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Stepwise membrane binding of extended synaptotagmins revealed by optical tweezers

Nature Chemical Biology volume 18, pages 313–320 (2022)Cite this article

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Abstract

Extended synaptotagmins (E-Syts) mediate lipid exchange between the endoplasmic reticulum (ER) and the plasma membrane (PM). Anchored on the ER, E-Syts bind the PM via an array of C2 domains in a Ca2+- and lipid-dependent manner, drawing the two membranes close to facilitate lipid exchange. How these C2 domains bind the PM and regulate the ER–PM distance is not well understood. Here, we applied optical tweezers to dissect PM binding by E-Syt1 and E-Syt2. We detected Ca2+- and lipid-dependent membrane-binding kinetics of both E-Syts and determined the binding energies and rates of individual C2 domains or pairs. We incorporated these parameters in a theoretical model to recapitulate salient features of E-Syt-mediated membrane contacts observed in vivo, including their equilibrium distances and probabilities. Our methods can be applied to study other proteins containing multiple membrane-binding domains linked by disordered polypeptides.

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Fig. 1: E-Syt C2 domains bind membranes in a stepwise manner as revealed by optical tweezers.
Fig. 2: E-Syt C2 domains reversibly and sequentially bind to membranes.
Fig. 3: Force-dependent probabilities and transition rates of different C2 binding states.
Fig. 4: Membrane binding of E-Syt1 C2CD and E-Syt2 C2AB is Ca2+ dependent, while binding of E-Syt1 C2E and E-Syt2 C2C is Ca2+ independent.
Fig. 5: Membrane binding of E-Syt1 C2CD and C2E differentially depends on PI(4,5)P2 and DOPS.
Fig. 6: Properties of E-Syt-mediated ER–PM contacts can be theoretically modeled.

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Data availability

Data supporting the findings of this work were deposited at https://github.com/zhanglabyale/E-Syt_membrane_tethering_simulation.

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Acknowledgements

We thank J. Jiao and A. Rebane for technical assistance. This work was supported by NIH grants R35GM131714, R01GM093341 and R01GM120193 to Y.Z.; R01NS113236 to E.K.; and NS36251 and DA018343 to P.D.C.

Author information

Author notes

  1. Xin Bian

    Present address: State Key Laboratory of Medicinal Chemical Biology, College of Life Sciences, Nankai University, Tianjin, China

  2. Lu Ma

    Present address: Beijing National Laboratory for Condensed Matter Physics and CAS Key Laboratory of Soft Matter Physics, Institute of Physics, Chinese Academy of Sciences, Beijing, China

  3. Yanghui Li

    Present address: College of Optical and Electronic Technology, China Jiliang University, Hangzhou, China

  4. These authors contributed equally: Jinghua Ge, Xin Bian, Lu Ma, Yiying Cai, Yanghui Li.

Authors and Affiliations

  1. Department of Cell Biology, Yale University School of Medicine, New Haven, CT, USA

    Jinghua Ge, Xin Bian, Lu Ma, Yiying Cai, Yanghui Li, Jie Yang, Pietro De Camilli & Yongli Zhang

  2. Department of Neuroscience, Yale University School of Medicine, New Haven, CT, USA

    Xin Bian, Yiying Cai & Pietro De Camilli

  3. Howard Hughes Medical Institute, Yale University School of Medicine, New Haven, CT, USA

    Xin Bian, Yiying Cai & Pietro De Camilli

  4. Program in Cellular Neuroscience, Neurodegeneration and Repair, Yale University School of Medicine, New Haven, CT, USA

    Xin Bian, Yiying Cai & Pietro De Camilli

  5. Department of Cellular and Molecular Physiology, Yale University School of Medicine, New Haven, CT, USA

    Lu Ma & Erdem Karatekin

  6. Nanobiology Institute, Yale University, West Haven, CT, USA

    Lu Ma & Erdem Karatekin

  7. Department of Molecular Biophysics and Biochemistry, Yale University, New Haven, CT, USA

    Erdem Karatekin & Yongli Zhang

  8. Université de Paris, Saints-Pères Paris Institute for the Neurosciences (SPPIN), Centre National de la Recherche Scientifique (CNRS), Paris, France

    Erdem Karatekin

  9. Kavli Institute for Neuroscience, Yale University School of Medicine, New Haven, CT, USA

    Pietro De Camilli

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  1. Jinghua Ge
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Contributions

All authors designed experiments and analyzed data; J.G., L.M. and Y.L. performed single-molecule experiments; J.G., X.B., L.M. and Y.C. prepared protein and DNA samples; Y.Z. performed the calculations shown in Fig. 6. J.G., X.B., E.K., P.D.C. and Y.Z. wrote the paper.

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Correspondence to Yongli Zhang.

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Peer review information Nature Chemical Biology thanks Raul Perez-Jimenez and other anonymous reviewer(s) for their contribution to the peer review of this work.

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Extended data

Extended Data Fig. 1 Representative proteins that contain multiple C2 domains.

The lengths (a.a.) of the predicted disordered linkers that join different protein domains or membranes are indicated.

Extended Data Fig. 2 Two conserved membrane binding motifs in the C2 domains of synaptotagmins (Syts) and extended synaptotagmins (E-Syts).

Alignments of the C2 amino acid sequences showing two conserved membrane binding motifs highlighted in bold and color: the Ca2+-binding motif (blue) and the basic patch (red).

Extended Data Fig. 3 The purified E-Syt1 C2ABCDE was sufficiently pure and biotinylated.

(a) SDS-PAGE of the purified E-Syt1 C2ABCDE stained with Coomassie blue. Streptavidin maintained its tetrameric structure in the SDS gel when it was loaded to the gel without heating to high temperature. Binding of streptavidin to the biotinylated E-Syt1 C2ABCDE shifted the migration of C2ABCDE to a position with a higher molecular weight. (b) Gel filtration chromatogram of E-Syt1 C2ABCDE in a Superdex-200 column. Similar results were obtained from three batches of purified proteins.

Extended Data Fig. 4 E-Syt1 C2AB only weakly binds to membranes enriched with negatively charged lipids.

(a, b) Diagrams showing no binding (a) and weak binding (b) of the E-Syt1 C2AB domain in the presence of 10% and 20% DOPS, respectively. (c) Force-extension curves showing no membrane binding of E-Syt1 C2AB domain in the absence of supported bilayer or in the presence of the supported bilayer containing 10% DOPS. Weak binding was detected in the presence of 20% DOPS, as indicated by the rip at low force (red arrow). (d) Extension-time trajectories at constant forces (black curves) and their idealized transitions derived from hidden-Markov modeling (red curves). (e) Unbinding probability and binding and unbinding rates (symbols) and their best model fits (lines). The fitting revealed an unbinding energy of 4.4 (±0.3) kBT for the E-Syt1 C2AB domain (Supplementary Table 2).

Extended Data Fig. 5 The microfluidic system to facilitate changes of the Ca2+ concentration in the single-molecule manipulation experiment.

(a) Schematics of the microfluidic system to change Ca2+ concentration when a single C2 repeat was being pulled. Two buffers containing 200 mM NaCl, 25 mM HEPES, pH 7.4, and 500 µM EGTA were prepared, one with CaCl2 (Ca2+ buffer) and another without CaCl2 (EGTA buffer). The two buffers were flowed through a mixer into the central flow cell. The two flows were independently controlled using computer-controlled pressure regulators (MS4-LR, Festo, NY) in combination with flow sensors (SLI-0430, Sensirion, Switzerland) that measure the flow rates. The constant flow rate was achieved by adjusting the pressure in the buffer vial through PID feedback control using a LabVIEW interface. The total calcium concentration in the flow cell ([Ca]), which consisted of both free and EGTA-chelated calcium, was determined by the total calcium concentration of the Ca2+ buffer ([Ca]=V1) and volume velocities of the two buffers (V1 and V2) before mixing. The free Ca2+ concentration ([Ca2+]) in the flow cell was calculated using Maxchelator (Web version v1.2) based on the total concentrations of calcium and EGTA. (b) The measured tracing dye concentration and predicted free Ca2+ concentration in the flow cell as the flow rate of the Ca2+ channel linearly increased from 0 to 12 µL/min while keeping the total flow rate of the two channels at 12 µL/min. To test the concentration change scheme, we added 100 nM rhodamine dye to the Ca2+ buffer and detected the concentration of the dye in the flow cell based on its fluorescence intensity measured by widefield fluorescence microscopy. We linearly increased the flow rate of the rhodamine-containing Ca2+ buffer from 0 to 12 µL/min and simultaneously decreased the flow rate of the EGTA buffer to keep the total flow rate of the two buffers to be 12 µL/min. The dye concentration linearly increased as expected, which justified our concentration change scheme. However, although the observation implied that the total calcium concentration in the flow cell varied linearly as predicted, the corresponding free Ca2+ concentration ([Ca2+]) responded in a nonlinear manner due to the buffering effect of EGTA. Combining with the flow control system, we detected C2 membrane binding transitions at constant force while changing Ca2+ concentration either continuously in the presence of a flow or stepwise in the absence of flow. While the former method allowed rapid [Ca2+] change at the expense of slight extra noise in force and extension measurements, the latter method permitted more accurate single-molecule measurement in the absence of flow after each [Ca2+] change (Fig. 4).

Extended Data Fig. 6 E-Syt1 C2ABCDE binds to the membrane containing 30% DOPS and 0% PI(4,5)P2 via its C2CD domain, but not its C2E domain.

C2CD unbinding probability (top panel) and binding and unbinding rates (bottom panel) as a function of force. The experimental data (symbols) were fit by a nonlinear model to yield the best-fits (lines).

Extended Data Fig. 7 E-Syt2 C2C undergoes a reversible force-dependent, but Ca2+-independent conformational change to inactivate its membrane binding.

(a-c) Extension-time trajectories at constant force in the absence (a) and presence (b) of Ca2+ for E-Syt2 C2ABC or in the presence of Ca2+ for E-Syt2 C2C (c). The long gaps in the unbound state highlighted blue represent the binding inactive state. (d) Diagram of the conformational transition of the C2 domain in the binding active and inactive states.

Extended Data Fig. 8 Cytosolic E-Syt1 containing the SMP domain binds to membranes in a manner like its C2 repeat C2ABCDE.

(a) Schematic diagram showing different E-Syt1 binding states. (b) Force-extension curves obtained by pulling E-Syt1 under different conditions. (c) Extension-time trajectories of E-Syt1 at constant force in the presence or absence of Ca2+.

Extended Data Fig. 9 Calculations of equilibrium membrane distance and membrane binding energy and kinetics.

(a) Diagram illustrating the equilibrium membrane distance determined by the balanced membrane repulsive force (equation 6 in the main text) and E-Syt tethering force (equation 1). The equilibrium distance was solved as a solution for the system of equations \(\left\{ {\begin{array}{*{20}{l}} {\frac{{k_BT}}{P}\left[ {\frac{1}{{4\left( {1 - \frac{x}{L}} \right)^2}} + \frac{x}{L} - \frac{1}{4}} \right] = - V\prime (d),} \hfill \\ {d = x + h,} \hfill \end{array}} \right.\) (7). where V′ is the derivative of V(d) and h is the total length of the folded C2 modules in the pulling direction1 (estimated as 2 nm for each C2 module2,3). (b) Energy landscape corresponding to the C2-membrane interaction with the C2 module free in the solution (blue curve), tethered to the membrane by a flexible and relaxed polypeptide linker (black), or tethered to the membrane with a stretched linker (red). The three key parameters of the energy landscape associated with the free C2 module are determined from our single-molecule measurements (Supplementary Table 1). The energy landscape with the tethered and relaxed C2 module (corresponding to the force-unloaded C2 module) is determined by equation 3, with an effective concentration of the C2 module around the membrane estimated by equation 4. Note that the tethering does not affect the unbinding rate of the C2 module4. Stretching the bound C2 module by moving the PM membrane away from the ER membrane increases the energy barrier for C2 binding by Estretch and decreases the energy barrier for C2 unbinding by FΔx as indicated by equation 5.

Extended Data Fig. 10 Calculated state probabilities, forces, and energy as a function of membrane separation due to potential trans-membrane binding of E-Syt1 (left panel) and E-Syt2 (right panel) lacking a membrane-binding C-terminal C2 module.

(a, c) Schematics of different C2 binding and membrane tethering states in the absence and presence of Ca2+ for E-Syt1 (a) or E-Syt2 (c). The calculations were to simulate the results of membrane contact formation from in vivo imaging using E-Syts with mutant C-terminal C2 domains (C2E in E-Syt1 or C2C in E-Syt2 and E-Syt3) that did not bind to membranes or with the domains truncated. (b, d) Calculated probabilities (top panel), average stretching force (middle), and free energy (bottom) of different states for truncated E-Syt1 (b) or E-Syt2 (d). Calculations corresponding to the presence of Ca2+ or the absence of Ca2+ are indicated by solid and dashed lines, respectively, with their colors indicating different states as shown in a or b: red for the bound state I and black for the unbound state iii. Stable and unstable states are indicated by solid and hollow circles, respectively. The derived equilibrium distances and free energy are shown in Supplementary Table 3.

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Ge, J., Bian, X., Ma, L. et al. Stepwise membrane binding of extended synaptotagmins revealed by optical tweezers. Nat Chem Biol 18, 313–320 (2022). https://doi.org/10.1038/s41589-021-00914-3

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