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. Author manuscript; available in PMC: 2008 Dec 8.
Published in final edited form as: Neuron. 2007 Sep 20;55(6):874–889. doi: 10.1016/j.neuron.2007.06.041

Figure 4. A Dyn3-Homer-Shank Protein Complex Couples the EZ and the PSD.

Figure 4

(A) Schematic diagram of dynamin family GTPases illustrating their domain organization. The percent amino acid conservation between dynamins 1–3 for each separate protein domain is indicated. PH, pleckstrin homology domain; GED, GTPase effector domain; PRD, proline-rich domain. Bottom numbers are amino acid positions corresponding to dynamin-3.

(B) Expression of Dyn3-PRD uncouples the EZ from the PSD. Images are of hippocampal neurons expressing PSD-95-GFP and clathrin-DsRed together with the indicated Flag-tagged dynamin constructs. Right panels correspond to regions in the dashed white boxes. Scale bars, 2 µm and 1 µm.

(C) Schematic representation of protein interactions linking dynamin-3, Homer1b/c, and Shank. PH, pleckstrin homology domain; GED, GTPase effector domain; PRD, proline-rich domain; EVH1, Ena/Vasp homology domain 1; PXXF, consensus EVH1-binding motifs; Ank, ankyrin repeats; PDZ, PSD-95/discs-large/ZO-1 homology domain; SAM, sterile alpha motif.

(D) Dynamin-3 forms a complex with Homer and Shank in rat brain. Immunoprecipitations (IP) were performed with antibodies against either dynamin-3 (Dyn3) or Shank or with control IgG and precipitated proteins subjected to immunoblot analysis (IB) for the indicated proteins. PNS, postnuclear supernatant.

(E) The Homer binding domain of dynamin-3 is required for PSD-EZ coupling. Images correspond to dendritic segments of hippocampal neurons expressing PSD-95-GFP (green) and clathrin-DsRed (red) along with either wildtype (Dyn3) or Homer-binding deficient mutant (Dyn3-P800L) dynamin-3 constructs. Scale bars, 2 µm and 1 µm. Arrowheads indicate EZ-negative PSD-95 puncta.

(F) Quantitative analysis of PSD-associated endocytic machinery. Data represent means ± SEM of the fraction of synapses lacking a clathrin-DsRed punctum within 0.7 µm (EZ-negative synapses) on hippocampal neurons expressing the indicated constructs. n = 134, 15, 20, 34, 27, 16, and 20 from left to right; ** p < 0.001 relative to control, t-test.

(G) Disruption of Homer and Shank interactions causes loss of the spine EZ. Data represent means ± SEM of the fraction of EZ-negative synapses on hippocampal neurons expressing the indicated constructs. n = 134, 37, 12, and 21 from left to right; ** p < 0.001 relative to control, t-test.

(H) GTPase-deficient mutant dynamins do not affect clathrin localization adjacent to the PSD. Data and analysis as in (F) and (G). n = 20, 15, 12, and 16, respectively; p > 0.05 relative to control, t-test.