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

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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Figure 1: Overview of the protocol.
Figure 2: Tools and reagents for preparing larval-stage C. elegans for time-lapse microscopy.
Figure 3: Anesthetizing and mounting worms for time-lapse microscopy.
Figure 4: Hardware systems and imaging perspectives for time-lapse analysis of AC invasion.
Figure 5: Re-alignment by free rotation and use of the Snapshot tool in Imaris.
Figure 6: Quantifying AC-invadopodia dynamics.
Figure 7: Using isosurfaces to make spatial measurements.
Figure 8: Quantification of BM-hole expansion over time.

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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.

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Authors

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.

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Correspondence to David R Sherwood.

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The authors declare no competing financial interests.

Integrated supplementary information

Supplementary Figure 1 Anchor-cell invasion through basement membrane in C. elegans.

(a) The schematic depicts the developmental time points during anchor cell (AC) invasion in C. elegans. The AC invades in tight synchrony with the 10 fated vulval precursor cell P6.p cell divisions during the L3 larval stage. During the P6.p one-cell stage, F-actin/endolysosomal membrane based invadopodia form along the invasive cell membrane (orange) within the AC (green) at the AC-basement membrane (BM) interface (magenta). AC invasion initiates during the late P6.p two-cell stage (the P6.p cell divides and gives rise to two descendants), when one or two invadopodium breaches the BM. A single breaching invadopodium transitions to an invasive protrusion that shuts down further invadopodia formation. As the invasive protrusion enlarges, it clears a large gap in the basement membrane and moves between the central P6.p vulval precursor cells by the P6.p four-cell stage (P6.p descendants divide giving rise to four cells). (b) AC invasion through BM. During invasion (0-53 minutes), the AC (F-actin; green) generates a protrusion that breaches the BM (laminin; magenta). While the BM opening expands, the protrusion grows, and reaches a mature size. Scale bar, 5 μm.

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Kelley, L., Wang, Z., Hagedorn, E. et al. Live-cell confocal microscopy and quantitative 4D image analysis of anchor-cell invasion through the basement membrane in Caenorhabditis elegans. Nat Protoc 12, 2081–2096 (2017). https://doi.org/10.1038/nprot.2017.093

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