A novel role for snapin in dendrite patterning: interaction with cypin - PubMed (original) (raw)

A novel role for snapin in dendrite patterning: interaction with cypin

Maxine Chen et al. Mol Biol Cell. 2005 Nov.

Abstract

Temporal and spatial assembly of signal transduction machinery determines dendrite branch patterning, a process crucial for proper synaptic transmission. Our laboratory previously cloned and characterized cypin, a protein that decreases PSD-95 family member localization and regulates dendrite number. Cypin contains zinc binding, collapsin response mediator protein (CRMP) homology, and PSD-95, Discs large, zona occludens-1 binding domains. Both the zinc binding and CRMP homology domains are needed for dendrite patterning. In addition, cypin binds tubulin via its CRMP homology domain to promote microtubule assembly. Using a yeast two-hybrid screen of a rat brain cDNA library with cypin lacking the carboxyl terminal eight amino acids as bait, we identified snapin as a cypin binding partner. Here, we show by affinity chromatography and coimmunoprecipitation that the carboxyl-terminal coiled-coil domain (H2) of snapin is required for cypin binding. In addition, snapin binds to cypin's CRMP homology domain, which is where tubulin binds. We also show that snapin competes with tubulin for binding to cypin, resulting in decreased microtubule assembly. Subsequently, overexpression of snapin in primary cultures of hippocampal neurons results in decreased primary dendrites present on these neurons and increased probability of branching. Together, our data suggest that snapin regulates dendrite number in developing neurons by modulating cypin-promoted microtubule assembly.

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Figures

Figure 1.

Figure 1.

The carboxyl terminal coiled-coil domain of snapin is required to bind to the CRMP homology region of cypin. (A) Schematic of cypin. (B) Detergent soluble extract of rat brain was incubated with glutathione-Sepharose bound to 25 μg of GST or GST-cypin. The Sepharose was washed and eluted, and proteins were resolved by 10% SDS-polyacrylamide electrophoresis and transferred to Immobilon-P. Western blotting of eluates demonstrates that snapin binds to cypin above background binding to GST. Similar results were found when the experiment was performed using COS-7 cells expressing snapin tagged with GFP at its carboxy terminus. (C) Amylose resin bound to 25 μg of maltose binding protein (MBP) fusions of the indicated regions of cypin were incubated with extracts from COS-7 cells expressing snapin tagged with GFP at its amino terminus. Western blotting reveals that snapin binds to cypin when the CRMP homology domain is present (221-end, 350-end) and not when it is absent (1-120, 1-220, 1-350) or deleted (Δ350-403). (D) COS-7 cells were transfected with cDNAs encoding amino acids 1–68 of snapin fused to GFP or GFP alone. Detergent-soluble extracts of these cells were incubated with glutathione-Sepharose bound to 25 μg of GST or GST-Cypin. The Sepharose was washed and eluted, and proteins were resolved by 10% SDS-polyacrylamide electrophoresis and transferred to Immobilon-P. Western blotting of eluates demonstrates that neither snapin(1-68) nor GFP binds to cypin. (E) Extracts from rat brain were incubated with GST fusions of full-length snapin, the first (1-68) and second (69-136) halves of snapin, and the carboxyl-terminal coiled-coil domain (H2) of snapin (defined as either 81-126 or 83-119). The data demonstrate that amino acids 81–126 represent the minimal binding domain of snapin that binds to cypin. Load noted represents the percentage of input material corresponding to the appropriate affinity chromatography.

Figure 2.

Figure 2.

Snapin coimmunoprecipitates with cypin. (A) Detergent-soluble brain extract was incubated with rabbit preimmune serum, rabbit IgG, or rabbit anti-snapin. Antibody complexes were collected with protein A-Sepharose, and the immunoprecipitated complexes were separated on a 10% SDS-polyacrylamide electrophoresis. Western blotting reveals that cypin, but not PSD-95 or SNAP-25, coimmunoprecipitates with snapin. (B) Snapin coimmunoprecipitates with cypin but not tubulin. Western blotting for snapin reveals a doublet at ∼15 and 18 kDa. The 18-kDa form is seen in load. The smaller form found in the immunoprecipitate may represent a degradation product recognized by the antibody. Neither form is seen in precipitates of rabbit or mouse IgG or mouse tubulin. Cypin is precipitated with the cypin antibody, and tubulin is precipitated with the tubulin antibody. Load noted represents the percentage of input material corresponding to the appropriate immunoprecipitation.

Figure 3.

Figure 3.

Snapin is enriched in the cell bodies of developing hippocampal neurons and is found in all synaptosomal fractions. (A) Cultures of primary hippocampal neurons were grown for 7 and 12 d.i.v. and immunostained for snapin or cypin. (B) Snapin, cypin, and actin protein expression were assayed in 15 μg of extracts from neuronal cultures at different developmental time points by Western blotting. Snapin protein expression is low at 2 and 4 d.i.v. when primary dendrites are forming and branching, and expression increases by 7 d.i.v. when primary dendrites have stopped forming and higher order branches are forming. Snapin protein expression remains through 17 d.i.v., when spine formation begins. Cypin protein is expressed at all time points assessed, consistent with the idea that cypin plays a role in dendrite formation and branching. Actin expression serves as a loading control. (C) Snapin is found in all synaptosomal fractions, including synaptic plasma membranes (which are enriched in GluR1), the synaptic vesicle fraction (which is enriched in synaptophysin), and the synaptic cytosol. Like snapin, cypin is enriched in all synaptosomal fractions. Ten micrograms of each fraction was loaded.

Figure 4.

Figure 4.

Snapin is membrane associated and cytosolic. (A) Snapin is evenly partitioned between membrane-associated (P2) and soluble fractions (S2). H, unfractionated homogenate. Twenty micrograms of each fraction was loaded. A representative of two different fractionations and Western blots is shown. (B–E) Hippocampal neurons cultured for 4, 7, and 12 d.i.v. were double labeled for snapin and EEA1, an early endosomal marker (B); TGN-38, a _trans_-Golgi marker (C); phalloidin, a plasma membrane marker (D); or synaptophysin, a synaptic marker (E). The majority of snapin does not colocalize with any of the markers and is absent from the nucleus (E). A fraction of snapin colocalizes with the plasma membrane (D). Bar, 20 μm. The confocal z-sections shown are optimized for organelle localization.

Figure 5.

Figure 5.

Snapin competes tubulin heterodimer binding to cypin and slows cypin-promoted microtubule assembly. (A) Purified cypin was mixed with tubulin heterodimers in the presence of increasing amounts of purified snapin and subjected to immunoprecipitation with an antibody raised against cypin. The immunoprecipitates were resolved by SDS-PAGE, and tubulin was detected by Western blotting. Full-length snapin but not the amino terminal half (1-68) competes tubulin binding to cypin. (B) Model for snapin and tubulin binding to cypin. In the absence of snapin (top), one tubulin binds to the CRMP homology domain of cypin. When snapin levels are increased (bottom), snapin competes with tubulin to bind to cypin. Snapin has higher affinity than tubulin for cypin and hence tubulin is released from cypin. (C) Purified snapin proteins were mixed with purified cypin and tubulin heterodimers, and absorbance at 340 nm was taken to assess microtubule polymerization. In the absence of snapin (GST, filled squares), cypin promoted polymerization, whereas the presence of snapin (open circles) slowed down cypin-promoted assembly. The amino terminal half of snapin (1-68, open squares) had no effect on cypin-promoted assembly.

Figure 6.

Figure 6.

Snapin affects dendrite patterning. (A) Representative neurons transfected with cDNA encoding GFP, GFP-snapin, GFP-snapin(1-68), GFP-snapin(69-136), or GFP-snapin(81-126). Bar, 10 μm. (B) Average number of primary and secondary dendrites in neurons that overexpress GFP (n = 74), GFP-snapin (n = 12), GFP-snapin(1-68) (n = 22), GFP-snapin(69-136) (n = 36), or GFP-snapin(81-126) (n = 29). Snapin proteins that bind to cypin [snapin, snapin(69-136), snapin(81-126)] decrease primary dendrite number but do not affect secondary dendrite number. *p < 0.05 and **p < 0.01 by ANOVA followed by Dunnett's multiple comparison test compared with GFP control. (C) Snapin, snapin(69-136), and snapin(81-126) but not snapin(1-68) increase probability of dendrite branching. Data from B were plotted as a distribution of primary dendrites that branch. The graph clearly indicates that the branching probability is higher for snapin (red line), snapin(69-136) (green line), and GFP-snapin(81-126) (yellow line) compared with snapin(1-68) (blue line) and GFP (black line). GFP and GFP-snapin(1-68) do not differ from each other (p > 0.05) nor do GFP-snapin, GFP-snapin(69-136), and GFP-snapin (81-26) differ from each other (p > 0.05), but the two groups differ from each other (p < 0.001).

Figure 7.

Figure 7.

Snapin is enriched in the cell body and does not affect cypin's guanine deaminase activity. (A) Representative confocal z-sections of hippocampal neurons (12 d.i.v.) immunostained for snapin. Z-sections were taken at 1-μm intervals. Bar, 20 μm. (B) Snapin does not influence the guanine deaminase activity of cypin. Lysate (50 μl) from COS-7 cells expressing cypin-GFP was mixed with lysates (0, 80, 120, or 200 μl) of COS-7 cells expressing snapin-GFP (N-terminal tagged) or snapin-DsRed (C-terminal tagged). The remaining volume (to total of 250-μl volume of cell lysate) was lysate from untransfected COS-7 cells. This mixture was then assayed for guanine deaminase activity using a colorometric assay. n = 4 assays, each performed in duplicate. p > 0.05 as determined by ANOVA. (C) Unphosphorylated snapin binds to cypin better than a phosphomimetic form of snapin. Extracts from COS-7 cells expressing either GFP-snapin(S50A) or GFP-snapin(S50D) were incubated with GST or GST-cypin bound to glutathione beads. Eluates were analyzed for the presence of snapin by SDS-PAGE and Western blotting using a polyclonal antibody to snapin. A representative blot is shown for three different experiments.

Figure 8.

Figure 8.

Model for snapin action. Snapin protein is expressed in the cell bodies of developing neurons where it can interact with cypin and inhibit microtubule assembly. This results in decreased primary dendrite production. Snapin is expressed at lower levels in the dendrites than in the cell bodies, where cypin can promote microtubule assembly in primary dendrites. Hence, branching occurs and secondary dendrites are formed.

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