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.
This is a preview of subscription content, access via your institution
Access options
Access Nature and 54 other Nature Portfolio journals
Get Nature+, our best-value online-access subscription
$29.99 / 30 days
cancel any time
Subscribe to this journal
Receive 12 print issues and online access
$259.00 per year
only $21.58 per issue
Rent or buy this article
Prices vary by article type
from$1.95
to$39.95
Prices may be subject to local taxes which are calculated during checkout
Similar content being viewed by others
References
Yurchenco, P.D. Basement membranes: cell scaffoldings and signaling platforms. Cold Spring Harb. Perspect. Biol. 3, a004911 (2011).
Pozzi, A., Yurchenco, P.D. & Iozzo, R.V. The nature and biology of basement membranes. Matrix Biol. 57-58, 1–11 (2017).
Madsen, C.D. & Sahai, E. Cancer dissemination: lessons from leukocytes. Dev. Cell 19, 13–26 (2010).
Kelley, L.C., Lohmer, L.L., Hagedorn, E.J. & Sherwood, D.R. Traversing the basement membrane in vivo: a diversity of strategies. J. Cell Biol. 204, 291–302 (2014).
Rowe, R.G. & Weiss, S.J. Breaching the basement membrane: who, when and how? Trends Cell Biol. 18, 560–574 (2008).
Hagedorn, E.J. & Sherwood, D.R. Cell invasion through basement membrane: the anchor cell breaches the barrier. Curr. Opin. Cell Biol. 23, 589–596 (2011).
Christofori, G. New signals from the invasive front. Nature 441, 444–450 (2006).
Paul, C.D., Mistriotis, P. & Konstantopoulos, K. Cancer cell motility: lessons from migration in confined spaces. Nat. Rev. Cancer 17, 131–140 (2017).
Schoumacher, M., Louvard, D. & Vignjevic, D. Cytoskeleton networks in basement membrane transmigration. Eur. J. Cell Biol. 90, 93–99 (2011).
Albini, A. & Noonan, D.M. The 'chemoinvasion' assay, 25 years and still going strong: the use of reconstituted basement membranes to study cell invasion and angiogenesis. Curr. Opin. Cell Biol. 22, 677–689 (2010).
Wang, Z. & Sherwood, D.R. Dissection of genetic pathways in C. elegans. Methods Cell Biol. 106, 113–157 (2011).
Beerling, E., Ritsma, L., Vrisekoop, N., Derksen, P.W.B. & van Rheenen, J. Intravital microscopy: new insights into metastasis of tumors. J. Cell Sci. 124, 299–310 (2011).
Schindler, A.J. & Sherwood, D.R. Morphogenesis of the Caenorhabditis elegans vulva. Wiley Interdiscip. Rev. Dev. Biol. 2, 75–95 (2013).
Sherwood, D.R. & Sternberg, P.W. Anchor cell invasion into the vulval epithelium in C. elegans. Dev. Cell 5, 21–31 (2003).
Lohmer, L.L. et al. A sensitized screen for genes promoting invadopodia function in vivo: CDC-42 and Rab GDI-1 direct distinct aspects of invadopodia formation. PLoS Genet. 12, e1005786 (2016).
Morrissey, M.A. et al. SPARC promotes cell invasion in vivo by decreasing type iv collagen levels in the basement membrane. PLoS Genet. 12, e1005905 (2016).
Matus, D.Q. et al. In vivo identification of regulators of cell invasion across basement membranes. Sci. Signal. 3, ra35 (2010).
Hagedorn, E.J. et al. ADF/cofilin promotes invadopodial membrane recycling during cell invasion in vivo. J. Cell Biol. 204, 1209–1218 (2014).
Hagedorn, E.J. et al. Integrin acts upstream of netrin signaling to regulate formation of the anchor cell's invasive membrane in C. elegans. Dev. Cell 17, 187–198 (2009).
Sherwood, D.R., Butler, J.A., Kramer, J.M. & Sternberg, P.W. FOS-1 promotes basement-membrane removal during anchor-cell invasion in C. elegans. Cell 121, 951–962 (2005).
Hohenester, E. & Yurchenco, P.D. Laminins in basement membrane assembly. Cell Adh. Migr. 7, 56–63 (2013).
Randles, M.J., Humphries, M.J. & Lennon, R. Proteomic definitions of basement membrane composition in health and disease. Matrix Biol. 57-58, 12–28 (2016).
Carreon, T.A., Edwards, G., Wang, H. & Bhattacharya, S.K. Segmental outflow of aqueous humor in mouse and human. Exp. Eye Res. 158, 59–66 (2016).
Benton, G., Arnaoutova, I., George, J., Kleinman, H.K. & Koblinski, J. Matrigel: from discovery and ECM mimicry to assays and models for cancer research. Adv. Drug Deliv. Rev. 79-80, 3–18 (2014).
Schoumacher, M., Glentis, A., Gurchenkov, V.V. & Vignjevic, D.M. Basement membrane invasion assays: native basement membrane and chemoinvasion assay. Methods Mol. Biol. 1046, 133–144 (2013).
Deryugina, E.I. & Quigley, J.P. Chick embryo chorioallantoic membrane model systems to study and visualize human tumor cell metastasis. Histochem. Cell Biol. 130, 1119–1130 (2008).
Chen, M.B. et al. On-chip human microvasculature assay for visualization and quantification of tumor cell extravasation dynamics. Nat. Protoc. 12, 865–880 (2017).
Hiramatsu, R. et al. External mechanical cues trigger the establishment of the anterior-posterior axis in early mouse embryos. Dev. Cell 27, 131–144 (2013).
Nakaya, Y., Sukowati, E.W. & Sheng, G. Epiblast integrity requires CLASP and Dystroglycan-mediated microtubule anchoring to the basal cortex. J. Cell Biol. 202, 637–651 (2013).
Nakaya, Y., Sukowati, E.W., Alev, C., Nakazawa, F. & Sheng, G. Involvement of dystroglycan in epithelial-mesenchymal transition during chick gastrulation. Cells Tissues Organs 193, 64–73 (2011).
Srivastava, A., Pastor-Pareja, J.C., Igaki, T., Pagliarini, R. & Xu, T. Basement membrane remodeling is essential for Drosophila disc eversion and tumor invasion. Proc. Natl. Acad. Sci. USA 104, 2721–2726 (2007).
Pastor-Pareja, J.C., Grawe, F., Martín-Blanco, E. & García-Bellido, A. Invasive cell behavior during Drosophila imaginal disc eversion is mediated by the JNK signaling cascade. Dev. Cell 7, 387–399 (2004).
Morin, X., Daneman, R., Zavortink, M. & Chia, W. A protein trap strategy to detect GFP-tagged proteins expressed from their endogenous loci in Drosophila. Proc. Natl. Acad. Sci. USA 98, 15050–15055 (2001).
Guha, A., Lin, L. & Kornberg, T.B. Regulation of Drosophila matrix metalloprotease Mmp2 is essential for wing imaginal disc:trachea association and air sac tubulogenesis. Dev. Biol. 335, 317–326 (2009).
Parton, R.M., Vallés, A.M., Dobbie, I.M. & Davis, I. Live cell imaging in Drosophila melanogaster. Cold Spring Harb. Protoc. 2010, pdb.top75 (2010).
Stoletov, K. et al. Visualizing extravasation dynamics of metastatic tumor cells. J. Cell Sci. 123, 2332–2341 (2010).
Stoletov, K., Montel, V., Lester, R.D., Gonias, S.L. & Klemke, R. High-resolution imaging of the dynamic tumor cell vascular interface in transparent zebrafish. Proc. Natl. Acad. Sci. USA 104, 17406–17411 (2007).
Kedrin, D. et al. Intravital imaging of metastatic behavior through a mammary imaging window. Nat. Methods 5, 1019–1021 (2008).
Pittet, M.J. & Weissleder, R. Intravital imaging. Cell 147, 983–991 (2011).
Antinucci, P. & Hindges, R. A crystal-clear zebrafish for in vivo imaging. Sci. Rep. 6, 29490 (2016).
Ben-Yakar, A., Chronis, N. & Lu, H. Microfluidics for the analysis of behavior, nerve regeneration, and neural cell biology in C. elegans. Curr. Opin. Neurobiol. 19, 561–567 (2009).
Chronis, N., Zimmer, M. & Bargmann, C.I. Microfluidics for in vivo imaging of neuronal and behavioral activity in Caenorhabditis elegans. Nat. Methods 4, 727–731 (2007).
Keil, W., Kutscher, L.M., Shaham, S. & Siggia, E.D. Long-term high-resolution imaging of developing C. elegans larvae with microfluidics. Dev. Cell 40, 202–214 (2017).
Chai, Y. et al. Live imaging of cellular dynamics during Caenorhabditis elegans postembryonic development. Nat. Protoc. 7, 2090–2102 (2012).
Hastie, E.L. & Sherwood, D.R. A new front in cell invasion: the invadopodial membrane. Eur. J. Cell Biol. 95, 441–448 (2016).
Ihara, S. et al. Basement membrane sliding and targeted adhesion remodels tissue boundaries during uterine–vulval attachment in Caenorhabditis elegans. Nat. Cell Biol. 13, 641–651 (2011).
Wang, Z. et al. UNC-6 (netrin) stabilizes oscillatory clustering of the UNC-40 (DCC) receptor to orient polarity. J. Cell Biol. 206, 619–633 (2014).
Hagedorn, E.J. et al. The netrin receptor DCC focuses invadopodia-driven basement membrane transmigration in vivo. J. Cell Biol. 201, 903–913 (2013).
Morrissey, M.A. et al. B-LINK: a hemicentin, plakin, and integrin-dependent adhesion system that links tissues by connecting adjacent basement membranes. Dev. Cell 31, 319–331 (2014).
Chen, X., Feng, X. & Guang, S. Targeted genome engineering in Caenorhabditis elegans. Cell Biosci. 6, 60 (2016).
Hochbaum, D., Ferguson, A.A. & Fisher, A.L. Generation of transgenic C. elegans by biolistic transformation. J. Vis. Exp. http://dx.doi.org/10.3791/2090 (2010).
Berkowitz, L.A., Knight, A.L., Caldwell, G.A. & Caldwell, K.A. Generation of stable transgenic C. elegans using microinjection. J. Vis. Exp. http://dx.doi.org/10.3791/833 (2008).
Pettitt, J., Wood, W.B. & Plasterk, R.H. cdh-3, a gene encoding a member of the cadherin superfamily, functions in epithelial cell morphogenesis in Caenorhabditis elegans. Development 122, 4149–4157 (1996).
Inoue, T. et al. Gene expression markers for Caenorhabditis elegans vulval cells. Mech. Dev. 119 (Suppl. 1), S203–S209 (2002).
Matus, D.Q. et al. Invasive cell fate requires G1 cell-cycle arrest and histone deacetylase-mediated changes in gene expression. Dev. Cell 35, 162–174 (2015).
McKay, S.J. et al. Gene expression profiling of cells, tissues, and developmental stages of the nematode C. elegans. Cold Spring Harb. Symp. Quant. Biol. 68, 159–169 (2003).
McClatchey, S.T. et al. Boundary cells restrict dystroglycan trafficking to control basement membrane sliding during tissue remodeling. elife 5, e17218 (2016).
Bettinger, J.C., Euling, S. & Rougvie, A.E. The terminal differentiation factor LIN-29 is required for proper vulval morphogenesis and egg laying in Caenorhabditis elegans. Development 124, 4333–4342 (1997).
Carisey, A., Stroud, M., Tsang, R. & Ballestrem, C. Fluorescence recovery after photobleaching. Methods Mol. Biol. 769, 387–402 (2011).
Chang, C.-W., Sud, D. & Mycek, M.A. Fluorescence lifetime imaging microscopy. Methods Cell Biol. 81, 495–524 (2007).
Gligorijevic, B., Kedrin, D., Segall, J.E., Condeelis, J. & van Rheenen, J. Dendra2 photoswitching through the mammary imaging window. J. Vis. Exp. http://dx.doi.org/10.3791/1278 (2009).
Zou, W., Yadav, S., DeVault, L., Nung Jan, Y. & Sherwood, D.R. RAB-10-dependent membrane transport is required for dendrite arborization. PLoS Genet. 11, e1005484 (2015).
Schindler, A.J., Baugh, L.R. & Sherwood, D.R. Identification of late larval stage developmental checkpoints in Caenorhabditis elegans regulated by insulin/IGF and steroid hormone signaling pathways. PLoS Genet. 10, e1004426 (2014).
Clay, M.R. & Sherwood, D.R. Basement membranes in the worm: a dynamic scaffolding that instructs cellular behaviors and shapes tissues. Curr. Top Membr. 76, 337–371 (2015).
Knobel, K.M., Jorgensen, E.M. & Bastiani, M.J. Growth cones stall and collapse during axon outgrowth in Caenorhabditis elegans. Development 126, 4489–4498 (1999).
McGee-Russell, S.M. & Allen, R.D. Reversible stabilization oflabile microtubules in the reticulopodial network of Allogromia. Adv. Cell Mol. Biol. 1, 153–184 (1971).
Wang, L. & Audhya, A. In vivo imaging of C. elegans endocytosis. Methods 68, 518–528 (2014).
Kim, E., Sun, L., Gabel, C.V. & Fang-Yen, C. Long-term imaging of Caenorhabditis elegans using nanoparticle-mediated immobilization. PLoS ONE 8, e53419 (2013).
Sulston, J. & Hodgkin, J. in The Nematode Caenorhabditis elegans (ed. Wood, W.B.) 587–606 (Cold Spring Harbor Laboratory, 1988).
Baugh, L.R. To grow or not to grow: nutritional control of development during Caenorhabditis elegans L1 arrest. Genetics 194, 539–555 (2013).
Maxwell, C.S., Antoshechkin, I., Kurhanewicz, N., Belsky, J.A. & Baugh, L.R. Nutritional control of mRNA isoform expression during developmental arrest and recovery in C. elegans. Genome Res. 22, 1920–1929 (2012).
Park, E.C. & Horvitz, H.R. Mutations with dominant effects on the behavior and morphology of the nematode Caenorhabditis elegans. Genetics 113, 821–852 (1986).
Stiernagle, T. Maintenance of C. elegans. in WormBook (ed. The C. elegans research community) http://dx.doi.org/10.1895/wormbook.1.101.1 (2006).
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
Authors and Affiliations
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.
Corresponding author
Ethics declarations
Competing interests
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.
Supplementary information
Supplementary Text and Figures
Supplementary Figure 1 and Supplementary Tables 1 and 2 (PDF 1815 kb)
41596_2017_BFnprot2017093_MOESM33_ESM.mov
Invasive protrusion growth by the AC (green) during basement membrane (magenta) transmigration (62 min; scale bar, 5 μm.) (MOV 1002 kb)
Rights and permissions
About this article
Cite this article
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
Published:
Issue Date:
DOI: https://doi.org/10.1038/nprot.2017.093
Comments
By submitting a comment you agree to abide by our Terms and Community Guidelines. If you find something abusive or that does not comply with our terms or guidelines please flag it as inappropriate.