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High-speed photothermal off-resonance atomic force microscopy reveals assembly routes of centriolar scaffold protein SAS-6


The self-assembly of protein complexes is at the core of many fundamental biological processes1, ranging from the polymerization of cytoskeletal elements, such as microtubules2, to viral capsid formation and organelle assembly3. To reach a comprehensive understanding of the underlying mechanisms of self-assembly, high spatial and temporal resolutions must be attained. This is complicated by the need to not interfere with the reaction during the measurement. As self-assemblies are often governed by weak interactions, they are especially difficult to monitor with high-speed atomic force microscopy (HS-AFM) due to the non-negligible tip–sample interaction forces involved in current methods. We have developed a HS-AFM technique, photothermal off-resonance tapping (PORT), which is gentle enough to monitor self-assembly reactions driven by weak interactions. We apply PORT to dissect the self-assembly reaction of SAS-6 proteins, which form a nine-fold radially symmetric ring-containing structure that seeds the formation of the centriole organelle. Our analysis reveals the kinetics of SAS-6 ring formation and demonstrates that distinct biogenesis routes can be followed to assemble a nine-fold symmetrical structure.

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Fig. 1: Assembly of the centriolar scaffolding protein CrSAS-6 cannot be observed with conventional HS-AM-AFM.
Fig. 2: Basic principle of HS-PORT.
Fig. 3: Imaging forces on mica in liquid comparing HS-AM-AFM and HS-PORT.
Fig. 4: Imaging and analysis of CrSAS-6 self-assembly using HS-PORT.


  1. Kushner, D. J. Self-assembly of biological structures. Bacteriol. Rev. 33, 302–345 (1969).

    CAS  Google Scholar 

  2. Mandelkow, E. M. Microtubule dynamics and microtubule caps: a time-resolved cryo-electron microscopy study. J. Cell Biol. 114, 977–991 (1991).

    CAS  Article  Google Scholar 

  3. Cameron, J. C., Wilson, S. C., Bernstein, S. L. & Kerfeld, C. A. Biogenesis of a bacterial organelle: the carboxysome assembly pathway. Cell 155, 1131–1140 (2013).

    CAS  Article  Google Scholar 

  4. Detrich, H. W.., & Williams, R. C.. Reversible dissociation of the αβ beta dimer of tubulin from bovine brain. Biochemistry 17, 3900–3907 (1978).

    CAS  Article  Google Scholar 

  5. Garzón, M. T. et al. The dimerization domain of the HIV-1 capsid protein binds a capsid protein-derived peptide: a biophysical characterization. Protein Sci. 13, 1512–1523 (2004).

    Article  Google Scholar 

  6. Kodera, N., Yamamoto, D., Ishikawa, R. & Ando, T. Video imaging of walking myosin V by high-speed atomic force microscopy. Nature 468, 72–76 (2010).

    CAS  Article  Google Scholar 

  7. Preiner, J. et al. IgGs are made for walking on bacterial and viral surfaces. Nat. Commun. 5, 4394 (2014).

    CAS  Article  Google Scholar 

  8. Oshima, H., Hayashi, T. & Kinoshita, M. Statistical thermodynamics for actin–myosin binding: the crucial importance of hydration effects. Biophys. J. 110, 2496–2506 (2016).

    CAS  Article  Google Scholar 

  9. Shibata, M., Yamashita, H., Uchihashi, T., Kandori, H. & Ando, T. High-speed atomic force microscopy shows dynamic molecular processes in photoactivated bacteriorhodopsin. Nat. Nanotech. 5, 208–212 (2010).

    CAS  Article  Google Scholar 

  10. Kitagawa, D. et al. Structural basis of the 9-fold symmetry of centrioles. Cell 144, 364–375 (2011).

    CAS  Article  Google Scholar 

  11. van Breugel, M. et al. Structures of SAS-6 suggest its organization in centrioles. Science 331, 1196–1199 (2011).

    Article  Google Scholar 

  12. Bornens, M. The centrosome in cells and organisms. Science 335, 422–426 (2012).

    CAS  Article  Google Scholar 

  13. Hirono, M. Cartwheel assembly. Philos. Trans. R. Soc. B 369, 20130458 (2014).

    Article  Google Scholar 

  14. Strnad, P. & Gönczy, P. Mechanisms of procentriole formation. Trends Cell Biol. 18, 389–396 (2008).

    CAS  Article  Google Scholar 

  15. Pfreundschuh, M., Alsteens, D., Hilbert, M., Steinmetz, M. O. & Müller, D. J. Localizing chemical groups while imaging single native proteins by high-resolution atomic force microscopy. Nano Lett. 14, 2957–2964 (2014).

    CAS  Article  Google Scholar 

  16. Hilbert, M. et al. SAS-6 engineering reveals interdependence between cartwheel and microtubules in determining centriole architecture. Nat. Cell Biol. 18, 393–403 (2016).

    CAS  Article  Google Scholar 

  17. Rosa-Zeiser, A., Weilandt, E., Hild, S. & Marti, O. The simultaneous measurement of elastic, electrostatic and adhesive properties by scanning force microscopy: pulsed-force mode operation. Meas. Sci. Technol. 8, 1333–1338 (1997).

    CAS  Article  Google Scholar 

  18. Ortega-Esteban, A. et al. Minimizing tip–sample forces in jumping mode atomic force microscopy in liquid. Ultramicroscopy 114, 56–61 (2012).

    CAS  Article  Google Scholar 

  19. Xu, X., Carrasco, C., de Pablo, P. J., Gomez-Herrero, J. & Raman, A. Unmasking imaging forces on soft biological samples in liquids when using dynamic atomic force microscopy: a case study on viral capsids. Biophys. J. 95, 2520–8 (2008).

    CAS  Article  Google Scholar 

  20. Ashby, P. D. Gentle imaging of soft materials in solution with amplitude modulation atomic force microscopy: Q control and thermal noise. Appl. Phys. Lett. 91, 254102 (2017).

    Article  Google Scholar 

  21. Kumar, B., Pifer, P. M., Giovengo, A. & Legleiter, J. The effect of set point ratio and surface Young’s modulus on maximum tapping forces in fluid tapping mode atomic force microscopy. J. Appl. Phys. 107, 044508 (2010).

    Article  Google Scholar 

  22. Ratcliff, G. C., Erie, D. A. & Superfine, R. Photothermal modulation for oscillating mode atomic force microscopy in solution. Appl. Phys. Lett. 72, 1911–1913 (1998).

    CAS  Article  Google Scholar 

  23. Nievergelt, A. P., Erickson, B. W., Hosseini, N., Adams, J. D. & Fantner, G. E. Studying biological membranes with extended range high-speed atomic force microscopy. Sci. Rep. 5, 11987 (2015).

    CAS  Article  Google Scholar 

  24. Johnson, K. L. Contact Mechanics (Cambridge Univ. Press, Cambridge, 1985).

  25. Guzman, H. V., Perrino, A. P. & Garcia, R. Peak forces in high-resolution imaging of soft matter in liquid. ACS Nano 7, 3198–3204 (2013).

    CAS  Article  Google Scholar 

  26. Kodera, N., Sakashita, M. & Ando, T. Dynamic proportional-integral-differential controller for high-speed atomic force microscopy. Rev. Sci. Instrum. 77, 83704 (2006).

    Article  Google Scholar 

  27. Amo, C. A. & Garcia, R. Fundamental high-speed limits in single-molecule, single-cell, and nanoscale force spectroscopies. ACS Nano 10, 7117–7124 (2016).

    CAS  Article  Google Scholar 

  28. Ando, T. et al. A high-speed atomic force microscope for studying biological macromolecules. Proc. Natl Acad. Sci. USA 98, 12468–12472 (2001).

    CAS  Article  Google Scholar 

  29. Schlecker, B. et al. Single-cycle-PLL detection for real-time FM-AFM applications. IEEE Trans. Biomed. Circuits Syst. 8, 206–215 (2014).

    Article  Google Scholar 

  30. Garcia, R. & Herruzo, E. T. The emergence of multifrequency force microscopy. Nat. Nanotech. 7, 217–226 (2012).

    CAS  Article  Google Scholar 

  31. Martin-Jimenez, D., Chacon, E., Tarazona, P. & Garcia, R. Atomically resolved three-dimensional structures of electrolyte aqueous solutions near a solid surface. Nat. Commun. 7, 12164 (2016).

    CAS  Article  Google Scholar 

  32. Keller, D. et al. Mechanisms of HsSAS-6 assembly promoting centriole formation in human cells. J. Cell Biol. 204, 697–712 (2014).

    CAS  Article  Google Scholar 

  33. Bauer, M., Cubizolles, F., Schmidt, A. & Nigg, E. A. Quantitative analysis of human centrosome architecture by targeted proteomics and fluorescence imaging. EMBO J. 35, 2152–2166 (2016).

    CAS  Article  Google Scholar 

  34. Zhang, Y., Zhang, Y. & Marcus, R. B. Thermally actuated microprobes for a new wafer probe card. J. Micro. Syst. 8, 43–49 (1999).

    Article  Google Scholar 

  35. Gönczy, P. Towards a molecular architecture of centriole assembly. Nat. Rev. Mol. Cell Biol. 13, 425–435 (2012).

    Article  Google Scholar 

  36. Nievergelt, A. P., Adams, J. D., Odermatt, P. D. & Fantner, G. E. High-frequency multimodal atomic force microscopy. Beilstein J. Nanotechnol. 5, 2459–2467 (2014).

    Article  Google Scholar 

  37. Guichard, P. et al. Cell-free reconstitution reveals centriole cartwheel assembly mechanisms. Nat. Commun. 8, 14813 (2017).

    CAS  Article  Google Scholar 

  38. Erickson, B. W., Coquoz, S., Adams, J. D., Burns, D. J. & Fantner, G. E. Large-scale analysis of high-speed atomic force microscopy data sets using adaptive image processing. Beilstein J. Nanotechnol. 3, 747–758 (2012).

    Article  Google Scholar 

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The authors thank C. Brillard, J. D. Adams and G. Hatzopoulos for assistance. We also thank F. Johann and J. Lopez from Oxford Instruments for help during the measurements with their Cypher VRS microscope. We thank the EPFL workshops ATPR and ATMX for the fabrication of research equipment. This work was funded by the European Union’s Seventh Framework Programme FP7/2007-2013 under grant agreement 286146, the European Union’s Seventh Framework Programme FP7/2007-2013/ERC grant agreements 307338 (to G.E.F.) and 340227 (to P.G.), and the European Union H2020 Framework Programme for Research & Innovation (2014-2020), ERC-2017-CoG, InCell, Project number 773091 (to G.E.F). N.B. was supported initially by a grant from the ERC to P.G. (AdG 340227), and then by the EPFL Fellows postdoctoral fellowship program funded by the European Union’s Horizon 2020 Framework Programme for Research and Innovation (Grant agreement 665667, MSCA-COFUND).

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Authors and Affiliations



A.P.N. and N.B. contributed equally to this work. A.P.N. designed and built the instrument, performed experiments, analysed data and wrote the paper. N.B. prepared samples, performed experiments, analysed data and wrote the paper. S.H.A. built the instrumentation. P.G. conceived the experiments and wrote the paper. G.E.F. designed the instruments, conceived experiments and wrote the paper. All the authors discussed the results and commented on the manuscript.

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Correspondence to Georg E. Fantner.

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Supplementary information

Supplementary Information

Supplementary Text, Supplementary Figures 1–12, Supplementary Table 1 and Supplementary References

Supplementary Video 1

Representative recording of CrSAS-6 assembly experiment when recorded with HS-AM-AFM. Intermediate assemblies form but no full rings can be seen forming

Supplementary Video 2

Disruption of preformed oligomers in HS-AM-AFM due to too high tip–sample forces

Supplementary Video 3

HS-PORT recording of CrSAS-6 assembly

Supplementary Video 4

Additional HS-PORT recording of CrSAS-6 assembly

Supplementary Video 5

Additional HS-PORT recording of CrSAS-6 assembly

Supplementary Video 6

Detail of one-by-one addition assembly of CrSAS-6 ring. Width of image is 116 nm

Supplementary Video 7

Detail of CrSAS-6 ring by topographical rearrangement of ring fragments. Width of image is 116 nm

Supplementary Video 8

Detail of CrSAS-6 ring by topographical rearrangement of ring fragments. Width of image is 116 nm

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Nievergelt, A.P., Banterle, N., Andany, S.H. et al. High-speed photothermal off-resonance atomic force microscopy reveals assembly routes of centriolar scaffold protein SAS-6. Nature Nanotech 13, 696–701 (2018).

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