Article Figures & Data
Figures
- Figure 1.
An ALPS motif within the B domain of Syn I. A, Schematics of rat Syn Ia, Syn IIa, and Syn IIIa indicating the putative ALPS. Multiple sequence alignment of putative ALPS within Syn orthologues from different species. B, Helical wheel projection of Syn Ia-derived ALPS peptide. Purple, polar hydrophilic residues; yellow, hydrophobic. C–E, CD spectra of Syn I-derived ALPS peptides in the absence (black) or presence of 30–100 nm (red) or 400 nm (blue) liposomes. C, WT ALPS peptide. D, 2P-ALPS inactive mutant. E, Scrambled peptide.
- Figure 2.
Membrane curvature sensing and liposome binding by Syn I involves an ALPS. A, Silver-stained gel illustrating purified eGFP-Syn Ia. Right, Molecular weight markers in kDa. B–F, Single liposome curvature assays. A mixture of DID-labeled fluorescent liposomes of various sizes (Ø 50–700 nm) immobilized on a glass support were incubated with proteins or peptides: WT-eGFP-Syn I (B); the pleckstrin homology domain of phospholipase Cδ (curvature-insensitive) (eGFP-PHPLCδ; C); ΔALPS-eGFP-Syn I (D); 2P-ALPS-eGFP-Syn I (E); Atto-labeled Syn I-derived ALPS peptide (WTALPS; F). Plots display protein/peptide density (arbitrary units, a.u.) versus liposome diameter (Ø range: 50–700 nm). G, Liposome association of WTALPS and of 2PALPS or 3EALPS mutant peptides expressed as a percentage of the input peptide. H, Binding of WT or ALPS mutants of Syn I (50 nm) to SV-like liposomes (Ø 50–70 nm; 20 μg of phospholipid/sample). Bound Syn I (left) is expressed in percentage of total Syn I added (mean ± SEM). *p < 0.05; **p < 0.01 versus WT, Dunnett's multiple-comparison test (right).
- Figure 3.
Syn I-mediated vesicle clustering is modulated by ALPS-mediated membrane curvature sensing. A, Representative images of liposome (Lipos) clusters induced by WT or mutant eGFP-Syn Ia (Δ). B, Quantitative analysis of data in A. C, Same as A, but liposomes were preincubated with peptides (100 μm). WTALPS, ALPS peptide from human or rat Syn Ia; WTlALPS, ALPS peptide from lamprey Syn I; 2PALPS, proline-containing mutant version of WTALPS; AP180, control peptide from AP180; Kes1pALPS, ALPS peptide from yeast Kes1p. D, Quantifications of data in C. Scale bar, 10 μm. ***p < 0.001(one-way ANOVA); n.s., non-significant.
- Figure 4.
Membrane curvature sensing by ALPS modulates Syn I dispersion and SV reassociation. A, B, Increasing concentrations (40–200 nm) of WT-eGFP-Syn I (squares), 2P-ALPS-eGFP-Syn I (diamonds), or ΔALPS-eGFP-Syn I (triangles) incubated with synapsin-depleted SVs. Reisolated SVs were immunblotted for eGFP-Syn Ia or synaptophysin (Syp). A, Representative immunoblot and quantitation of densitometric scans fitted with a one-site binding function. B, Percentage of bound eGFP-Syn I (mean ± SEM; n = 4 independent experiments; **p < 0.01; ***p < 0.001 vs WT, Dunnett's multiple-comparison test). C, Representative images illustrating presynaptic localization of eGFP-Syn I (WT or ΔALPS, green). Boutons were labeled with bassoon (red). Scale bar, 2 μm. D, Reduced presynaptic expression of eGFP-Syn Ia (ΔALPS). Left, Expression level of WT and ΔALPS eGFP-Syn Ia quantified by total GFP fluorescence (mean ± SEM; n = 2 independent experiments). Right, Presynaptic expression measured by averaging basal fluorescence (F0) in 10 frames preceding stimulation (mean ± SEM; 9 coverslips/construct; 20 boutons/coverslip; n = 2 independent experiments; **p < 0.01, Student's t test). E, Time courses of changes in eGFP fluorescence before, during, and after stimulation (black horizontal bar) in neurons expressing eGFP-Syn Ia (black dots) or eGFP-Syn Ia (ΔALPS; red dots) (mean ± SEM) (9 coverslips/construct; 20 boutons/coverslip; n = 2 independent experiments). F, Rate of dispersion (τ; left) during stimulation fitted to single exponential. The dispersion plateau (ΔF/ F0; right) was significantly lower for ΔALPS compared with WT (*p < 0.05, Student's t test). G, Rate of reassociation (τ; left) from the end of the stimulation to the end of the recording fitted to single exponential. The amplitude of recovery (i.e., the ΔF/ F0 plateau; right) was significantly reduced for ΔALPS (*p < 0.05, Student's t test). Reassociation kinetics (left) were unaffected. H, Depolarization-induced dispersion and recovery of WT- and ΔALPS-Syn I in nerve terminals of SynI/II/III−/− neurons (mean ± SEM; n = 5–6 experiments). *p < 0.05; **p < 0.01; Student's t test; NS, non-significant.
