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

Abstract

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.

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Acknowledgements

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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Contributions

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.

Corresponding author

Correspondence to Georg E. Fantner.

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The authors declare no competing financial interests.

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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). https://doi.org/10.1038/s41565-018-0149-4

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