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Nanoscale manipulation of membrane curvature for probing endocytosis in live cells

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Abstract

Clathrin-mediated endocytosis (CME) involves nanoscale bending and inward budding of the plasma membrane, by which cells regulate both the distribution of membrane proteins and the entry of extracellular species1,2. Extensive studies have shown that CME proteins actively modulate the plasma membrane curvature1,3,4. However, the reciprocal regulation of how the plasma membrane curvature affects the activities of endocytic proteins is much less explored, despite studies suggesting that membrane curvature itself can trigger biochemical reactions5,6,7,8. This gap in our understanding is largely due to technical challenges in precisely controlling the membrane curvature in live cells. In this work, we use patterned nanostructures to generate well-defined membrane curvatures ranging from +50 nm to −500 nm radius of curvature. We find that the positively curved membranes are CME hotspots, and that key CME proteins, clathrin and dynamin, show a strong preference towards positive membrane curvatures with a radius <200 nm. Of ten CME-related proteins we examined, all show preferences for positively curved membrane. In contrast, other membrane-associated proteins and non-CME endocytic protein caveolin1 show no such curvature preference. Therefore, nanostructured substrates constitute a novel tool for investigating curvature-dependent processes in live cells.

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Figure 1: Vertical nanopillars generate well-defined membrane curvatures that induce local accumulation of endocytic proteins.
Figure 2: Engineered 3D nanostructures for versatile control of membrane curvatures and endocytic protein accumulations.
Figure 3: Probing curvature sensitivity of various endocytic proteins using nanobar arrays.
Figure 4: Pre-curved membranes are preferred sites for endocytosis.

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Acknowledgements

We thank Y. Miao and S.H. Hong of the D.G.D. group in UC Berkeley for valuable discussion as well as helpful comments on genome-edited cell lines and endocytic lifetime analysis; K. Shen in Stanford for generous support on spinning disk confocal microscopy, M. Galic of the T. Meyer group in Stanford for suggestions and amphiphysin1-YFP plasmid; S. Guo of the B.C. group for constructing mCherry-CAAX plasmid, as well as A. McGuire, C. Xie and Z. Lin of the B.C. group in Stanford for advice and help on the nanostructure fabrication. We also thank Q. Ong and L. Kaplan of the B.C. group for comments on the manuscript. Fabrication and characterization of nanostructures were conducted in Stanford Nanofabrication Facility (SNF) and Stanford Nano Shared Facilities (SNSF). Spinning disk confocal with perfect focus for lifetime measurement was conducted in Cell Science Imaging Facility (CSIF) at Stanford University. This work was supported by the National Science Foundation (CAREER award no. 1055112), the National Institutes of Health (NIH; grant no. NS057906), a Searle Scholar award, a Packard Science and Engineering Fellowship (to B.C.), NIH fellowship 1F32 GM113379-01A1 (to J.R.M.), Studying Abroad Scholarship (to H.-Y. L.), Arnold O. Beckman Postdoctoral Fellowship (to M.A.), Heart Rhythm Research Fellowship (to F.S.) and the NIH grant R35GM118149 (to D.G.D.).

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Contributions

W.Z., B.C., Y.C. and D.G.D conceived the study and designed the experiment. W.Z. fabricated the nanostructure substrates, and performed most of experiments. L.H. performed TEM measurements. F.S. conducted the FIB-SEM characterization. H.-Y.L. performed most of the endocytic protein test on nanobar arrays and the quantification and statistical analysis. W.Z., P.D.C. and B.C. developed the Matlab code for the dynamic analysis. W.Z. analysed most of the data. M.A. analysed the AP2/Dynamin2 movies. A.G. and J.R.M. provided and characterized the genome-edited cell line. W.Z. and B.C. wrote the manuscript. All the authors discussed the results and commented on the manuscript.

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Correspondence to David G. Drubin, Yi Cui or Bianxiao Cui.

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The authors declare no competing financial interests.

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Zhao, W., Hanson, L., Lou, HY. et al. Nanoscale manipulation of membrane curvature for probing endocytosis in live cells. Nature Nanotech 12, 750–756 (2017). https://doi.org/10.1038/nnano.2017.98

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