Presynaptic calcium channels and α3-integrins are complexed with synaptic cleft laminins, cytoskeletal elements and active zone components - PubMed (original) (raw)
Presynaptic calcium channels and α3-integrins are complexed with synaptic cleft laminins, cytoskeletal elements and active zone components
Steven S Carlson et al. J Neurochem. 2010 Nov.
Abstract
At chemical synapses, synaptic cleft components interact with elements of the nerve terminal membrane to promote differentiation and regulate function. Laminins containing the β2 subunit are key cleft components, and they act in part by binding the pore-forming subunit of a pre-synaptic voltage-gated calcium channel (Ca(v)α) (Nishimune et al. 2004). In this study, we identify Ca(v)α-associated intracellular proteins that may couple channel-anchoring to assembly or stabilization of neurotransmitter release sites called active zones. Using Ca(v)α-antibodies, we isolated a protein complex from Torpedo electric organ synapses, which resemble neuromuscular junctions but are easier to isolate in bulk. We identified 10 components of the complex: six cytoskeletal proteins (α2/β2 spectrins, plectin 1, AHNAK/desmoyokin, dystrophin, and myosin 1), two active zone components (bassoon and piccolo), synaptic laminin, and a calcium channel β subunit. Immunocytochemistry confirmed these proteins in electric organ synapses, and PCR analysis revealed their expression by developing mammalian motor neurons. Finally, we show that synaptic laminins also interact with pre-synaptic integrins containing the α3 subunit. Together with our previous finding that a distinct synaptic laminin interacts with SV2 on nerve terminals (Son et al. 2000), our results identify three paths by which synaptic cleft laminins can send developmentally important signals to nerve terminals.
© 2010 The Authors. Journal of Neurochemistry © 2010 International Society for Neurochemistry.
Figures
Figure 1. The VGCC protein complex isolated by preparative immunoprecipitation and components separated by SDS-PAGE
The immunoprecipitate was prepared from TX-100 solubilized electric organ synaptosmes with an anti-Cavα antibody (CP15) and subjected to SDS-PAGE on a 2.7–6% polyacrylamide gradient gel stained for protein with Coomassie Blue. The visualized proteins are labeled A–N. Bands M and N correspond to the IgG heavy (M) and light (N) chains, respectively, of the immunoprecipitating antibody. The remaining proteins were identified by MS/MS and/or Western blotting.
Figure 2. Components of the Immunoprecipitated VGCC protein complex identified by Western blots
Electric organ synaptosomes (s lanes) solubilized in Triton X100 were incubated with immunobeads coated with an anti-Cavα antibody (+ lanes) (CP15) or a secondary antibody only (− lanes), and the resulting immunoprecipitates subjected to Western blot. The blots were probed with antibodies to Plectin 1, Dystrophin, α-spectrin, AHNAK/Desmoyokin, laminin β2 and γ1 chains, Cavα, and myosin 1. The laminin chains, which co-migrate on the gel, were detected with antibody to the HNK-1 carbohydrate. Specificity of this antibody for laminins is documented in Sunderland et al. 2000. Letter in parentheses correspond to bands in Figure 1.
Figure 3. Immunoprecipitation of the VGCC protein complex by anti-Cavβ antibodies
a) Electric organ synaptosomes were subjected to Western blot and probed with an anti-Cavβ antibody. b) Electric organ synaptosomes (s lanes) solubilized in Triton X100 were incubated with immunobeads coated with an anti-Cavβ antibody (+ lanes) or a secondary antibody only (− lanes), and the resulting immunoprecipitates subjected to Western blot. The blots were probed with antibodies to the Cavα subunit or HNK1, which identifies the laminin β2 and γ1 chains (see Sunderland et al., 2000). On this 2.7–14% acrylaminde gel, the laminin essentially chains migrate together.
Figure 4. The VGCC protein complex contains Bassoon and Piccolo
TX-100 solubilized electric organ synaptosomes (s lanes) were subjected to imunoprecipitation with anti-Cavα (a, + lanes), anti-Bassoon/Piccolo antibody (b, + lanes), or secondary antibody only (− lanes). The resulting immunoprecipitates were subjected to Western blot probed with an antibody that recognizes both Bassoon and Piccolo (Bass/Pic), an anti-Piccolo antibody, an anti-Cavα antibody, or an anti-HNK-1 antibody specific for laminins (Sunderland et al., 2000). The black arrowhead indicates the protein identified by the anti-Piccolo antibody. The Cavα migrates as two bands at ~190 kD (arrow) as seen previously and ~180 kD (*). The 180 kD protein is probably due to the α subunit being nicked by a protease during the preparation of the synaptosomes. The synaptosomes used for the immunoprecipitations in a) are the same as those shown in lane “s” of b).
Figure 5. Laminin α4β2γ1 is associated with α3-Integrin in electric organ nerve terminals
a) In electric organ synaptosomes an antibody prepared to the 14 residue carboxy-terminal region of the α3-integrin chain identifies a ~150 kD protein under non-reducing conditions and a ~30 kD protein after reduction. A peptide containing the 14 residue antigenic sequence blocks the reactivity of the anti- α3-integrin antibody on both the ~150 kD protein and ~30 kD fragment. b) Starting with the standard preparation, synaptosomes (s lanes) were immuno-purified away from membranes of the postsynaptic cell as previously demonstrated (Son et al., 2000; Sunderland et al., 2000). This immunoprecipitation was done with a mouse monclonal antibody to a luminal domain (SV1) of the synaptic vesicle protein SV2 present on the nerve terminal surface. The control immunoprecipitation contained a monoclonal antibody of the same isotype (IgG1) with an unknown specificity. The immunopurified synaptosomes contain α3-integrin along with two laminins at 900 kD (grey arrowhead) and 740 kD (arrow) shown to be an α5-containing laminin and α4β2γ1 laminin, respectively (Son et al., 2000; Sunderland et al., 2000). c) Electric organ synaptosomes (the standard preparation) were applied directly to a Western blot (s lanes), or solubilized in TX100 and imunoprecipitated with immunobeads containing an anti-α3-integrin antibody (+ lanes) or a secondary antibody only (− lanes). Lanes were stained with anti-α3-integrin (α3I), anti-laminin 1 (Ln1), anti-HNK1, anti-α spectrin, anti-Cavα, anti-syntaxin, and anti-SV2 antibodies. The Western blots for detecting laminin were prepared from non-reducing SDS-PAGE so that laminin would migrate as a trimer. Torpedo α4β2γ1 migrates as 740 kD protein which stains with anti-laminin 1 and HNK-1 antibodies (Sunderland et al., 2000).
Figure 6. Immunocytochemical localization of VGCC- associated proteins at the electric organ synapse
Longitudinal (ɑ3I, AH, Myo) or cross-sections (Pl) of electric organ were stained for α3-Integrin (α3I), AHNAK/Desmoyokin (AH), myosin 1 (Myo), or plectin (Pl). Sections were counterstained with anti-SV2 to label nerve terminals (exSV2 or SV2) and bungartoxin to label AchRs (AR). Colored panels show merged images. Arrows or arrowheads indicate nerve terminals. The SV2 staining in panel exSV2 is on the surface of the nerve terminal; unfixed electric organ was incubated in the anti-SV2 mAb and then washed before being fixed, sectioned, and stained for integrin. In all other panels sections were stained with anti-SV2 mAb after sectioning. Scale bars are 2 μm.
Figure 7. Presynaptic localization of VGCC-associated proteins seen with collagenase-digested electric organ
Electric organ was digested with collagenase to allow pre- and postsynaptic membranes to separate, then fixed and cryo-sectioned. Sections were stained with antibodies to α3-integrin (α3I), plectin1 (Pl), AHNAK/Desmoyokin (AH), dystrophin (Dys), or myosin 1 (Myo). Sections were counterstained with antibodies to synaptic vesicles (anti-SV2 mAb – SV or anti-synaptotagmin Ab - Sy) and bungarotoxin to label AChRs (AR). Panels on right show merged images. Arrowheads indicate examples of co-localization between a VGCC associated protein and a presynaptic marker in dissociated nerve terminals. Scale bars are 3 μm.
Figure 8. E13 mouse motor neurons express VGCC binding partners
Motor neurons were purified from E13 spinal cords and immediately processed for RNA extraction. Gene-specific primers were used to detect VGCC binding partner’s transcripts after cDNA synthesis (A) or after RNase-treatment to control for genomic carry-over (B).
Figure 9. Known and potential associations between the components of the VGCC protein complex
The figure shows a Cavα protein complex containing all the cytoskeletal and active zone proteins described here. We have shown that the Cavα, synaptic laminin, Piccolo and bassoon are all in one complex. However, the other cytoskeletal proteins might be associated with Cavα in one complex or several. For example, Cavα might be bound to spectrin in one complex and AHNAK/Desmoyokin in another. See text for additional details.
References
- Carlson SS, Wagner JA, Kelly RB. Purification of synaptic vesicles from elasmobranch electric organ and the use of biophysical criteria to demonstrate purity. Biochemistry. 1978;17:1188–1199. -PubMed
- Catterall WA, Few AP. Calcium channel regulation and presynaptic plasticity. Neuron. 2008;59:882–901. -PubMed
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