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Fluorescent and photo-oxidizing TimeSTAMP tags track protein fates in light and electron microscopy

Nature Neuroscience volume 15, pages 1742–1751 (2012)Cite this article

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Abstract

Protein synthesis is highly regulated throughout nervous system development, plasticity and regeneration. However, tracking the distributions of specific new protein species has not been possible in living neurons or at the ultrastructural level. Previously we created TimeSTAMP epitope tags, drug-controlled tags for immunohistochemical detection of specific new proteins synthesized at defined times. Here we extend TimeSTAMP to label new protein copies by fluorescence or photo-oxidation. Live microscopy of a fluorescent TimeSTAMP tag reveals that copies of the synaptic protein PSD95 are synthesized in response to local activation of growth factor and neurotransmitter receptors, and preferentially localize to stimulated synapses in rat neurons. Electron microscopy of a photo-oxidizing TimeSTAMP tag reveals new PSD95 at developing dendritic structures of immature neurons and at synapses in differentiated neurons. These results demonstrate the versatility of the TimeSTAMP approach for visualizing newly synthesized proteins in neurons.

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Figure 1: Development of TS:YFP, a TimeSTAMP tag with a YFP output.
Figure 2: Tracking of basal and activity-induced PSD95 production with TimeSTAMP.
Figure 3: BDNF-dependent local accumulation of new PSD95 in neurons.
Figure 4: Local new PSD95 accumulation is induced by local metabotropic glutamate receptor activation.
Figure 5: Design and development of photo-oxidizing TimeSTAMPs.
Figure 6: Tracking new PSD95 populations using TS:YSOG1.
Figure 7: Pulse labeling of older PSD95 populations using TS:YSOG2.

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Acknowledgements

We thank members of the Tsien and Ellisman laboratories for helpful discussion, especially V. Lev-Ram, S. Adams and T. Deerinck. This work was supported by a US National Institutes of Health (NIH) Pharmacology Training grant (M.T.B.), the US National Science Foundation Graduate Research Fellowships Program (M.T.B.), the Lucille Packard Children's Hospital Pediatric Research Fund (Y.G.), World Class University Program grant R31-2008-000-10083-0 from the Korea Research Foundation of the South Korea Ministry of Education, Science and Technology (N.L.J.), the Howard Hughes Medical Institute and NIH grant 4R37NS027177-23 (R.Y.T.), NIH grant P41GM103412-24 (M.H.E.), NIH grant R01NS076860 (M.Z.L.), and the Burroughs Wellcome Fund (M.Z.L.). M.Z.L. is funded as a Rita Allen Foundation Scholar.

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Authors and Affiliations

  1. Department of Pharmacology and Howard Hughes Medical Institute, University of California San Diego, La Jolla, California, USA

    Margaret T Butko, Jin Yang, Xiaokun Shu, Roger Y Tsien & Michael Z Lin

  2. Department of Pediatrics, Stanford University, Palo Alto, California, USA

    Yang Geng & Michael Z Lin

  3. Department of Bioengineering, Stanford University, Palo Alto, California, USA

    Yang Geng & Michael Z Lin

  4. School of Mechanical and Aerospace Engineering, Seoul National University, Seoul, South Korea

    Hyung Joon Kim & Noo Li Jeon

  5. Department of Pharmaceutical Chemistry, University of California San Francisco, San Francisco, California, USA

    Xiaokun Shu

  6. Cardiovascular Research Institute, University of California San Francisco, San Francisco, California, USA

    Xiaokun Shu

  7. National Center for Microscopy and Imaging Research, Center for Research on Biological Systems, University of California San Diego, La Jolla, California, USA

    Mason R Mackey & Mark H Ellisman

  8. Department of Neurosciences, University of California San Diego, La Jolla, California, USA

    Mason R Mackey & Mark H Ellisman

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  1. Margaret T Butko
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Contributions

M.T.B. conceived, designed and performed electron microscopy experiments and co-wrote the manuscript. J.Y. designed and validated constructs. Y.G. designed and performed dendritic stimulation experiments. H.J.K. and N.L.J. designed and fabricated microfluidic chambers. X.S. assisted with the study design. M.R.M. assisted with electron microscopy sample preparation and imaging. M.H.E. and R.Y.T. supervised the project and provided advice. M.Z.L. conceived, designed and performed dendritic stimulation experiments, co-wrote the manuscript, and provided supervision and advice.

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Correspondence to Roger Y Tsien or Michael Z Lin.

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Supplementary information

Supplementary Text and Figures

Supplementary Figures 1–8 (PDF 20095 kb)

Supplementary Video 1

Time-lapse imaging of neurons expressing PSD95-TS2:YFP cultured in compartmentalized chambers. BILN-2061 (1 μM) was added to both chambers and 100 μM DHPG was added to the top chamber at the time of acquisition of the first frame. Frames were acquired at 2.5-h intervals. Asterisks mark TS2:YFP signal in distal dendrites from neurons residing in the bottom chamber that cross the microchannels. (AVI 2261 kb)

Supplementary Video 2

Time-lapse imaging of neurons expressing PSD95-TS2:YFP cultured in compartmentalized chambers. BILN-2061 (1 μM) was added to both chambers and 100 μM DHPG was added to the top chamber at the time of acquisition of the first frame. Frames were acquired at 2.5-h intervals. Asterisks mark TS2:YFP signal in distal dendrites from neurons residing in the bottom chamber that cross the microchannels. (AVI 209 kb)

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Butko, M., Yang, J., Geng, Y. et al. Fluorescent and photo-oxidizing TimeSTAMP tags track protein fates in light and electron microscopy. Nat Neurosci 15, 1742–1751 (2012). https://doi.org/10.1038/nn.3246

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