Protocol | Published:

Live-cell confocal microscopy and quantitative 4D image analysis of anchor-cell invasion through the basement membrane in Caenorhabditis elegans

Nature Protocols volume 12, pages 20812096 (2017) | Download Citation

Abstract

Cell invasion through basement membrane (BM) barriers is crucial in development, leukocyte trafficking and the spread of cancer. The mechanisms that direct invasion, despite their importance in normal and disease states, are poorly understood, largely because of the inability to visualize dynamic cell–BM interactions in vivo. This protocol describes multichannel time-lapse confocal imaging of anchor-cell invasion in live Caenorhabditis elegans. Methods presented include outline-slide preparation and worm growth synchronization (15 min), mounting (20 min), image acquisition (20–180 min), image processing (20 min) and quantitative analysis (variable timing). The acquired images enable direct measurement of invasive dynamics including formation of invadopodia and cell-membrane protrusions, and removal of BM. This protocol can be combined with genetic analysis, molecular-activity probes and optogenetic approaches to uncover the molecular mechanisms underlying cell invasion. These methods can also be readily adapted by any worm laboratory for real-time analysis of cell migration, BM turnover and cell-membrane dynamics.

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Acknowledgements

We thank P. Maddox (University of North Carolina, Chapel Hill) for advice and reagents during the initiation of this project; N. Devos (Duke University) for assistance with video editing; M. Morrissey, D. Keeley and K. Naegeli for comments on the manuscript; and all former and current members of the laboratory of D.R.S. for their support. This work was supported by The Pew Scholars Program in the Biomedical Sciences, NIGMS R01 GM079320, R21 HD84290 and NIGMS R35 MIRA GM118049 to D.R.S.

Author information

Author notes

    • Laura C Kelley
    • , Zheng Wang
    •  & Elliott J Hagedorn

    These authors contributed equally to this work.

Affiliations

  1. Department of Biology, Regeneration Next, Duke University, Durham, North Carolina, USA.

    • Laura C Kelley
    • , Zheng Wang
    •  & David R Sherwood
  2. Center for Tissue Engineering and Regenerative Medicine, Union Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, China.

    • Zheng Wang
    • , Lin Wang
    • , Wanqing Shen
    •  & Shijun Lei
  3. Department of Gastrointestinal Surgery, Union Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, China.

    • Zheng Wang
  4. Department of Pediatrics, Boston Children's Hospital, Harvard Medical School, Howard Hughes Medical Institute, Boston, Massachusetts, USA.

    • Elliott J Hagedorn
  5. Department of Clinical Laboratory, Union Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, China.

    • Lin Wang
  6. Light Microscopy Core Facility, Duke University, Durham, North Carolina, USA.

    • Sam A Johnson

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Contributions

L.C.K., Z.W. and E.J.H. contributed equally to this work. E.J.H. performed the experiments and designed the protocol. L.C.K., E.J.H., Z.W. and D.R.S. contributed to writing the manuscript. L.C.K., E.H.H. and Z.W. prepared the figures and tables. S.A.J. advised on imaging. L.W., W.S. and S.L. provided supplementary materials.

Competing interests

The authors declare no competing financial interests.

Corresponding author

Correspondence to David R Sherwood.

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https://doi.org/10.1038/nprot.2017.093

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