 59, 575–599 (1997).
Orr, A.W., Helmke, B.P., Blackman, B.R. & Schwartz, M.A. Mechanisms of mechanotransduction. Dev. Cell 10, 11–20 (2006).
Chen, C.S. Mechanotransduction—a field pulling together? J. Cell Sci. 121, 3285–3292 (2008).
Henderson, E., Haydon, P.G. & Sakaguchi, D.S. Actin filament dynamics in living glial cells imaged by atomic force microscopy. Science 257, 1944–1946 (1992).
Charras, G.T. & Horton, M.A. Single cell mechanotransduction and its modulation analyzed by atomic force microscope indentation. Biophys. J. 82, 2970–2981 (2002).
Prass, M., Jacobson, K., Mogilner, A. & Radmacher, M. Direct measurement of the lamellipodial protrusive force in a migrating cell. J. Cell Biol. 174, 767–772 (2006).
Dai, J. & Sheetz, M.P. Mechanical properties of neuronal growth cone membranes studied by tether formation with laser optical tweezers. Biophys. J. 68, 988–996 (1995).
Wang, N., Butler, J.P. & Ingber, D.E. Mechanotransduction across the cell surface and through the cytoskeleton. Science 260, 1124–1127 (1993).
Laurent, V.M. et al. Assessment of mechanical properties of adherent living cells by bead micromanipulation: comparison of magnetic twisting cytometry vs optical tweezers. J. Biomech. Eng. 124, 408–421 (2002).
Huang, H. et al. Three-dimensional cellular deformation analysis with a two-photon magnetic manipulator workstation. Biophys. J. 82, 2211–2223 (2002).
Marcy, Y., Prost, J., Carlier, M.-F. & Sykes, C. Forces generated during actin-based propulsion: a direct measurement by micromanipulation. Proc. Natl. Acad. Sci. USA 101, 5992–5997 (2004).
Hochmuth, R.M. Micropipette aspiration of living cells. J. Biomech. 33, 15–22 (2000).
Evans, E., Ritchie, K. & Merkel, R. Sensitive force technique to probe molecular adhesion and structural linkages at biological interfaces. Biophys. J. 68, 2580–2587 (1995).
Pelham, R.J. Jr. & Wang, Y. Cell locomotion and focal adhesions are regulated by substrate flexibility. Proc. Natl. Acad. Sci. USA 94, 13661–13665 (1997).
Banes, A.J. et al. Mechanoreception at the cellular level: the detection, interpretation, and diversity of responses to mechanical signals. Biochem. Cell Biol. 73, 349–365 (1995).
Sniadecki, N.J. et al. Magnetic microposts as an approach to apply forces to living cells. Proc. Natl. Acad. Sci. USA 104, 14553–14558 (2007).
Hui, E.E. & Bhatia, S.N. Micromechanical control of cell-cell interactions. Proc. Natl. Acad. Sci. USA 104, 5722–5726 (2007).
Tanase, M. et al. Assembly of multicellular constructs and microarrays of cells using magnetic nanowires. Lab Chip 5, 598–605 (2005).
Fink, J. et al. External forces control mitotic spindle positioning. Nat. Cell Biol. 13, 771–778 (2011).
Gao, J. et al. Intracellular spatial control of fluorescent magnetic nanoparticles. J. Am. Chem. Soc. 130, 3710–3711 (2008).
de Vries, A.H., Krenn, B.E., van Driel, R. & Kanger, J. Micro magnetic tweezers for nanomanipulation inside live cells. Biophys. J. 88, 2137–2144 (2005).
Tseng, P., Di Carlo, D. & Judy, J.W. Rapid and dynamic intracellular patterning of cell-internalized magnetic fluorescent nanoparticles. Nano Lett. 9, 3053–3059 (2009).
Dobson, J. Remote control of cellular behaviour with magnetic nanoparticles. Nat. Nanotechnol. 3, 139–143 (2008).
Mannix, R.J. et al. Nanomagnetic actuation of receptor-mediated signal transduction. Nat. Nanotechnol. 3, 36–40 (2008).
Huang, H., Delikanli, S., Zeng, H., Ferkey, D.M. & Pralle, A. Remote control of ion channels and neurons through magnetic-field heating of nanoparticles. Nat. Nanotechnol. 5, 602–606 (2010).
Glickman, M. et al. High-performance lateral-actuating magnetic MEMS switch. J. Microelectromech. Syst. 20, 842–851 (2011).
Pai, J.-H. Photoresist with low fluorescence for bioanalytical applications. Anal. Chem. 79, 8774–8780 (2007).
Schäffer, E., Nørrelykke, S.F. & Howard, J. Surface forces and drag coefficients of microspheres near a plane surface measured with optical tweezers. Langmuir 23, 3654–3665 (2007).
Abraham, V.C., Krishnamurthi, V., Taylor, D.L. & Lanni, F. The actin-based nanomachine at the leading edge of migrating cells. Biophys. J. 77, 1721–1732 (1999).
Berg, J.S. & Cheney, R.E. Myosin-X is an unconventional myosin that undergoes intrafilopodial motility. Nat. Cell Biol. 4, 246–250 (2002).
Papakonstanti, E.A. & Stournaras, C. Association of PI-3 kinase with PAK1 leads to actin phosphorylation and cytoskeletal reorganization. Mol. Biol. Cell 13, 2946–2962 (2002).
Dharmawardhane, S., Brownson, D., Lennartz, M. & Bokoch, G.M. Localization of p21-activated kinase 1 (PAK1) to pseudopodia, membrane ruffles, and phagocytic cups in activated human neutrophils. J. Leukoc. Biol. 66, 521–527 (1999).
Hahn, C. & Schwartz, M.A. Mechanotransduction in vascular physiology and atherogenesis. Nat. Rev. Mol. Cell Biol. 10, 53–62 (2009).
Tzima, E. et al. Activation of Rac1 by shear stress in endothelial cells mediates both cytoskeletal reorganization and effects of gene expression. EMBO J. 21, 6791–6800 (2002).
Zhang, H. et al. A tension-induced mechanotransduction pathway promotes epithelial morphogenesis. Nature 471, 99–103 (2011).
Delorme-Walker, V.D. et al. Pak1 regulates focal adhesion strength, myosin IIA distribution, and actin dynamics to optimize cell migration. J. Cell Biol. 193, 1289–1303 (2011).
Van den Broeke, C. et al. Alphaherpesvirus US3-mediated reorganization of the actin cytoskeleton is mediated by group A p21-activated kinases. Proc. Natl. Acad. Sci. USA 106, 8707–8712 (2009).
Théry, M. et al. The extracellular matrix guides the orientation of the cell division axis. Nat. Cell Biol. 7, 947–953 (2005).
Théry, M., Jiménez-Dalmoroni, A., Racine, V., Bornens, M. & Jülicher, F. Experimental and theoretical study of mitotic spindle orientation. Nature 447, 493–496 (2007).
Pande, A.N., Kohler, R.H., Aikawa, E., Weissleder, R. & Jaffer, F.A. Detection of macrophage activity in atherosclerosis in vivo using multichannel, high-resolution laser scanning fluorescence microscopy. J. Biomed. Opt. 11, 021009 (2006).
Guillou, H. et al. Lamellipodia nucleation by filopodia depends on integrin occupancy and downstream Rac1 signaling. Exp. Cell Res. 314, 478–488 (2008).
Nolen, B.J. et al. Characterization of two classes of small molecule inhibitors of Arp2/3 complex. Nature 460, 1031–1034 (2009).
Deacon, S.W. et al. An isoform-selective, small-molecule inhibitor targets the autoregulatory mechanism of p21-activated kinase. Chem. Biol. 15, 322–331 (2008).
This work was partially supported through the US National Institutes of Health Director's New Innovator Award (1DP2OD007113). The authors thank M. Bachman and N. Gunn (University of California, Irvine) for samples of PSR; J. Harrison, M. Glickman and I. Goldberg for assistance with the permalloy electroplating bath; members of the UCLA Advanced Light Microscopy Spectroscopy facility for assistance with confocal microscopy; K. Lin for high-speed imaging assistance; I. Williams for running FACS; and engineers of the UCLA Nanolab for processing assistance.
The authors declare no competing financial interests.
Supplementary Figures 1–9 (PDF 1355 kb)
Magnetic fluorescent nanoparticles coalesce quickly under high magnetic field stimulation (resin thickness is 0.5 μm).
Nanoparticle assembly occurs over a period of ~30 min at the left edge of the cell. As nanoparticles begin coalescing at the membrane edge, small clusters of nanoparticles enter temporary filopodial protrusions that extend beyond the edge. This effect continues until around the 1-h mark, when the cell membrane begins to yield under the high tension until finally the nanoparticle cluster exhibits a 'pull-in' instability, and the entire nanoparticle cluster protrudes from the edge of the cell membrane. Cell cytoplasm is labeled with calcein AM. (MOV 1472 kb)
Video displays the trajectories of two magnetic beads moving along the substrate surface towards the magnetized elements using resins with thicknesses of 2.5 and 5.3 μm, respectively. (AVI 970 kb)
Confocal microscopy z slices of a single cell under moderate magnetic nanoparticle–mediated tension.
At the z planes where the cell membrane is under magnetic nanoparticle–induced tension, local effects are observed including (i) local deformation of the flanking stress fiber caused by the applied mechanical tension and (ii) flanking actin-rich protrusions emanating from the regions of highest mechanical deformation. Positive myosin-X staining at the tips of protrusions indicate induced active, ECM-attachable filopodia. (AVI 479 kb)
Confocal microscopy z slices showing the rich band of stress fiber–localized phospho-PAK progressing through the cortical regions of high deformation.
This colocalization occurs whether or not the particular cell is expressing a high filopodial asymmetry and is distinct at regions directly above where the nanoparticles are localized. (AVI 1042 kb)
The time-lapse video shows a single cell adhering to an I-shaped fibronectin pattern as it divides under high nanoparticle-induced tension. As the cell undergoes and completes mitosis, the cell divides biased in the direction of the force generated by the magnetic nanoparticles. Upon successful division, both cells adhere and move normally. The nanoparticles remain only in one of the daughter cells. (MOV 936 kb)
About this article
Cite this article
Tseng, P., Judy, J. & Di Carlo, D. Magnetic nanoparticle–mediated massively parallel mechanical modulation of single-cell behavior. Nat Methods 9, 1113–1119 (2012). https://doi.org/10.1038/nmeth.2210
Angewandte Chemie International Edition (2021)
ACS Applied Materials & Interfaces (2021)
ACS Applied Materials & Interfaces (2021)
A review on microwell and microfluidic geometric array fabrication techniques and its potential applications in cellular studies
The Canadian Journal of Chemical Engineering (2021)
Investigate the effect of the Nano zero valent iron and Nano titanium in the genetic materials of the living cell
Materials Today: Proceedings (2021)