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Lateral organization of endocytic machinery in dendritic spines

Nature Neuroscience volume 7, pages 917–918 (2004)Cite this article

Abstract

Postsynaptic membrane trafficking plays an important role in synaptic plasticity, but the organization of trafficking machinery within dendritic spines is poorly understood. We use immunocytochemical analysis of rat hippocampal neurons to show that proteins mediating endocytosis are systematically arrayed within dendritic spines, tangential to the synapse. Thus, previously unrecognized lateral domains of the spine organize endocytic protein machinery at sites removed from the postsynaptic density.

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Figure 1: Electron micrographs, from CA1 hippocampus of adult rat (experimental protocols approved by University of North Carolina at Chapel Hill animal use committee).
Figure 2: Quantitative analysis of immunogold labeling.

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Acknowledgements

We thank I. Perez-Otano and M. Pucak for comments on the text and H. Zhang, I. Lebedeva and K. Phend for technical support. Supported by the National Alliance for Research on Schizophrenia and Depression (T.A.B.), Christopher Reeve Paralysis Foundation (M.D.E.), Broad Foundation Scholar Award (M.D.E.) and US National Institutes of Health (M.D.E., R.J.W.).

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Authors and Affiliations

  1. Department of Cell & Developmental Biology, CB #7090, University of North Carolina, Chapel Hill, 27599, North Carolina, USA

    Bence Rácz & Richard J Weinberg

  2. Department of Neuroscience Center, CB #7090, University of North Carolina, Chapel Hill, 27599, North Carolina, USA

    Richard J Weinberg

  3. Department of Neurobiology, Box 3209, Duke University Medical Center, Durham, 27710, North Carolina, USA

    Thomas A Blanpied & Michael D Ehlers

  4. Departments of Cell Biology and of Pharmacology and Cancer Biology and Neuroproteomics Laboratory, Box 3209, Duke University Medical Center, Durham, 27710, North Carolina, USA

    Michael D Ehlers

Authors
  1. Bence Rácz
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  2. Thomas A Blanpied
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  3. Michael D Ehlers
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  4. Richard J Weinberg
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Corresponding author

Correspondence to Michael D Ehlers.

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Supplementary information

Supplementary Fig. 1

Coated pits in postsynaptic regions of cultured hippocampal neurons. (a) Coated pits (arrowheads) in spines and dendrites of cultured hippocampal neurons, as revealed by EM. Note that coated pits typically lie away from the PSD. (b) Confocal image of a cultured hippocampal neuron co-transfected with clathrin-DsRed and PSD-95-GFP. PSD-95 often clustered into puncta at presumptive excitatory synapses (arrowheads); in these regions, clathrin puncta were also prevalent. Stretches of dendrite without PSD-95 puncta (brackets) usually lacked clathrin puncta. (c) Linescan analysis of the image in (b), showing mean intensity along an 8 pixel-wide line traced along the center of the dendrite. (JPG 264 kb)

Supplementary Fig. 2

AP-2 and dynamin colocalize with clathrin in dendritic spines. (a) Projected confocal z-stack of a dendrite from a 28 DIV cultured hippocampal neuron co-transfected with the AP-2 subunit β2-adaptin-YFP (green) and clathrin-CFP (red). β2-adaptin colocalized with clathrin puncta throughout the dendrite and in spines (arrowheads) and also displayed diffuse cytosolic fluorescence outside of the clathrin-positive puncta. The magnified region (bottom panels) shows a single spine extending upwards from a dendritic shaft, highlighting the precise colocalization of clathrin and β2-adaptin in the spine head (arrow). The thin spine neck is barely visible. (b) A pair of dendrites from a 28 DIV neuron co-transfected for 36 hrs with dynamin 2-GFP (green), clathrin-DsRed (red), and PSD-95-CFP (not shown). Dynamin localized strongly to clathrin puncta (arrowheads). High magnification view shown in lower panels. Arrows point to double-labeled spine heads. Scale bar: 5 μm upper panels; 1 μm lower panels. (JPG 85 kb)

Supplementary Fig. 3

Double labeling immuno-EM for endocytic proteins. Immunoperoxidase staining for one antigen combined with pre-embedding immunogold labeling for another show endocytic proteins co-expressed in dendritic spines: (a,b) clathrin (immunogold, arrowheads) and AP-2 (electron-dense immunoperoxidase precipitate). (c) dynamin (immunoperoxidase) and AP-2 (immunogold, arrowhead). Scale bar: 200 nm (JPG 91 kb)

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Rácz, B., Blanpied, T., Ehlers, M. et al. Lateral organization of endocytic machinery in dendritic spines. Nat Neurosci 7, 917–918 (2004). https://doi.org/10.1038/nn1303

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