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Region-directed phototransfection reveals the functional significance of a dendritically synthesized transcription factor

Nature Methods volume 3, pages 455–460 (2006)Cite this article

Abstract

Multiple nuclear transcription factors including E-26–like protein 1 (Elk-1) have been found in neuronal dendrites, yet the functional significance of such localization has not yet been explained. Here we use a focal transfection procedure, 'phototransfection', to introduce Elk1 mRNA into specific regions of live, intact primary rat neurons. Introduction and translation of Elk1 mRNA in dendrites produced cell death, whereas introduction and translation of Elk1 mRNA in cell bodies did not produce cell death. Elk-1 translated in dendrites was transported to the nucleus, and cell death depended upon transcription, supporting the dendritic imprinting hypothesis and highlighting the importance of the dendritic environment on protein function. Our demonstration of the utility of phototransfection for spatially controlled introduction of mRNAs opens the broader opportunity to use this method to introduce selected quantities of small molecules into discrete regions of live cells to assess their biological functions.

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Figure 1: Elk-1 protein and Elk1 mRNA are localized in dendrites.
Figure 2: Phototransfection successfully introduces exogenous constructs into neurons.
Figure 3: Phototransfection of Elk1 mRNA into neuronal dendrites initiates cell death.
Figure 4: Cell death initiated by Elk1 mRNA depends on transcription.

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Acknowledgements

We thank C. Garner for providing the polyclonal MAP2 antibody, M. Maronski for preparation of primary neuronal cultures, and R. Pittman and C. Lai as well as members of the Eberwine laboratory for helpful discussions. This work was funded by grants MH74169 (to L.E.B.), DA015395 and DA09082 (to E.V.B.), P30NS047321, P20MH071705, R01NS043142 and R37NS037585 (to P.G.H.), and AG9900 and MH58561 (to J.H.E.).

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Author notes

  1. Lindy E Barrett and Jai-Yoon Sul: These authors contributed equally to this work.

Authors and Affiliations

  1. Department of Neuroscience, University of Pennsylvania School of Medicine, Philadelphia, 19104, Pennsylvania, USA

    Lindy E Barrett, Jai-Yoon Sul, Hajime Takano & Philip G Haydon

  2. Silvio Conte Center for Integration at the Tripartite Synapse, University of Pennsylvania School of Medicine, Philadelphia, 19104, Pennsylvania, USA

    Jai-Yoon Sul, Hajime Takano & Philip G Haydon

  3. Department of Neurosurgery, Thomas Jefferson University, Farber Institute for Neurosciences, Philadelphia, 19107, Pennsylvania, USA

    Elisabeth J Van Bockstaele

  4. Department of Pharmacology, University of Pennsylvania School of Medicine, Philadelphia, 19104, Pennsylvania, USA

    James H Eberwine

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  1. Lindy E Barrett
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  3. Hajime Takano
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  4. Elisabeth J Van Bockstaele
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  6. James H Eberwine
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Contributions

L.E.B. and J.-Y.S. planned and performed experiments, H.T. performed experiments, E.J.B. planned and interpreted the electron microscopy, and P.G.H. and J.H.E. planned and interpreted experiments.

Corresponding author

Correspondence to James H Eberwine.

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Competing interests

L.E.B., J.Y.S., P.G.H. and J.H.E. have applied for a patent covering the phototransfection technology.

P.G.H. has an equity interest in Prairie Technologies, the company that manufactures the microscopes used in this study.

Supplementary information

Supplementary Fig. 1

Additional controls for Elk-1 localization and translation. (PDF 5817 kb)

Supplementary Methods (PDF 31 kb)

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Barrett, L., Sul, JY., Takano, H. et al. Region-directed phototransfection reveals the functional significance of a dendritically synthesized transcription factor. Nat Methods 3, 455–460 (2006). https://doi.org/10.1038/nmeth885

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