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Correlative cryo-ET identifies actin/tropomyosin filaments that mediate cell–substrate adhesion in cancer cells and mechanosensitivity of cell proliferation

Abstract

The actin cytoskeleton is the primary driver of cellular adhesion and mechanosensing due to its ability to generate force and sense the stiffness of the environment. At the cell’s leading edge, severing of the protruding Arp2/3 actin network generates a specific actin/tropomyosin (Tpm) filament population that controls lamellipodial persistence. The interaction between these filaments and adhesion to the environment is unknown. Using cellular cryo-electron tomography we resolve the ultrastructure of the Tpm/actin copolymers and show that they specifically anchor to nascent adhesions and are essential for focal adhesion assembly. Re-expression of Tpm1.8/1.9 in transformed and cancer cells is sufficient to restore cell–substrate adhesions. We demonstrate that knock-out of Tpm1.8/1.9 disrupts the formation of dorsal actin bundles, hindering the recruitment of α-actinin and non-muscle myosin IIa, critical mechanosensors. This loss causes a force-generation and proliferation defect that is notably reversed when cells are grown on soft surfaces. We conclude that Tpm1.8/1.9 suppress the metastatic phenotype, which may explain why transformed cells naturally downregulate this Tpm subset during malignant transformation.

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Fig. 1: Tropomyosins link cellular motility initiation with adhesion assembly.
Fig. 2: Ultrastructure of Tpm/actin filaments tethered to adhesions.
Fig. 3: Tropomyosins restore cell adhesion in normal and cancer cells.
Fig. 4: Myosin and α-actinin dorsal stress fibre recruitment depend on Tpm1.8/1.9.
Fig. 5: Tpm1.8/1.9 suppress growth on soft surfaces via mechanosensing.

Data availability

The authors declare that all the data supporting the findings of this study are available within the article and the Supplementary Information. Requests for resources including plasmids should be directed to and will be fulfilled by the corresponding author. The publicly available mRNA sequence of mouse Tpm1.8/1.9 used in this study has the access code NCBI M_001164253.1. Source data are provided with this paper.

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Acknowledgements

We would like to thank the Katharina Gaus Light Microscopy Facility, the Flow Cytometry Facility and the Electron Microscopy Unit of the Mark Wainwright Analytical Centre at UNSW, and the Cryo Electron Microscopy Facility through the Victor Chang Innovation Centre for their valuable help. Thanks to J. Hook and Y. Yao for their technical assistance. P.W.G. and E.C.H. were supported by grants from the ARC (DP160101623), the Australian NHMRC (APP1100202, APP1079866) and The Kid’s Cancer Project. N.A. was supported by the Australian NHMRC (APP1102730).

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M.L.C. conceptualized and wrote the manuscript. M.L.C., N.A. and S.B. performed the data acquisition and analysis. P.W.G., E.C.H., N.S.B. and N.A. reviewed the manuscript, supervised the work and acquired funding.

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Correspondence to Peter W. Gunning.

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Supplementary Information

Supplementary Figs. 1–7 and Video 1.

Reporting Summary

Cryo-tomogram of a nascent adhesion site

Supplementary Video 1 . Related to Fig. 2. Notice the increased cellular density at the filaments/adhesion Z-plane compared to the cytoplasm above

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Cagigas, M.L., Bryce, N.S., Ariotti, N. et al. Correlative cryo-ET identifies actin/tropomyosin filaments that mediate cell–substrate adhesion in cancer cells and mechanosensitivity of cell proliferation. Nat. Mater. (2021). https://doi.org/10.1038/s41563-021-01087-z

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