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Imaging dynamic cell-cell junctional coupling in vivo using Trojan-LAMP

Nature Methods volume 5, pages 835–841 (2008)Cite this article

Abstract

To study the physiological regulation and function of cell-cell gap junction communication in vivo, we developed a bioconjugate of caged dye, named dextran-CANPE-HCC, for imaging cell coupling in small model organisms. In vitro, the compound was photolyzed efficiently with robust fluorescence enhancement. Dextran-CANPE-HCC delivered into Caenorhabditis elegans oocytes was retained in cells throughout development. Using local uncaging, we photolyzed dextran-CANPE-HCC to release the small HCC dye and imaged the dynamics of intercellular dye transfer through gap junction channels, a technique we named Trojan–local activation of molecular fluorescent probes (LAMP). Early during embryonic development, the pattern of cell coupling undergoes dramatic remodeling and imaging revealed that the germ cell precursors, P2, P3 and P4, were isolated from the somatic cell communication compartment. As dextran-CANPE-HCC is chemically and metabolically stable, labeled worms showed very bright signal upon photoactivation after hatching, which allowed us to examine cell coupling in living worms noninvasively.

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Figure 1: Bioconjugates of caged probes.
Figure 2: Synthesis and characterization of dextran-CANPE-HCC.
Figure 3: Dye transfer in early embryos.
Figure 4: Dye transfer in early 4-cell embryos.
Figure 5: Patterns of cell coupling in late 4-cell embryos.
Figure 6: Cell-cell dye transfer in developing C. elegans embryos is through gap junction channels.
Figure 7: Dynamic gap junction communication network in early developing embryos.

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Acknowledgements

Imaging experiments involving two-photon excitation were performed at the Live Cell Imaging Core Facility of University of Texas Southwestern Medical Center and directed by K. Luby-Phelps, who also provided critical comments on the manuscript. We thank the Caenorhabditis Genetics Center for providing worm strains and X. Wang (University of Texas Southwestern) for providing the microinjection apparatus. We thank the Welch Foundation (I-1510) and the US National Institutes of Health for financial support.

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Author notes

  1. Yan-Ming Guo and Shiuhwei Chen: These authors contributed equally to this work.

Authors and Affiliations

  1. Departments of Cell Biology and of Biochemistry, University of Texas Southwestern Medical Center, 5323 Harry Hines Blvd., Dallas, 75390, Texas, USA

    Yan-Ming Guo, Shiuhwei Chen, Genhua Zheng & Wen-hong Li

  2. Department of Molecular Biology, University of Texas Southwestern Medical Center, 5323 Harry Hines Blvd., Dallas, 75390, Texas, USA

    Premnath Shetty & Rueyling Lin

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Corresponding author

Correspondence to Wen-hong Li.

Supplementary information

Supplementary Text and Figures

Supplementary Figure 1–8 , Supplementary Methods (PDF 1579 kb)

Supplementary movie 1.

Dye transfer in an early 4-cell C. elegans embryo after uncaging EMS. (MOV 776 kb)

Supplementary movie 2.

Dye transfer in a late 4-cell C. elegans embryo after uncaging EMS. (MOV 1088 kb)

Supplementary movie 3.

Restricted dye diffusion into the P3 cell (interphase) of developing C. elegans embryos. (MOV 2528 kb)

Supplementary movie 4.

Restricted dye diffusion into the dividing P3 cell of developing C. elegans embryos. (MOV 1975 kb)

Supplementary movie 5.

Restricted dye diffusion into the P4 and D cells of developing C. elegans embryos. (MOV 2568 kb)

Supplementary movie 6.

Fluorescence enhancement after a global uncaging of a L1 larva labeled with dextran-CANPE-HCC. (MOV 1966 kb)

Supplementary movie 7.

Image dynamic cell-cell coupling in a living worm (L1 larva). (MOV 704 kb)

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Guo, YM., Chen, S., Shetty, P. et al. Imaging dynamic cell-cell junctional coupling in vivo using Trojan-LAMP. Nat Methods 5, 835–841 (2008). https://doi.org/10.1038/nmeth.1238

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