Skip to main content

Three-Dimensional Reconstruction Imaging Method to Study the Function of EFHD2 in Invadopodia Formation

  • Protocol
  • First Online:
Calcium-Binding Proteins of the EF-Hand Superfamily

Part of the book series: Methods in Molecular Biology ((MIMB,volume 1929))

Abstract

Calcium and calcium-binding proteins play crucial roles in the regulation of actin dynamics, which contributes to cancer cell migration and invasion. In this chapter, we have focused on a three-dimensional imaging method to explore the pathophysiological function of EF-hand domain-containing protein D2 (EFHD2), a novel actin-binding protein. To overcome the limitations of two-dimensional imaging on substrate-coated cover glass for examination of invasive protrusions of cancer cells, we suggest three-dimensional reconstruction from optical z-sections of cells cultured on substrate-impregnated membrane filters of Transwell.

This is a preview of subscription content, log in via an institution to check access.

Access this chapter

Protocol
USD 49.95
Price excludes VAT (USA)
  • Available as PDF
  • Read on any device
  • Instant download
  • Own it forever
eBook
USD 169.00
Price excludes VAT (USA)
  • Available as EPUB and PDF
  • Read on any device
  • Instant download
  • Own it forever
Hardcover Book
USD 219.99
Price excludes VAT (USA)
  • Durable hardcover edition
  • Dispatched in 3 to 5 business days
  • Free shipping worldwide - see info

Tax calculation will be finalised at checkout

Purchases are for personal use only

Institutional subscriptions

References

  1. Greka A, Mundel P (2012) Calcium regulates podocyte actin dynamics. Semin Nephrol 32(4):319–326. https://doi.org/10.1016/j.semnephrol.2012.06.003

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  2. Rottner K, Stradal TE (2011) Actin dynamics and turnover in cell motility. Curr Opin Cell Biol 23(5):569–578. https://doi.org/10.1016/j.ceb.2011.07.003

    Article  CAS  PubMed  Google Scholar 

  3. Kwon MS, Park KR, Kim YD et al (2013) Swiprosin-1 is a novel actin bundling protein that regulates cell spreading and migration. PLoS One 8(8):e71626. https://doi.org/10.1371/journal.pone.0071626

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  4. Huh YH, Kim SH, Chung KH et al (2013) Swiprosin-1 modulates actin dynamics by regulating the F-actin accessibility to cofilin. Cell Mol Life Sci 70(24):4841–4854. https://doi.org/10.1007/s00018-013-1447-5

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  5. Huh YH, Oh S, Yeo YR et al (2015) Swiprosin-1 stimulates cancer invasion and metastasis by increasing the Rho family of GTPase signaling. Oncotarget 6(15):13060–13071. https://doi.org/10.18632/oncotarget.3637

    Article  PubMed  PubMed Central  Google Scholar 

  6. Wang Z, Liang X, Cai M et al (2016) Analysis of invadopodia formation in breast cancer cells. Methods Mol Biol 1406:203–210. https://doi.org/10.1007/978-1-4939-3444-7_18

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  7. Linder S, Wiesner C, Himmel M (2011) Degrading devices: invadosomes in proteolytic cell invasion. Annu Rev Cell Dev Biol 27:85–211. https://doi.org/10.1146/annurev-cellbio-092910-154216

    Article  CAS  Google Scholar 

  8. Murphy DA, Courtneidge SA (2011) The ‘ins’ and ‘outs’ of podosomes and invadopodia: characteristics, formation and function. Nat Rev Mol Cell Biol 12:413–426. https://doi.org/10.1038/nrm3141

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  9. Gawden-Bone C, Zhou Z, King E et al (2010) Dendritic cell podosomes are protrusive and invade the extracellular matrix using metalloproteinase MMP-14. J Cell Sci 123(9):1427–1437. https://doi.org/10.1242/jcs.056515

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  10. Baranov M, Ter Beest M, Reinieren-Beeren I et al (2014) Podosomes of dendritic cells facilitate antigen sampling. J Cell Sci 127(5):1052–1064. https://doi.org/10.1242/jcs.141226

    Article  CAS  PubMed  PubMed Central  Google Scholar 

Download references

Acknowledgments

This research was supported by the Bio Imaging and Cell Logistics Research Center (NRF-2016R1A5A1007318), Brain Research Program NRF-2014M3C7A1046050, NRF-2016M3C7A1905475, and NRF-2016R1C1B1013237 to YHH through the National Research Foundation of Korea funded by the Ministry of Science, ICT and Future Planning. The Bio Imaging and Cell Logistics Research Center provided access to microscopes.

Author information

Authors and Affiliations

Authors

Corresponding author

Correspondence to Woo Keun Song .

Editor information

Editors and Affiliations

Rights and permissions

Reprints and permissions

Copyright information

© 2019 Springer Science+Business Media, LLC, part of Springer Nature

About this protocol

Check for updates. Verify currency and authenticity via CrossMark

Cite this protocol

Huh, Y.H., Yeo, Y.R., Song, W.K. (2019). Three-Dimensional Reconstruction Imaging Method to Study the Function of EFHD2 in Invadopodia Formation. In: Heizmann, C. (eds) Calcium-Binding Proteins of the EF-Hand Superfamily. Methods in Molecular Biology, vol 1929. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-9030-6_38

Download citation

  • DOI: https://doi.org/10.1007/978-1-4939-9030-6_38

  • Published:

  • Publisher Name: Humana Press, New York, NY

  • Print ISBN: 978-1-4939-9029-0

  • Online ISBN: 978-1-4939-9030-6

  • eBook Packages: Springer Protocols

Publish with us

Policies and ethics