Abstract
Membrane curvatures are involved in essential cellular processes, such as endocytosis and exocytosis, in which they are believed to act as microdomains for protein interactions and intracellular signaling. These membrane curvatures appear and disappear dynamically, and their locations are difficult or impossible to predict. In addition, the size of these curvatures is usually below the diffraction limit of visible light, making it impossible to resolve their values using live-cell imaging. Therefore, precise manipulation of membrane curvature is important to understanding how membrane curvature is involved in intracellular processes. Recent studies show that membrane curvatures can be induced by surface topography when cells are in direct contact with engineered substrates. Here, we present detailed procedures for using nanoscale structures to manipulate membrane curvatures and probe curvature-induced phenomena in live cells. We first describe detailed procedures for the design of nanoscale structures and their fabrication using electron-beam (E-beam) lithography. The fabrication process takes 2 d, but the resultant chips can be cleaned and reused repeatedly over the course of 2 years. Then we describe how to use these nanostructures to manipulate local membrane curvatures and probe intracellular protein responses, discussing surface coating, cell plating, and fluorescence imaging in detail. Finally, we describe a procedure to characterize the nanostructure–cell membrane interface using focused ion beam and scanning electron microscopy (FIB–SEM). Nanotopography-based methods can induce stable membrane curvatures with well-defined curvature values and locations in live cells, which enables the generation of a library of curvatures for probing curvature-related intracellular processes.
This is a preview of subscription content, access via your institution
Access options
Access Nature and 54 other Nature Portfolio journals
Get Nature+, our best-value online-access subscription
$29.99 / 30 days
cancel any time
Subscribe to this journal
Receive 12 print issues and online access
$259.00 per year
only $21.58 per issue
Rent or buy this article
Prices vary by article type
from$1.95
to$39.95
Prices may be subject to local taxes which are calculated during checkout
Data availability
The data that support the findings of this study are available from the corresponding author upon reasonable request.
Code availability
The MATLAB script for fluorescence protein image analysis on nanostructures is available as Supplementary Data 2 of this article.
References
Di Fiore, P. P. & von Zastrow, M. Endocytosis, signaling, and beyond. Cold Spring Harb. Perspect. Biol. 6, a016865 (2014).
Sorkin, A. & von Zastrow, M. Endocytosis and signalling: intertwining molecular networks. Nat. Rev. Mol. Cell Biol. 10, 609–622 (2009).
Mattila, P. K. & Lappalainen, P. Filopodia: molecular architecture and cellular functions. Nat. Rev. Mol. Cell Biol. 9, 446–454 (2008).
Mitchison, H. M. & Valente, E. M. Motile and non-motile cilia in human pathology: from function to phenotypes. J. Pathol. 241, 294–309 (2017); erratum 241, 564 (2017).
Lou, H.-Y., Zhao, W., Zeng, Y. & Cui, B. The role of membrane curvature in nanoscale topography-induced intracellular signaling. Acc. Chem. Res. 51, 1046–1053 (2018).
McMahon, H. T. & Gallop, J. L. Membrane curvature and mechanisms of dynamic cell membrane remodelling. Nature 438, 590–596 (2005).
Veland, I. R., Awan, A., Pedersen, L. B., Yoder, B. K. & Christensen, S. T. Primary cilia and signaling pathways in mammalian development, health and disease. Nephron Physiol. 111, 39–53 (2009).
Habermann, B. The BAR-domain family of proteins: a case of bending and binding? EMBO Rep. 5, 250–255 (2004).
Mim, C. & Unger, V. M. Membrane curvature and its generation by BAR proteins. Trends Biochem. Sci. 37, 526–533 (2012).
Simunovic, M., Voth, G. A., Callan-Jones, A. & Bassereau, P. When physics takes over: BAR proteins and membrane curvature. Trends Cell Biol 25, 780–792 (2015).
Dawson, J. C., Legg, J. A. & Machesky, L. M. Bar domain proteins: a role in tubulation, scission and actin assembly in clathrin-mediated endocytosis. Trends Cell Biol 16, 493–498 (2006).
Ma, Y. et al. The inverse F-BAR domain protein srGAP2 acts through srGAP3 to modulate neuronal differentiation and neurite outgrowth of mouse neuroblastoma cells. PLoS ONE 8, e57865 (2013).
Kooijman, E. E. et al. Spontaneous curvature of phosphatidic acid and lysophosphatidic acid. Biochemistry 44, 2097–2102 (2005).
Abbott, J. et al. CMOS nanoelectrode array for all-electrical intracellular electrophysiological imaging. Nat. Nanotechnol. 12, 460–466 (2017).
Xie, C., Lin, Z., Hanson, L., Cui, Y. & Cui, B. Intracellular recording of action potentials by nanopillar electroporation. Nat. Nanotechnol 7, 185–190 (2012).
Wang, M. et al. Nanotechnology and nanomaterials for improving neural interfaces. Adv. Funct. Mater. 28, 1700905 (2017).
Limongi, T. et al. Fabrication and applications of micro/nanostructured devices for tissue engineering. Nanomicro Lett. 9, 1 (2016).
Feller, L. et al. Cellular responses evoked by different surface characteristics of intraosseous titanium implants. Biomed. Res. Int. 2015, 171945 (2015).
Wennerberg, A. & Albrektsson, T. Effects of titanium surface topography on bone integration: a systematic review. Clin. Oral Implants Res. 20(Suppl. 4), 172–184 (2009).
Teo, B. K. K. et al. Nanotopography modulates mechanotransduction of stem cells and induces differentiation through focal adhesion kinase. ACS Nano 7, 4785–4798 (2013).
Rebollar, E. et al. Proliferation of aligned mammalian cells on laser-nanostructured polystyrene. Biomaterials 29, 1796–1806 (2008).
Park, J. et al. Directed migration of cancer cells guided by the graded texture of the underlying matrix. Nat. Mater. 15, 792–801 (2016).
Brammer, K. S., Choi, C., Frandsen, C. J., Oh, S. & Jin, S. Hydrophobic nanopillars initiate mesenchymal stem cell aggregation and osteo-differentiation. Acta Biomater. 7, 683–690 (2011).
Luu, T. U., Gott, S. C., Woo, B. W. K., Rao, M. P. & Liu, W. F. Micro- and nanopatterned topographical cues for regulating macrophage cell shape and phenotype. ACS Appl. Mater. Interfaces 7, 28665–28672 (2015).
Sorre, B. et al. Nature of curvature coupling of amphiphysin with membranes depends on its bound density. Proc. Natl. Acad. Sci. USA 109, 173–178 (2012).
Shi, Z. & Baumgart, T. Membrane tension and peripheral protein density mediate membrane shape transitions. Nat. Commun. 6, 5974 (2015).
Bhatia, V. K., Hatzakis, N. S. & Stamou, D. A unifying mechanism accounts for sensing of membrane curvature by BAR domains, amphipathic helices and membrane-anchored proteins. Semin. Cell Dev. Biol. 21, 381–390 (2010).
Simunovic, M. & Voth, G. A. Membrane tension controls the assembly of curvature-generating proteins. Nat. Commun. 6, 7219 (2015).
Tsujita, K., Takenawa, T. & Itoh, T. Feedback regulation between plasma membrane tension and membrane-bending proteins organizes cell polarity during leading edge formation. Nat. Cell Biol. 17, 749–758 (2015).
Suetsugu, S. The proposed functions of membrane curvatures mediated by the BAR domain superfamily proteins. J. Biochem. 148, 1–12 (2010).
Kim, W., Ng, J. K., Kunitake, M. E., Conklin, B. R. & Yang, P. Interfacing silicon nanowires with mammalian cells. J. Am. Chem. Soc. 129, 7228–7229 (2007).
Xie, C. et al. Noninvasive neuron pinning with nanopillar arrays. Nano Lett. 10, 4020–4024 (2010).
Xu, A. M. et al. Quantification of nanowire penetration into living cells. Nat. Commun. 5, 3613 (2014).
Shalek, A. K. et al. Vertical silicon nanowires as a universal platform for delivering biomolecules into living cells. Proc. Natl. Acad. Sci. USA 107, 1870–1875 (2010).
Berthing, T. et al. Cell membrane conformation at vertical nanowire array interface revealed by fluorescence imaging. Nanotechnology 23, 415102 (2012).
Santoro, F. et al. Revealing the cell-material interface with nanometer resolution by focused ion beam/scanning electron microscopy. ACS Nano 11, 8320–8328 (2017).
Dipalo, M. et al. Cells adhering to 3D vertical nanostructures: cell membrane reshaping without stable internalization. Nano Lett. 18, 6100–6105 (2018).
Hanson, L. et al. Vertical nanopillars for in situ probing of nuclear mechanics in adherent cells. Nat. Nanotechnol. 10, 554–562 (2015).
Zhao, W. et al. Nanoscale manipulation of membrane curvature for probing endocytosis in live cells. Nat. Nanotechnol. 12, 750–756 (2017).
Frost, A. et al. Structural basis of membrane invagination by F-BAR domains. Cell 132, 807–817 (2008).
Zhao, H., Pykäläinen, A. & Lappalainen, P. I-BAR domain proteins: linking actin and plasma membrane dynamics. Curr. Opin. Cell Biol. 23, 14–21 (2011).
Antonny, B. Mechanisms of membrane curvature sensing. Annu. Rev. Biochem. 80, 101–123 (2011).
Galic, M. et al. External push and internal pull forces recruit curvature-sensing N-BAR domain proteins to the plasma membrane. Nat. Cell Biol. 14, 874–881 (2012).
Bugnicourt, G., Brocard, J., Nicolas, A. & Villard, C. Nanoscale surface topography reshapes neuronal growth in culture. Langmuir 30, 4441–4449 (2014).
Kang, K. et al. Axon-first neuritogenesis on vertical nanowires. Nano Lett. 16, 675–680 (2016).
Rasmussen, C. H. et al. Enhanced differentiation of human embryonic stem cells toward definitive endoderm on ultrahigh aspect ratio nanopillars. Adv. Funct. Mater. 26, 815–823 (2015).
Wierzbicki, R. et al. Mapping the complex morphology of cell interactions with nanowire substrates using FIB-SEM. PLoS ONE 8, e53307 (2013).
Santoro, F. et al. Interfacing electrogenic cells with 3D nanoelectrodes: position, shape, and size matter. ACS Nano 8, 6713–6723 (2014).
Hochmuth, R. M. Micropipette aspiration of living cells. J. Biomech. 33, 15–22 (2000).
Sen, S., Subramanian, S. & Discher, D. E. Indentation and adhesive probing of a cell membrane with AFM: theoretical model and experiments. Biophys. J. 89, 3203–3213 (2005).
Xie, C., Hanson, L., Cui, Y. & Cui, B. X. Vertical nanopillars for highly localized fluorescence imaging. Proc. Natl. Acad. Sci. USA 108, 3894–3899 (2011).
Kim, W., Ng, J. K., Kunitake, M. E., Conklin, B. R. & Yang, P. Interfacing silicon nanowires with mammalian cells. J. Am. Chem. Soc. 129, 7228–7229 (2007).
Acknowledgements
This work was supported by NIH grant 1R01GM125737 to B.C. and an NTU Start-up Grant and NTU-NNI Neurotechnology Fellowship to W. Zhao. X.L. thanks the Banting Postdoctoral Fellowships program, administered by the Government of Canada. We thank the D. Drubin lab, University of California, Berkeley, for the gift of U2OS human bone osteosarcoma epithelial cells, and the V. Siciliano lab, Istituto Italiano di Tecnologia, for the gift of HEK 293FT human embryonic kidney cells. B.C. and X.L. thank the Stanford Nanofabrication Facility and Stanford Nano Shared Facilities for their help with nanofabrication. F.S. and L.M. thank V. Mollo for help with the embedding procedure and A. Qualtieri for help with SEM–FIB in Lecce, Italy.
Author information
Authors and Affiliations
Contributions
X.L., A.F.M., W. Zhao, and B.C. designed the nanochips and conceived the nanofabrication process. X.L. fabricated the nanochips. L.M., C.L., and F.S. performed FIB–SEM imaging. W. Zhang and L.K. performed cell culture and imaging. All the authors wrote the manuscript.
Corresponding authors
Ethics declarations
Competing interests
The authors declare no competing interests.
Additional information
Journal peer review information: Nature Protocols thanks Orit Shefi, Bozhi Tian and other anonymous reviewer(s) for their contribution to the peer review of this work.
Publisher’s note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
Related links
Key references using this protocol
Zhao, W. et al. Nat. Nanotechnol. 12, 750–756 (2017): https://www.nature.com/articles/nnano.2017.98
Hanson, L. et al. Nat. Nanotechnol. 10, 554–562 (2015): https://www.nature.com/articles/nnano.2015.88
Santoro, F. et al. ACS Nano 11, 8320–8328 (2017): https://pubs.acs.org/doi/abs/10.1021/acsnano.7b03494
Integrated supplementary information
Supplementary Figure 1 Curvature measurements on the nanostructures.
The measurements of the curvatures designed in the nanostructures are labeled in the top view. “D” denotes diameter. The values of the curvatures are listed in Supplementary Table 1. There are also flat surfaces as a control in the design, such as the side walls of the nanobar (b) and the I in CUI (c).
Supplementary Figure 2 Microbubbles in FIB–SEM.
Air trapped in resin deforms cellular structure. Scale, 1 µm.
Supplementary Figure 3 Optical micrograph of defects after liftoff.
The regions of missing Cr masks are indicated by the red arrows. Scale bar, 5 µm.
Supplementary Figure 4 Substrate piece placed on a carrier wafer for dry etching.
The substrate piece is cut into four pieces. Vacuum pump oil is applied between the substrate piece and dummy wafer.
Supplementary Figure 5
Surface chemistry for immobilizing ECM proteins.
Supplementary Figure 6 Selection of ends and centers on a nanobar.
This is a crop from U2-OS DNM2-EGFP fluorescence image (5 µm by 5 µm), generated. The small blue masks select the centers on both sides of the nanobar. The small red masks select the ends of the nanobar. The intensity values are read in a customized MATLAB program. The total intensity of the ends is divided by the total intensity of the centers to obtain a ratio for a given nanobar. Scale bar, 1 µm.
Supplementary Figure 7 Cracks in samples in FIB–SEM.
Scale bar, 5 µm.
Supplementary Figure 8 Charging effect in FIB–SEM imaging.
Scale bar, 5 µm.
Supplementary Figure 9 Cross-sectioning in FIB–SEM.
a, Secondary electron imaging. b, Location of a region of interest. d, Pt deposition. d, Trenching. Scale bars, 1 µm.
Supplementary Figure 10 Curtaining effect.
Sample is tilted to 52°.
Supplementary Figure 11 Collapsed nanopillars after dry etching.
Nanopillars are 200 nm in diameter and 1 µm in height. Scale bar, 5 µm.
Supplementary information
Supplementary Information
Supplementary Figures 1–11 and Supplementary Table 1
Supplementary Data 1
Nanostructure design examples.
Supplementary Data 2
MATLAB code and image examples for protein accumulation analysis.
Rights and permissions
About this article
Cite this article
Li, X., Matino, L., Zhang, W. et al. A nanostructure platform for live-cell manipulation of membrane curvature. Nat Protoc 14, 1772–1802 (2019). https://doi.org/10.1038/s41596-019-0161-7
Received:
Accepted:
Published:
Issue Date:
DOI: https://doi.org/10.1038/s41596-019-0161-7
This article is cited by
-
Electroactive nanoinjection platform for intracellular delivery and gene silencing
Journal of Nanobiotechnology (2023)
-
Curved adhesions mediate cell attachment to soft matrix fibres in three dimensions
Nature Cell Biology (2023)
-
Role of actin cytoskeleton in cargo delivery mediated by vertically aligned silicon nanotubes
Journal of Nanobiotechnology (2022)
-
Membrane curvature regulates the spatial distribution of bulky glycoproteins
Nature Communications (2022)
-
Insights into Membrane Curvature Sensing and Membrane Remodeling by Intrinsically Disordered Proteins and Protein Regions
The Journal of Membrane Biology (2022)
Comments
By submitting a comment you agree to abide by our Terms and Community Guidelines. If you find something abusive or that does not comply with our terms or guidelines please flag it as inappropriate.