Skip to main content

Live-Cell Imaging of F-Actin Dynamics During Fertilization in Arabidopsis thaliana

  • Protocol
  • First Online:
Plant Germline Development

Part of the book series: Methods in Molecular Biology ((MIMB,volume 1669))

Abstract

Fertilization comprises a complex series of cellular processes leading to the fusion of a male and female gamete. Many studies have been carried out to investigate each step of fertilization in plants; however, our comprehensive understanding of all the sequential events during fertilization is still limited. This is largely due to difficulty in investigating events in the female gametophyte, which is deeply embedded in the maternal tissue. Recent advances in confocal microcopy assisted by fluorescent marker lines have contributed to visualizing subcellular dynamics in real time during fertilization in vivo. In this chapter, we describe a method focusing on the investigation of F-actin dynamics in the central cell during male gamete nuclear migration. This method also allows the study of a wide range of early sexual reproduction events, from pollen tube guidance to the early stage of seed development.

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 109.00
Price excludes VAT (USA)
  • Available as EPUB and PDF
  • Read on any device
  • Instant download
  • Own it forever
Softcover Book
USD 139.00
Price excludes VAT (USA)
  • Compact, lightweight edition
  • Dispatched in 3 to 5 business days
  • Free shipping worldwide - see info
Hardcover Book
USD 169.99
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. Kawashima T, Berger F (2011) Green love talks; cell-cell communication during double fertilization in flowering plants. AoB Plants 2011:plr015

    Article  PubMed  PubMed Central  Google Scholar 

  2. Dresselhaus T, Sprunck S, Wessel GM (2016) Fertilization mechanisms in flowering plants. Curr Biol 26:R125–R139

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  3. Higashiyama T, Takeuchi H (2015) The mechanism and key molecules involved in pollen tube guidance. Annu Rev Plant Biol 66:393–413

    Article  CAS  PubMed  Google Scholar 

  4. Palanivelu R, Preuss D (2006) Distinct short-range ovule signals attract or repel Arabidopsis thaliana pollen tubes in vitro. BMC Plant Biol 6:7

    Article  PubMed  PubMed Central  Google Scholar 

  5. Jeong S, Lukowitz W (2015) Embryos, camera, laser, action. Dev Cell 34:137–138

    Article  CAS  PubMed  Google Scholar 

  6. Hamamura Y, Saito C, Awai C et al (2011) Live-cell imaging reveals the dynamics of two sperm cells during double fertilization in Arabidopsis thaliana. Curr Biol 21:497–502

    Article  CAS  PubMed  Google Scholar 

  7. Rotman N, Rozier F, Boavida L et al (2003) Female control of male gamete delivery during fertilization in Arabidopsis thaliana. Curr Biol 13:432–436

    Article  CAS  PubMed  Google Scholar 

  8. Ngo QA, Vogler H, Lituiev DS et al (2014) A calcium dialog mediated by the FERONIA signal transduction pathway controls plant sperm delivery. Dev Cell 29:491–500

    Article  CAS  PubMed  Google Scholar 

  9. Iwano M, Ngo QA, Entani T et al (2012) Cytoplasmic Ca2+ changes dynamically during the interaction of the pollen tube with synergid cells. Development 139:4202–4209

    Article  CAS  PubMed  Google Scholar 

  10. Hamamura Y, Nishimaki M, Takeuchi H et al (2014) Live imaging of calcium spikes during double fertilization in Arabidopsis. Nat Commun 5:4722

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  11. Denninger P, Bleckmann A, Lausser A et al (2014) Male-female communication triggers calcium signatures during fertilization in Arabidopsis. Nat Commun 5:4645

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  12. Kawashima T, Maruyama D, Shagirov M et al (2014) Dynamic F-actin movement is essential for fertilization in Arabidopsis thaliana. Elife 3:e04501

    Article  PubMed Central  Google Scholar 

  13. Kimata Y, Higaki T, Kawashima T et al (2016) Cytoskeleton dynamics control the first asymmetric cell division in Arabidopsis zygote. Proc Natl Acad Sci U S A 113:14157–14162

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  14. Gooh K, Ueda M, Aruga K et al (2015) Live-cell imaging and optical manipulation of Arabidopsis early embryogenesis. Dev Cell 34:242–251

    Article  CAS  PubMed  Google Scholar 

  15. Mizuta Y, Kurihara D, Higashiyama T (2015) Two-photon imaging with longer wavelength excitation in intact Arabidopsis tissues. Protoplasma 252:1231–1240

    Article  CAS  PubMed  Google Scholar 

  16. Riedl J, Crevenna AH, Kessenbrock K et al (2008) Lifeact: a versatile marker to visualize F-actin. Nat Methods 5:605–607

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  17. Ingouff M, Hamamura Y, Gourgues M et al (2007) Distinct dynamics of HISTONE3 variants between the two fertilization products in plants. Curr Biol 17:1032–1037

    Article  CAS  PubMed  Google Scholar 

  18. Kawashima T, Berger F (2015) The central cell nuclear position at the micropylar end is maintained by the balance of F-actin dynamics, but dispensable for karyogamy in Arabidopsis. Plant Reprod 28:103–110

    Article  CAS  PubMed  Google Scholar 

  19. Schindelin J, Arganda-Carreras I, Frise E et al (2012) Fiji: an open-source platform for biological-image analysis. Nat Methods 9:676–682

    Article  CAS  PubMed  Google Scholar 

  20. Schneider CA, Rasband WS, Eliceiri KW (2012) NIH image to ImageJ: 25 years of image analysis. Nat Methods 9:671–675

    Article  CAS  PubMed  PubMed Central  Google Scholar 

Download references

Acknowledgments

D.M. is supported by Toyoaki Scholarship Foundation and JSPS KAKENHI grant, number 15K14541 (Grant-in-Aid for Challenging Exploratory Research) and 16H06173 (Grant-in-Aid for Young Scientists).

Author information

Authors and Affiliations

Authors

Corresponding author

Correspondence to Tomokazu Kawashima .

Editor information

Editors and Affiliations

Rights and permissions

Reprints and permissions

Copyright information

© 2017 Springer Science+Business Media LLC

About this protocol

Cite this protocol

Susaki, D., Maruyama, D., Yelagandula, R., Berger, F., Kawashima, T. (2017). Live-Cell Imaging of F-Actin Dynamics During Fertilization in Arabidopsis thaliana . In: Schmidt, A. (eds) Plant Germline Development. Methods in Molecular Biology, vol 1669. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-7286-9_4

Download citation

  • DOI: https://doi.org/10.1007/978-1-4939-7286-9_4

  • Published:

  • Publisher Name: Humana Press, New York, NY

  • Print ISBN: 978-1-4939-7285-2

  • Online ISBN: 978-1-4939-7286-9

  • eBook Packages: Springer Protocols

Publish with us

Policies and ethics