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A nanostructure platform for live-cell manipulation of membrane curvature

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.

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Fig. 1: Overview of the nanochip-based cell culture platform for manipulating cell membrane curvature.
Fig. 2: Screenshot of the pattern design and conversion.
Fig. 3: Workflow of the nanofabrication process.
Fig. 4: E-beam writing system.
Fig. 5: Inspections through the nanofabrication process.
Fig. 6: SEM images of nanostructures after fabrication.
Fig. 7: Optical microscopy setups.
Fig. 8: Micrographs of U2OS cells cultured on nanochips.
Fig. 9: Schematics of the FIB–SEM workflow.
Fig. 10: Curvature response of DNM2–EGFP on nanobar arrays in different cell types.
Fig. 11: FIB–SEM images of cells cultured on nanostructures.

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.

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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

Authors

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

Correspondence to Wenting Zhao, Francesca Santoro or Bianxiao Cui.

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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

Reporting Summary

Supplementary Data 1

Nanostructure design examples.

Supplementary Data 2

MATLAB code and image examples for protein accumulation analysis.

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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

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