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Imaging diacylglycerol dynamics at organelle membranes

Nature Methods volume 3, pages 797–799 (2006)Cite this article

Abstract

Fluorescence imaging is a powerful technique to visualize spatiotemporal dynamics of biomolecules in living cells. We describe fluorescent indicators for a lipid second messenger, diacylglycerol (DAG), which allow the localized analysis of DAG dynamics at subcellular membranes. We have thus pinpointed that DAG concentrations increase and/or decrease at not only the plasma membrane but also organelle membranes such as endomembranes and mitochondrial outer membranes.

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Figure 1: Fluorescent indicators for DAG in living cells.
Figure 2: Imaging the FRET response of Daglas.
Figure 3: Subcellular dynamics of DAG in living cells.

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Acknowledgements

This work was supported by grants from Japan Science and Technology Agency (JST) and Japan Society for the Promotion of Science (JSPS). We thank S. Ohno for providing cDNA encoding rabbit protein kinase Cβ.

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Author notes

  1. Moritoshi Sato and Yoshibumi Ueda: These authors contributed equally to this work.

Authors and Affiliations

  1. Department of Chemistry, School of Science, The University of Tokyo, Hongo, Bunkyo-ku, 113-0033, Tokyo, Japan

    Moritoshi Sato, Yoshibumi Ueda & Yoshio Umezawa

  2. PRESTO, Japan Science and Technology Agency, 4-1-8 Honcho Kawaguchi, Saitama, Japan

    Moritoshi Sato

Authors
  1. Moritoshi Sato
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  2. Yoshibumi Ueda
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  3. Yoshio Umezawa
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Corresponding author

Correspondence to Yoshio Umezawa.

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The authors declare no competing financial interests.

Supplementary information

Supplementary Fig. 1

Subcellular localization of Daglas-pm1 in MDCK cells. (PDF 458 kb)

Supplementary Fig. 2

Time courses of Daglas-PHD(R284C)-pm1, -em1 and -mit1 responses upon PDBu stimulation in MDCK cells. (PDF 387 kb)

Supplementary Fig. 3

PMA-dependent FRET responses of Daglas-pm1, -em1 and -mit1. (PDF 265 kb)

Supplementary Fig. 4

pH titrations of the fluorescence of Daglas-pm1, -em1 and -mit1. (PDF 29 kb)

Supplementary Fig. 5

Photobleaching study of Daglas. (PDF 653 kb)

Supplementary Fig. 6

Fluorescence images of a MDCK cell expressed with a tandem array of GFP and both cysteine rich domains (CRDα and CRDβ) of protein kinase Cγ before (time 0s) and at 30s, 200s and 400s after 100 μM ATP stimulation. (PDF 62 kb)

Supplementary Methods (DOC 24 kb)

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Sato, M., Ueda, Y. & Umezawa, Y. Imaging diacylglycerol dynamics at organelle membranes. Nat Methods 3, 797–799 (2006). https://doi.org/10.1038/nmeth930

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