Synaptotagmin-1 and Doc2b Exhibit Distinct Membrane-Remodeling Mechanisms

Abstract

Synaptotagmin-1 (Syt1) is a calcium sensor protein that is critical for neurotransmission and is therefore extensively studied. Here, we use pairs of optically trapped beads coated with SNARE-free synthetic membranes to investigate Syt1-induced membrane remodeling. This activity is compared with that of Doc2b, which contains a conserved C₂AB domain and induces membrane tethering and hemifusion in this cell-free model. We find that the soluble C₂AB domain of Syt1 strongly affects the probability and strength of membrane-membrane interactions in a strictly Ca²⁺- and protein-dependent manner. Single-membrane loading of Syt1 yielded the highest probability and force of membrane interactions, whereas in contrast, Doc2b was more effective after loading both membranes. A lipid-mixing assay with confocal imaging reveals that both Syt1 and Doc2b are able to induce hemifusion; however, significantly higher Syt1 concentrations are required. Consistently, both C₂AB fragments cause a reduction in the membrane-bending modulus, as measured by a method based on atomic force microscopy. This lowering of the energy required for membrane deformation may contribute to Ca²⁺-induced fusion.

Figure 1

Membrane interactions captured with optical tweezers and confocal fluorescence microscopy. (a) A schematic (not to scale) of the dual-beam optical trapping setup used to manipulate two polystyrene microbeads (gray) coated with a phospholipid bilayer (blue) in the presence of proteins (green) and Ca²⁺ ions (yellow dots) is shown in panel 1. The liposomes are brought into contact for 5 s (panel 2) and moved away (panel 3) with a constant velocity by an automated approach and separation method. The force on the left fixed trap is measured. (b) A typical force-time plot showing eight consecutive interactions is shown. The force is zero when the liposomes are apart (1). A positive force occurs during membrane contact (2), and a negative force occurs during bead separation (3), indicative of tether formation. The rupture force (indicated by a gray arrow) is used to quantify the strength of each tether. (c) Shown are confocal fluorescence images of membrane tethers visualized in the presence of labeled proteins (Syt1-C₂AB-mCherry, top panel) or labeled phospholipids (1% Rhodamine-PE, bottom panel) on both beads. Scale bars, 1 μm. To see this figure in color, go online.

Figure 2

Membrane interactions induced by Syt1-C₂AB are dependent on Ca²⁺ and protein concentration. (a) Shown is the probability of membrane interactions with increasing Ca²⁺ concentration (in the absence of proteins or presence of 0.2 μM Syt1-C₂AB, gray and orange histogram, respectively). Numbers of approach and separation cycles (N) are indicated. At least 10 bead pairs were measured for each sample, with 10 cycles for each bead pair. Error bars indicate statistical error. (b) Median tether rupture forces at different Ca²⁺ concentrations are shown. Inset: shown are normalized cumulative distribution functions (CDF) of the rupture forces (color-coded from yellow to red as the Ca²⁺ increases) from which the median forces are calculated. (c) Shown is the probability of membrane interactions with increasing Syt1-C₂AB concentration (at fixed 0.5 mM CaCl₂). Numbers of interactions (N) are indicated. Error bars are statistical error. (d) Shown are median rupture forces with increasing Syt1-C₂AB concentration. Inset: normalized CDF of the rupture forces recorded (color-coded from light to dark green as the Syt1 concentration increases). In (b) and (d), the gray background gradient marks the maximum force that can be determined with the optical trap at the set laser power (5 W) and bead size (3.84 μm diameter). Error bars in rupture force plots indicate the standard deviation of the bootstrapped median rupture force values. The Kolmogorov-Smirnov test for all data both for (b) and (d) shows significantly different distribution at the p < 0.0005 level. To see this figure in color, go online.

Figure 3

Optimal tethering by Syt1 and Doc2b loaded on single and dual membranes, respectively. (a) Shown is a schematic of the two experimental configurations used. Asymmetric: protein was bound to a single membrane-coated bead (upper panel), as confirmed by confocal imaging in which Syt1-C₂AB-mCherry (0.5 μM Syt1, 0.25 mM CaCl₂) was bound to the left bead and decorated the tether structure but not the other bead. Note that at other times, the tether was drawn from the dark bead, and no bias of tether extension toward labeled lipids was observed. Symmetric: proteins were bound to both beads (bottom panel, see Fig 1c for confocal image). Scale bars, 1 μm. (b) Left: probability of Syt1-mediated membrane interactions in the asymmetric (dark gray) and symmetric (green) configurations. Error bars indicate statistical error. Right: median rupture force in the two configurations is shown. (c) Left: probability of Doc2b-mediated membrane interactions in the asymmetric (light gray) and symmetric (magenta) configurations is shown (0.05 μM Doc2b, 0.25 mM CaCl₂). Error bars are statistical error. Right: the median rupture force in the two configurations is shown. The color gradient in the symmetric configuration indicates a lower limit of this quantification because it reached the upper limit of our trapping force. Error bars indicate standard deviation of the bootstrapped median rupture force values. Note: the lower concentration of Doc2b compared with Syt1 is chosen to be within the range of break forces that can be experimentally quantified. To see this figure in color, go online.

Figure 4

Cholesterol significantly increases strength and probability for Doc2b-mediated tethers. Probability of interactions and median rupture forces for membranes containing only PC/PS (80:20) and in the presence of cholesterol (30%, +Chol). (a) Shown are Syt1-mediated interactions tested in the asymmetric configuration (0.5 μM, 0.25 mM CaCl₂). (b) Shown are Doc2b-mediated interactions tested in the symmetric configuration (0.05 μM, 0.25 mM CaCl₂). Numbers of approach and separation cycles (N) are indicated. Error bars in probability plots indicate statistical error; error bars in rupture force plots indicate standard deviations of the bootstrapped median rupture force values. Tether break forces with and without added cholesterol in the membrane are significantly different for both Doc2b- and Syt1, with p < 0.005. To see this figure in color, go online.

Figure 5

Membrane interactions in the presence of PI(4,5)P2. (a) Shown is a schematic illustration of the experimental configurations used: Syt1-C₂AB-mCherry (0.5 μM Syt1, 0.25 mM CaCl₂) was bound to a single membrane-coated PC/PS (80:20) bead (left); the opposite bead was coated with an additional 1% PIP₂, and it was left protein-free (right bead) (+PIP₂ configuration). We compare this configuration with the same asymmetric setting but in the absence of PIP₂ from both membrane-coated beads (−PIP₂ configuration). (b) Left plot: shown is the probability of Syt1-mediated membrane interactions in the absence of PIP₂ (white bar) and in the presence of PIP₂ (as schematically depicted in (a), orange bar). Error bars indicate statistical error. Right plot: median rupture force in the two configurations. Error bars indicate standard deviations of the bootstrapped median rupture force values. A Kolmogorov-Smirnov test showed no statistically significant difference at p < 0.05. To see this figure in color, go online.

Figure 6

Membrane bridging and hemifusion induced by Syt1 and Doc2b. (a) Shown is a schematic of possible protein-mediated membrane interactions (proteins bound on the membrane are represented in green). Left panel: shown are membranes bridging in which the bilayers stay separated and therefore labeled phospholipids (represented in red) remain on one membrane. Right panel: hemifusion is shown in which the proximal membrane leaflets have fused, causing lipid mixing. (b) Shown are confocal images in which only one membrane (left bead) was fluorescently labeled (1% Rhodamine-PE). Imaging was initiated as soon as a tether was detected. Upper panels: shown are images recorded at t = 0 s. Lower panels: shown are images recorded after 250 s. Shown are observed interactions mediated by Syt1 (left panel) and by Doc2b (right panel). (c) Shown are confocal images recorded after a 250-s interaction in the presence of 20 μM Syt1 in the symmetric (Symm) configuration (upper panel) and in the asymmetric (Asymm) configuration (bottom panel). (d) Shown is the fluorescence signal recorded from the dark bead over time in the presence of 0.7 μM Doc2b and 0.5 or 20 μM Syt1 in (b). (e) Shown are hemifusion probabilities under different conditions. To see this figure in color, go online.

Figure 7

High concentrations (20 μM) of Syt1-C₂AB and lower concentrations (0.9 μM) of Doc2b-C₂AB reduce the membrane-bending modulus. (a) Shown is an AFM image of a typical vesicle: 44% cholesterol, 20% porcine brain PS, 21% egg PC, and 15% egg SM. (b) A typical force plot obtained by nanoindentation of a vesicle is shown. From the slope of the initial linear part, the stiffness of the vesicle is obtained. Two rupture events of the two bilayers can be observed, and a tether is formed upon tip retraction. Dark blue: approach; light blue: retraction. Inset: zoom in on the tether rupture force, which is used to calculate the osmotic pressure in the vesicle as described in Vorselen et al. (49). The red arrow shows the difference in force. (c) Bending modulus values for various concentrations of Syt1-C₂AB and Doc2b-C₂AB, with N values indicating the numbers of indented vesicles. Error bars mark 68% confidence intervals determined by bootstrapping. To see this figure in color, go online.