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First published online 1 March 2005
doi: 10.1242/jcs.01711

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A dynamin-3 spliced variant modulates the actin/cortactin-dependent morphogenesis of dendritic spines

Noah W. Gray^1,*, Anne E. Kruchten², Jing Chen² and Mark A. McNiven^1,2,

¹ Molecular Neuroscience Program and Graduate School, Mayo Clinic, Rochester, MN 55905, USA
² Department of Biochemistry and Molecular Biology, Mayo Clinic, Rochester, MN 55905, USA

View larger version (114K): [in a new window]   Fig. 1. The expression of distinct Dyn3 variants induces profoundly different postsynaptic morphologies. (A,C) Neurons transfected with Dyn3aaa-GFP produce numerous mushroom-shaped dendritic spines by 18DIV (arrows in A). (B,D) Neurons expressing Dyn3baa-GFP exhibit morphologically immature dendritic spines at a mature culture age, with long filopodia remaining as the dominant structures instead of spines (arrows in B). (E,F) Although Dyn3aaa- and Dyn3baa-expressing neurons appear different at 18DIV, they exhibit remarkably similar morphologies at 11DIV, when filopodia normally persist. Images in C-F represent CFP fluorescence from Dyn3-spliceoform-co-transfected cells captured by confocal microscopy. Scale bars, 5 µm.

View larger version (26K): [in a new window]   Fig. 2. Dyn3baa inhibits the development of normal neuronal morphology. Neuron morphology was quantified using two morphological criteria to distinguish between filopodia and spines. 18DIV Dyn3baa-expressing cells remained similar to 11DIV cultures with respect to protrusion length (A) and presence of a spine head (B), indicating that filopodia at 11DIV do not mature into spines by 18DIV in these neurons. By contrast, control neurons expressing Dyn3aaa-GFP or GFP exhibit this normal progression during development from filopodia to spines. Expression of the GTPase-deficient Dyn3baaKA-GFP did not affect the morphology of transfected neurons at 18DIV but did cause immature (11DIV) transfected neurons to contain morphologically mature spines. All error bars represent s.d.

View larger version (66K): [in a new window]   Fig. 3. Synaptogenesis is impaired in Dyn3baa-expressing neurons. Neurons transfected with Dyn3aaa-GFP (A,C) or Dyn3baa-GFP (B,D) at 18DIV were labeled for the presynaptic marker synapsin (A,B) or the postsynaptic marker PSD-95 (C,D). Dyn3aaa-GFP was found to be directly juxtaposed to synapsin-positive puncta (arrows in A) and colocalized with PSD-95 (arrows in C). Dyn3baa-GFP and dendritic filopodia were neither directly juxtaposed to synapsin-positive puncta (arrows in B) nor colocalized with PSD-95 (arrows in D). (E,F) Quantitation of association between dendritic protrusions and synaptic markers revealed that control neurons exhibited little association with synaptic markers at 11DIV but extensive association at 18DIV. By contrast, neurons transfected with Dyn3baa-GFP exhibited impaired dendritic spine synaptogenesis, with 20% of filopodia containing PSD-95 (E) and less than 10% of filopodia juxtaposed to synapsin (F) at 18DIV. Neurons expressing a GTPase-deficient mutant Dyn3baa-GFP (KA mutant) did not exhibit this maturation defect and progressed normally through synaptogenesis with culture age. All error bars represent s.d. Scale bar, 5 µm.

View larger version (69K): [in a new window]   Fig. 4. Dyn3baa-GFP induces filopodial outgrowth in non-neuronal cells. COS-7 cells transfected with Dyn3aaa-GFP (A) maintained a normal morphology, with a smooth lamellar perimeter (arrows in A, close-up in A') and a rounded shape. By contrast, COS-7 cells transfected with Dyn3baa-GFP exhibited radical shape changes (B), with the smooth perimeter often replaced with numerous large membranous protrusions and long filopodia that were remarkably similar to the filopodia in neurons (arrows in B, close-up in B'). Transfection with Dyn3baa(KA)-GFP caused a phenotype resembling Dyn3aaa-GFP transfection, causing cells to maintain their normal smooth perimeter (C). Interestingly, mutant Dyn3baa remained at the membrane perimeter (arrows in C, close-up in C') but did not induce the shape changes observed with Dyn3baa wild-type transfection. Scale bar, 10 µm.

View larger version (25K): [in a new window]   Fig. 5. Dyn3baa directly binds with a greater affinity than Dyn3aaa to the actin-associated protein cortactin. (A) Comparing the known cortactin binding site of Dyn2 with the correlative site in the Dyn3-PRD reveal that these sequences are almost identical. (B) Cortactin directly binds to both Dyn3aaa and Dyn3baa. GST alone failed to pull down the cortactin protein. (C) Dyn3aaa and Dyn3baa directly bind to cortactin in a blot overlay experiment using a range of recombinant Dyn3 protein amounts. (D) Binding curves representing densitometry measurements demonstrating the greater affinity for cortactin of Dyn3baa compared with Dyn3aaa. Results are normalized to the average maximum in each independent experiment and plotted on a scale of 0 to 1. Error bars represent the s.d. (E) Fold differences in binding of cortactin to Dyn3baa compared with Dyn3aaa. Results were from the average of three experiments and error bars represent the s.e.m.

View larger version (55K): [in a new window]   Fig. 6. Dyn3baa-induced filopodial outgrowth in mature neurons requires both the PRD of Dyn3 and full-length cortactin. (A) Dyn3aaa and Dyn3baa were truncated by 115 amino acids to remove the PRD and putative cortactin binding site. (B) Dyn3aaaPRD does not affect synaptogenesis. Neurons transfected with Dyn3aaaPRD-GFP show normal spine morphology at 18DIV. (C) Dyn3baaPRD does not induce filopodial outgrowth in mature neurons. Neurons transfected with Dyn3baaPRD-GFP exhibit normal spine morphology at 18DIV. (D) Cortactin was truncated by 73 amino acids, removing the SH3 domain and putative dynamin binding site. (E) Neurons co-transfected with Dyn3aaa and CortSH3-RFP display no morphological defects in mature spine development. (F) Dyn3baa requires full-length cortactin to cause filopodial outgrowth in mature neurons. Neurons co-transfected with Dyn3baa-GFP and CortSH3-RFP exhibit normal spine morphology at 18DIV, unlike neurons singly transfected with Dyn3baa-GFP. Scale bar, 5 µm.

View larger version (84K): [in a new window]   Fig. 7. Dyn3 transcripts are differentially regulated during neuronal development. (A) Isolated hippocampal neurons: primers flanking a 30-nucleotide spliced insert in Dyn3 were used in RT-PCR reactions to assess the relative levels of Dyn3aaa and Dyn3baa in maturing cultured neurons at each age as labeled (`DIV' means `days in vitro'). The Dyn3aaa product is 370 bp, whereas the Dyn3baa product is 400 bp. The PCR product at 430 bp is an RT-PCR artefact representing a heteroduplex of strands from each spliceoform. GAPDH RT-PCR demonstrates the use of equal amounts of template in each reaction. MW, molecular weight markers; Br, brain; –con, negative control. (B) Identical RT-PCR reactions were prepared using template derived from whole rat hippocampus at various developmental stages as listed above (P, postnatal day). GAPDH RT-PCR provides evidence for equal transcript copy number in the loading of template for each reaction. (C) RT-PCR using the cultured neuron template set and primers designed to exhibit total levels of all Dyn3 transcripts. (D) RT-PCR using the whole hippocampi template set and primers designed to exhibit total levels of all Dyn3 transcripts.

View larger version (30K): [in a new window]   Fig. 8. Dyn3 interacts with PSD proteins to regulate dendritic spine maturation. (A) During synaptogenesis and early development, enhancing the expression of one of two PSD binding partners can lead to opposite dendritic spine phenotypes. Expression of Homer causes a supermaturation of spines, with these protrusions becoming larger and fully functional at an immature stage [11DIV (Sala et al., 2001)]. Conversely, expression of Dyn3baa inhibits the neuron from maturing and assembling spine synapses, leaving the cell in an immature state into the later stages of development (18DIV). An overabundance of another Dyn3 spliced variant (Dyn3aaa) or the GTPase-deficient Dyn3baa (Dyn3baaKA) has little effect on the normal spine developmental program; filopodia are eventually replaced by mushroom-shaped spines. (B,C) Theoretical modeling of Dyn3aaa (B) and Dyn3baa (C) of 100 amino acids around the PH domain based on the solved structure of Rac. Proline and charged residues in the insert [red in Dyn3baa (C)] might give rise to a protruding turned region extending away from the compact structure of the PH domain, as seen for Dyn3aaa in (B).

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