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Cited by 51Cellular transport and secretionNeuroscience and Neuropharmacology ResearchExtracellular vesicles in disease

Membrane compression by synaptic vesicle exocytosis triggers ultrafast endocytosis

Open access
Ogunmowo, Tyler H., Jing, Haoyuan, Raychaudhuri, Sumana, Kusick, Grant F., Imoto, Yuuta, Li, Shuo, Itoh, Kie, Ma, Ye +6 more·2023·Nature Communications
DOI 10.1038/s41467-023-38595-2PMC PMC10199930
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Abstract

Compensatory endocytosis keeps the membrane surface area of secretory cells constant following exocytosis. At chemical synapses, clathrin-independent ultrafast endocytosis maintains such homeostasis. This endocytic pathway is temporally and spatially coupled to exocytosis; it initiates within 50 ms at the region immediately next to the active zone where vesicles fuse. However, the coupling mechanism is unknown. Here, we demonstrate that filamentous actin is organized as a ring, surrounding the active zone at mouse hippocampal synapses. Assuming the membrane area conservation is due to this actin ring, our theoretical model suggests that flattening of fused vesicles exerts lateral compression in the plasma membrane, resulting in rapid formation of endocytic pits at the border between the active zone and the surrounding actin-enriched region. Consistent with model predictions, our data show that ultrafast endocytosis requires sufficient compression by exocytosis of multiple vesicles and does not initiate when actin organization is disrupted, either pharmacologically or by ablation of the actin-binding protein Epsin1. Our work suggests that membrane mechanics underlie the rapid coupling of exocytosis to endocytosis at synapses.

AI snapshot

Written from the paper’s full text.

TL;DR

Ultrafast endocytosis at mouse hippocampal synapses is triggered by lateral membrane compression: fused synaptic vesicles flatten against an F-actin ring surrounding the active zone, causing buckling and endocytic pit formation at the active-zone/periactive-zone border. The paper combines STED/gSTED imaging, flash-and-freeze electron microscopy, and a Helfrich-like membrane model to argue that stable F-actin, not dynamic actin polymerization, is required for this coupling.

Key findings

  • F-actin forms a ring around the active zone: 64% of synapses had a complete ring, 27% a partial ring, and 9% a patch; F-actin signal peaked 100–200 nm from the active zone edge, adjacent to Dyn1xA/endocytic-zone signals.
  • The lateral membrane compression model predicts endocytic pits form at the active-zone/periactive-zone interface by ~30 ms, with a curvature threshold >0.03 nm⁻¹; two fused vesicles are the minimal requirement, whereas one vesicle gave curvature = 0.009 nm⁻¹ at 7 ms and did not form pits.
  • Ultrafast endocytosis required intact F-actin but not dynamic polymerization: Latrunculin A blocked endocytic pits at 100 ms and ferritin-positive endocytic structures at 1 s, while Jasplakinolide did not block ultrafast endocytosis.
  • Epsin1 localized with F-actin (Pearson’s correlation coefficient = 0.64), and Epsin1 knockdown reduced presynaptic F-actin by 41% and blocked endocytic pits at 100 ms and ferritin-positive structures at 1 s; Doc2α knockout and EGTA-AM also blocked ultrafast endocytosis after a single action potential.

Methods

  • Mouse hippocampal cultures were used for 2D STED, 3D gSTED, Airyscan fluorescence imaging, Western blot, shRNA knockdown, pharmacology, and high-pressure freezing/flash-and-freeze or zap-and-freeze electron microscopy.
  • Exocytosis was triggered by light pulses in ChetaTC-expressing neurons or by 1 ms electrical pulses; samples were frozen at 100 ms or 1 s, with ferritin as a fluid-phase endocytic marker.
  • A Helfrich-like mechanical energy model simulated membrane shape changes with Surface Evolver, treating membrane tension as a Lagrange multiplier for area conservation and using bending moduli for vesicle, active-zone, and periactive-zone membrane domains.

Limitations

  • Stated: measuring the membrane stiffness of synaptic vesicles, active zones, and periactive zones is intractable with current technologies; the model does not describe fusion or endocytic pinching off; and it is unclear whether Epsin1 is the sole organizer of presynaptic F-actin.
  • Stated: UtrCH may not visualize all F-actin pools uniformly, and Phalloidin staining cannot readily distinguish presynaptic from postsynaptic actin pools.
  • Evident: the model predicted fewer endocytic events at low calcium than observed experimentally—ultrafast endocytosis occurred at similar frequency in 1.2 mM and 4 mM extracellular calcium—and the authors attribute this disparity partly to asynchronous release, while noting that not all exocytic events may trigger successful ultrafast endocytosis.

Model-written summary. Check it against the paper before relying on it.

Integrity signals

Registry

Crossref · 10.1038/s41467-023-38595-2

Cites

102 of 102 references verified0 citation errors · 0 not checkable

Statistics

Nothing to recompute.

Claims the paper makes

72 of 96 cited sources checked · 35 supported · 3 flagged · accuracy not yet measured

  • Interpretationreasoning

    A reduction in membrane tension after exocytosis cannot account for the kinetics and spatial precision of ultrafast endocytosis.

    “Therefore, even if membrane tension alters upon exocytosis, this model cannot account for the kinetics and spatial precision of ultrafast endocytosis.”
    Quote in the paperSubstantial claim2 of 2 sources checked
    • cites [10] Shi, Z. & Baumgart, T. Membrane tension and peripheral protein density mediate membrane shape transitions. Nat. Commun. 6, 5974 (2015). source

      The Discussion (page unavailable) says the opposite about kinetics: lowering membrane tension after exocytic vesicle fusion could trigger endocytosis rapidly enough to be a plausible signal for ultrafast endocytosis. The Abstract also describes tension reduction as a way to activate endocytosis rapidly at remote sites. The paper does not establish the claimed failure of spatial precision. (LLM, conf=0.90)

      Checked by a reasoning model — may be wrong.

      Experimental — accuracy not yet measured
    • cites [11] Gomis Perez, C. et al. Rapid propagation of membrane tension at retinal bipolar neuron presynaptic terminals. Sci. Adv. 8, eabl4411 (2022). source

      The Introduction says slowly propagating tension could not trigger rapid endocytosis, but presents that as a problem the study investigates, not its conclusion. The Discussion instead says fast equilibration of membrane tension in presynaptic terminals likely contributes to coupling exocytosis and endocytosis. The paper does not establish that this mechanism cannot account for ultrafast endocytosis’s kinetics or spatial precision. (LLM, conf=0.86)

      Checked by a reasoning model — may be wrong.

      Experimental — accuracy not yet measured
  • Premisecitation

    In ultrafast endocytosis, endocytic invaginations always form at the edge of the active zone.

    “In this pathway, endocytic invaginations always form at the edge of the active zone5–7.”
    Quote in the paperNot a substantial claim3 of 3 sources checked
    • cites [5] Watanabe, S. et al. Ultrafast endocytosis at mouse hippocampal synapses. Nature 504, 242–247 (2013). source

      The Discussion says ultrafast endocytosis takes place at the lateral edge of the active zone. The Results report that invaginations were outside the active zone and were often observed flanking it, but do not establish that they always form at the edge. (LLM, conf=0.86)

      Experimental — accuracy not yet measured
    • cites [6] Watanabe, S. et al. Ultrafast endocytosis at Caenorhabditis elegans neuromuscular junctions. Elife 2, e00723 (2013). source

      The Results describe invaginations next to the dense projection 20 ms after stimulation and others at flanking adherens junctions later. The Discussion (‘Two endocytic sites?’) explicitly identifies both as endocytic sites, contrary to ‘always’ at the edge of the active zone. No page numbers are available in our copy. (LLM, conf=0.87)

      Checked by a reasoning model — may be wrong.

      Experimental — accuracy not yet measured
    • cites [7] Watanabe, S. et al. Clathrin regenerates synaptic vesicles from endosomes. Nature 515, 228–233 (2014). source

      The Results section (‘Ultrafast endocytosis is intact after clathrin knock-down’; page unavailable) reports endocytic pits at the edges of the active zone in both control and clathrin-knock-down cells. Extended Data Fig. 1 also describes membrane retrieval at the lateral edge. Neither passage establishes the claim’s absolute ‘always’ across conditions. (LLM, conf=0.84)

      Experimental — accuracy not yet measured
  • Premisecitation

    Ultrafast endocytosis is tightly coupled to exocytosis in time and space.

    “Several recent studies suggest that synaptic vesicle endocytosis is mediated by clathrin-independent ultrafast endocytosis at mammalian central synapses and is tightly coupled to exocytosis in time and space5–9.”
    Quote in the paperSubstantial claim4 of 5 sources checked
    • cites [5] Watanabe, S. et al. Ultrafast endocytosis at mouse hippocampal synapses. Nature 504, 242–247 (2013). source
    • cites [6] Watanabe, S. et al. Ultrafast endocytosis at Caenorhabditis elegans neuromuscular junctions. Elife 2, e00723 (2013). source
    • cites [7] Watanabe, S. et al. Clathrin regenerates synaptic vesicles from endosomes. Nature 515, 228–233 (2014). source
    • cites [8] Soykan, T. et al. Synaptic vesicle endocytosis occurs on multiple timescales and is mediated by Formin-dependent actin assembly. Neuron 93, 854–866.e4 (2017). source
    • cites [9] Delvendahl, I., Vyleta, N. P., von Gersdorff, H. & Hallermann, S. Fast, temperature-sensitive and clathrin-independent endocytosis at central synapses. Neuron 90, 492–498 (2016). source

      The Abstract reports that single action potentials trigger clathrin-independent endocytosis with a 470-ms time constant at mature central synapses, supporting rapid temporal coupling to exocytosis. The cited passages do not establish that endocytosis occurs close to exocytosis sites; the Discussion’s reference to a spatiotemporal calcium signal concerns regulation of endocytosis speed, not spatial coupling of the two processes. (LLM, conf=0.86)

      Experimental — accuracy not yet measured
  • Premisecitation

    After exocytosis, vesicle membrane must be retrieved from the plasma membrane through endocytosis to keep the area of the plasma membrane constant.

    “After exocytosis, vesicle membrane must be retrieved from the plasma membrane through endocytosis to keep the area of the plasma membrane constant and maintain a sufficient number of synaptic vesicles at presynapses1–4.”
    Quote in the paperSubstantial claim2 of 4 sources checked
    • cites [1] Chanaday, N. L., Cousin, M. A., Milosevic, I., Watanabe, S. & Morgan, J. R. The Synaptic Vesicle Cycle Revisited: New Insights into the Modes and Mechanisms. J. Neurosci. 39, 8209–8216 (2019). source
    • cites [2] Saheki, Y. & De Camilli, P. Synaptic vesicle endocytosis. Cold Spring Harb. Perspect. Biol. 4, a005645 (2012). source
    • cites [3] Dittman, J. & Ryan, T. A. Molecular circuitry of endocytosis at nerve terminals. Annu. Rev. Cell Dev. Biol. 25, 133–160 (2009). source
    • cites [4] Kononenko, N. L. & Haucke, V. Molecular mechanisms of presynaptic membrane retrieval and synaptic vesicle reformation. Neuron 85, 484–496 (2015). source
  • Premisecitation

    Synaptic vesicle endocytosis at mammalian central synapses is mediated by clathrin-independent ultrafast endocytosis.

    “Several recent studies suggest that synaptic vesicle endocytosis is mediated by clathrin-independent ultrafast endocytosis at mammalian central synapses and is tightly coupled to exocytosis in time and space5–9.”
    Quote in the paperSubstantial claim4 of 5 sources checked
    • cites [5] Watanabe, S. et al. Ultrafast endocytosis at mouse hippocampal synapses. Nature 504, 242–247 (2013). source

      The Abstract (page not provided) reports ultrafast endocytosis at cultured mouse hippocampal synapses, supporting the proposed pathway in a mammalian central synapse. But the Discussion explicitly says it is premature to rule out clathrin’s involvement, so the paper does not establish that the pathway is clathrin-independent. (LLM, conf=0.88)

      Experimental — accuracy not yet measured
    • cites [6] Watanabe, S. et al. Ultrafast endocytosis at Caenorhabditis elegans neuromuscular junctions. Elife 2, e00723 (2013). source

      The Discussion reports ultrafast endocytosis after stimulation at C. elegans neuromuscular junctions, not at mammalian central synapses. In “Clathrin-mediated or kiss-and run?” (page unavailable), it says the observed pits lack clathrin coats but explicitly does not exclude a role for clathrin. The source therefore supports the ultrafast aspect, but not the claimed setting or an established clathrin-independent mechanism there. (LLM, conf=0.90)

      Experimental — accuracy not yet measured
    • cites [7] Watanabe, S. et al. Clathrin regenerates synaptic vesicles from endosomes. Nature 515, 228–233 (2014). source
    • cites [8] Soykan, T. et al. Synaptic vesicle endocytosis occurs on multiple timescales and is mediated by Formin-dependent actin assembly. Neuron 93, 854–866.e4 (2017). source
    • cites [9] Delvendahl, I., Vyleta, N. P., von Gersdorff, H. & Hallermann, S. Fast, temperature-sensitive and clathrin-independent endocytosis at central synapses. Neuron 90, 492–498 (2016). source

      The Results section reports rapid, clathrin-independent endocytosis after single action potentials at mature central synapses. But the Abstract and Results also report a slower, clathrin-dependent mode after stronger stimuli, so the unqualified claim that synaptic vesicle endocytosis is mediated by the clathrin-independent mode overstates the finding. Our copy provides section labels but no page numbers for these passages. (LLM, conf=0.88)

      Experimental — accuracy not yet measured
  • Classified as probably not a substantial claim. 37
  • May combine two claims. 33
  • The quote only partly states this claim. 1

  • Classified as probably not a substantial claim.

    Premisecitation

    Endocytosis after exocytosis maintains a sufficient number of synaptic vesicles at presynapses.

    “After exocytosis, vesicle membrane must be retrieved from the plasma membrane through endocytosis to keep the area of the plasma membrane constant and maintain a sufficient number of synaptic vesicles at presynapses1–4.”
    Quote in the paperSubstantial claim2 of 4 sources checked
    • cites [1] Chanaday, N. L., Cousin, M. A., Milosevic, I., Watanabe, S. & Morgan, J. R. The Synaptic Vesicle Cycle Revisited: New Insights into the Modes and Mechanisms. J. Neurosci. 39, 8209–8216 (2019). source
    • cites [2] Saheki, Y. & De Camilli, P. Synaptic vesicle endocytosis. Cold Spring Harb. Perspect. Biol. 4, a005645 (2012). source
    • cites [3] Dittman, J. & Ryan, T. A. Molecular circuitry of endocytosis at nerve terminals. Annu. Rev. Cell Dev. Biol. 25, 133–160 (2009). source
    • cites [4] Kononenko, N. L. & Haucke, V. Molecular mechanisms of presynaptic membrane retrieval and synaptic vesicle reformation. Neuron 85, 484–496 (2015). source

  • May combine two claims.

    Premisecitation

    Ultrafast endocytosis initiates as fast as 20-50 ms after exocytosis and completes within 100 ms.

    “Indeed, ultrafast endocytosis initiates as fast as 20–50 ms after exocytosis and completes within 100 ms5–7.”
    Quote in the paperSubstantial claim3 of 3 sources checked
    • cites [5] Watanabe, S. et al. Ultrafast endocytosis at mouse hippocampal synapses. Nature 504, 242–247 (2013). source

      The Results section “Endocytosis peaks at 100 ms” (page unavailable) reports shallow pits, deep pits, and fully internalized vesicles within 50 ms after stimulation, supporting very rapid initiation and completion of some events. It does not establish initiation specifically 20 ms after exocytosis or completion of the process within 100 ms: deep pits remained at 100 ms, and the paper says a majority of added membrane had been retrieved by then. (LLM, conf=0.83)

      Experimental — accuracy not yet measured
    • cites [6] Watanabe, S. et al. Ultrafast endocytosis at Caenorhabditis elegans neuromuscular junctions. Elife 2, e00723 (2013). source
    • cites [7] Watanabe, S. et al. Clathrin regenerates synaptic vesicles from endosomes. Nature 515, 228–233 (2014). source

      The Introduction reports endocytic pits appearing about 50 ms after stimulation, and the Discussion says ultrafast endocytosis removes excess membrane 30–100 ms after stimulation. These passages support the rapid onset and completion by about 100 ms, but not initiation as early as 20 ms. Page numbers are unavailable in our copy. (LLM, conf=0.86)

      Experimental — accuracy not yet measured

  • May combine two claims.

    Premisecitation

    Synaptic vesicles can fuse synchronously within a few milliseconds of an action potential and asynchronously thereafter over tens and hundreds of milliseconds.

    “Synaptic vesicles can fuse synchronously within a few milliseconds of an action potential and asynchronously thereafter over tens and hundreds of miliseconds65.”
    Quote in the paperSubstantial claim1 of 1 sources checked
    • cites [65] Kaeser, P. S. & Regehr, W. G. Molecular mechanisms for synchronous, asynchronous, and spontaneous neurotransmitter release. Annu. Rev. Physiol. 76, 333–363 (2014). source

  • May combine two claims.

    Premisecitation

    Membrane tension in neurons propagates at ~20 μm/s and is expected to equilibrate at the active zone within 10 ms of exocytosis, well before endocytic pit formation.

    “However, membrane tension in neurons seems to propagate at ~20 µm/s11, and therefore, is expected to equilibrate at the active zone within 10 ms of exocytosis, well before endocytic pit formation.”
    Quote in the paperSubstantial claim1 of 1 sources checked
    • cites [11] Gomis Perez, C. et al. Rapid propagation of membrane tension at retinal bipolar neuron presynaptic terminals. Sci. Adv. 8, eabl4411 (2022). source

      The Results (‘Membrane tension dynamics are very different in retinal bipolar neurons and adrenal chromaffin cells’; page unavailable) report that tension changes traveled 4–11 μm within at most a few seconds. This supports rapid propagation in the studied neuronal terminals, but the passages do not establish the cited ~20 μm/s speed. The 10-ms active-zone estimate is presented as the citing authors’ inference, not as a finding attributed to this source. (LLM, conf=0.78)

      Experimental — accuracy not yet measured

  • May combine two claims.

    Premisecitation

    Latrunculin A treatment completely blocks ultrafast endocytosis, with no induction of membrane pit formation.

    “When neurons are treated with Latrunculin A, which sequesters monomeric actin (G-actin) and perturbs F-actin formation12,13, ultrafast endocytosis fails completely, with no induction of membrane pit formation5.”
    Quote in the paperSubstantial claim2 of 3 sources checked
    • cites [5] Watanabe, S. et al. Ultrafast endocytosis at mouse hippocampal synapses. Nature 504, 242–247 (2013). source
    • cites [12] Morton, W. M., Ayscough, K. R. & McLaughlin, P. J. Latrunculin alters the actin-monomer subunit interface to prevent polymerization. Nat. Cell Biol. 2, 376–378 (2000). source
    • cites [13] Spector, I., Shochet, N. R., Kashman, Y. & Groweiss, A. Latrunculins: Novel marine toxins that disrupt microfilament organization in cultured cells. Science 219, 493–495 (1983). source

  • May combine two claims.

    Findingown data

    DMSO control did not affect ultrafast endocytosis, while Latrunculin A completely blocked it.

    “Consistent with our previous work5, treatment with DMSO (control) did not affect ultrafast endocytosis, while Latrunculin A treatment completely blocked it (Fig. 2c, d; more micrographs in Supplementary Fig. 4).”
    Quote in the paperSubstantial claim1 of 1 sources checked
    • cites [5] Watanabe, S. et al. Ultrafast endocytosis at mouse hippocampal synapses. Nature 504, 242–247 (2013). source

  • Classified as probably not a substantial claim.

    Premisecitation

    Endocytosis typically requires force generation by active actin polymerization at the maturing endocytic pit.

    “Endocytosis typically requires force generation by active actin polymerization at the maturing endocytic pit52–57.”
    Quote in the paperSubstantial claim1 of 6 sources checked
    • cites [52] Akamatsu, M. et al. Principles of self-organization and load adaptation by the actin cytoskeleton during clathrin-mediated endocytosis. eLife 9, e49840 (2020). source

      The Introduction says that, in metazoan cells under high membrane tension, pits stall without functional actin, implying that actin-generated force is needed late in clathrin-mediated endocytosis. The Results also say that cells under low membrane tension can carry out endocytosis with low Arp2/3 activity. The source therefore supports a requirement under particular conditions, not the unqualified claim that endocytosis typically requires active actin polymerization. (LLM, conf=0.85)

      Experimental — accuracy not yet measured
    • cites [53] Kaksonen, M., Sun, Y. & Drubin, D. G. A pathway for association of receptors, adaptors, and actin during endocytic internalization. Cell 115, 475–487 (2003). source
    • cites [54] Kaksonen, M., Toret, C. P. & Drubin, D. G. A modular design for the clathrin- and actin-mediated endocytosis machinery. Cell 123, 305–320 (2005). source
    • cites [55] Kaksonen, M. & Roux, A. Mechanisms of clathrin-mediated endocytosis. Nat. Rev. Mol. Cell Biol. 19, 313–326 (2018). source
    • cites [56] Merrifield, C. J., Perrais, D. & Zenisek, D. Coupling between clathrin-coated-pit invagination, cortactin recruitment, and membrane scission observed in live cells. Cell 121, 593–606 (2005). source
    • cites [57] Merrifield, C. J., Feldman, M. E., Wan, L. & Almers, W. Imaging actin and dynamin recruitment during invagination of single clathrin-coated pits. Nat. Cell Biol. 4, 691–698 (2002). source

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Premisecitation

    A substantial spatial gradient of membrane tension is thought to persist due to attachment of cytoskeleton to the membrane.

    “Indeed, a substantial spatial gradient of membrane tension is thought to persist due to attachment of cytoskeleton to the membrane28.”
    Quote in the paperNot a substantial claim1 of 1 sources checked
    • cites [28] Shi, Z., Graber, Z. T., Baumgart, T., Stone, H. A. & Cohen, A. E. Cell Membranes Resist Flow. Cell 175, 1769–1779.e13 (2018). source

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Premisecitation

    In yeast, active force generation by actin polymerization has been long established as the mechanism by which actin supports endocytosis.

    “In yeast, active force generation by actin polymerization has been long established as the mechanism by which actin supports endocytosis85,86.”
    Quote in the paperNot a substantial claim1 of 2 sources checked
    • cites [85] Nickaeen, M., Berro, J., Pollard, T. D. & Slepchenko, B. M. Actin assembly produces sufficient forces for endocytosis in yeast. MBoC 30, 2014–2024 (2019). source

      The Introduction says actin assembly is necessary for membrane invagination in yeast and that polymerizing actin was believed to produce the required forces. The Discussion says it had remained unclear whether actin assembly generated enough force to overcome turgor pressure. Thus the force-generating mechanism is supported, but describing it as long established overstates how settled the paper says it was. (LLM, conf=0.84)

      Experimental — accuracy not yet measured
    • cites [86] Engqvist-Goldstein, A. E. Y. & Drubin, D. G. Actin assembly and endocytosis: from yeast to mammals. Annu. Rev. Cell Dev. Biol. 19, 287–332 (2003). source

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Premisecitation

    This spatial arrangement of F-actin at mouse hippocampal boutons is similar to presynaptic terminals of lamprey recticulospinal cord and Drosophila neuromuscular junctions.

    “This spatial arrangement of F-actin at mouse hippocampal boutons is similar to presynaptic terminals of lamprey recticulospinal cord83 and Drosophila neuromuscular junctions84.”
    Quote in the paperNot a substantial claim1 of 2 sources checked
    • cites [83] Shupliakov, O. et al. Impaired recycling of synaptic vesicles after acute perturbation of the presynaptic actin cytoskeleton. Proc. Natl Acad. Sci. USA. 99, 14476–14481 (2002). source
    • cites [84] Dunaevsky, A. & Connor, E. A. F-actin is concentrated in nonrelease domains at frog neuromuscular junctions. J. Neurosci. 20, 6007–6012 (2000). source

      The Discussion describes F-actin in nonrelease domains at frog neuromuscular junctions. Searches for lamprey reticulospinal terminals and Drosophila neuromuscular junctions found no passage comparing their F-actin arrangement with mouse hippocampal boutons. Our copy therefore does not address the comparison attributed to this source. (LLM, conf=0.78)

      Experimental — accuracy not yet measured

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Interpretationcitation

    The presence of F-actin in such artificially-formed active zones may reflect cytoskeletal response due to changes in cortical tension, since a bead is much stiffer than a postsynaptic terminal.

    “The presence of F-actin in such artificially-formed active zones may reflect cytoskeletal response due to changes in cortical tension, since a bead is much stiffer than a postsynaptic terminal82.”
    Quote in the paperNot a substantial claim1 of 1 sources checked
    • cites [82] Schiffhauer, E. S. et al. Mechanoaccumulative elements of the mammalian Actin cytoskeleton. Curr. Biol. 26, 1473–1479 (2016). source

      The Results and Discussion (page unavailable) show that actin-binding proteins accumulate in mechanically stressed regions of mammalian cells, supporting the proposed cytoskeletal response in general. The paper does not examine artificially formed active zones, compare bead stiffness with postsynaptic terminals, or establish that cortical-tension changes explain F-actin at those zones. (LLM, conf=0.84)

      Experimental — accuracy not yet measured

  • May combine two claims.

    Premisecitation

    Epsin1 has been implicated in endocytosis at synapses and can interact with both the plasma membrane and F-actin through its N-terminal ENTH domain and C-terminal tail, respectively.

    “Epsin1 has been implicated in endocytosis at synapses71 and can interact with both the plasma membrane and F-actin through its N-terminal ENTH domain and C-terminal tail, respectively (Supplementary Fig. 7a)72.”
    Quote in the paperSubstantial claim2 of 2 sources checked
    • cites [71] Kyung, J. W., Bae, J. R., Kim, D.-H., Song, W. K. & Kim, S. H. Epsin1 modulates synaptic vesicle retrieval capacity at CNS synapses. Sci. Rep. 6, 31997 (2016). source
    • cites [72] Messa, M. et al. Epsin deficiency impairs endocytosis by stalling the actin-dependent invagination of endocytic clathrin-coated pits. Elife 3, e03311 (2014). source

  • May combine two claims.

    Premisecitation

    In a previous study, ultrafast endocytosis failed when neurons were incubated at room temperature for 5 min prior to experiments, and this failure was compensated by clathrin-mediated endocytosis.

    “In our previous study, we demonstrated that ultrafast endocytosis fails when neurons are incubated at room temperature for 5 min prior to experiments – this failure is compensated by clathrin-mediated endocytosis7.”
    Quote in the paperSubstantial claim1 of 1 sources checked
    • cites [7] Watanabe, S. et al. Clathrin regenerates synaptic vesicles from endosomes. Nature 515, 228–233 (2014). source

  • Probably not a substantial claim: may not pass S3 (load-bearing). May combine two claims.

    Premisecitation

    In a recent study using single-molecule localization microscopy of presynaptic terminals formed on polystyrene microbeads, three pools of F-actin were apparent: actin rails between the active zone and vesicles, actin corrals around the terminal, and an actin mesh at the active zone.

    “In that system, three pools of F-actin were apparent: actin rails between the active zone and vesicles, actin corrals around the terminal22, and in contrast to our results, an actin mesh at the active zone.”
    Quote in the paperNot a substantial claim1 of 1 sources checked
    • cites [22] Bingham, D. et al. Distinct nano-structures support a multifunctional role of actin at presynapses. 2022.05.18.492480 Preprint at https://doi.org/10.1101/2022.05.18.492480 (2022). source

      Split into 2 atoms — 1 confirmed, 0 partially supported, 0 disputed, 0 misattributed, 0 unverifiable, 1 not checked. Expand the claim to see per-atom verdicts.

      Experimental — accuracy not yet measured

  • Probably not a substantial claim: may not pass S3 (load-bearing). May combine two claims.

    Premisecitation

    Epsin1 interacts with other endocytic proteins such as Eps15 and Hip1R, both of which interact either directly or indirectly with membranes and actin.

    “However, it is not clear whether Epsin1 is the sole organizer of F-actin at synapses, since it interacts with other endocytic proteins such as Eps15 and Hip1R72,88, both of which interact either directly or indirectly with membranes and actin.”
    Quote in the paperNot a substantial claim2 of 2 sources checked
    • cites [72] Messa, M. et al. Epsin deficiency impairs endocytosis by stalling the actin-dependent invagination of endocytic clathrin-coated pits. Elife 3, e03311 (2014). source
    • cites [88] Chen, H., Slepnev, V. I., Di Fiore, P. P. & De Camilli, P. The interaction of epsin and Eps15 with the clathrin adaptor AP-2 is inhibited by mitotic phosphorylation and enhanced by stimulation-dependent dephosphorylation in nerve terminals. J. Biol. Chem. 274, 3257–3260 (1999). source

      The introductory passage identifies epsin as a major binding partner of Eps15, supporting that interaction. It does not address Hip1R or establish that both proteins interact with membranes and actin; the cited paper therefore supports only part of the attributed claim. (LLM, conf=0.86)

      Experimental — accuracy not yet measured

  • Probably not a substantial claim: may not pass S3 (load-bearing). May combine two claims.

    Premisecitation

    The first alpha helix (α0) of the N-terminal ENTH domain can insert into the membrane and induce curvature formation.

    “In addition, the first alpha helix (α0) of the N-terminal ENTH domain can insert into the membrane and induce curvature formation (Fig. 4a)89,90 – this function of Epsin1 is thought to be important for endocytosis.”
    Quote in the paperNot a substantial claim1 of 2 sources checked
    • cites [89] Hyman, J., Chen, H., Di Fiore, P. P., De Camilli, P. & Brunger, A. T. Epsin 1 undergoes nucleocytosolic shuttling and its eps15 interactor NH(2)-terminal homology (ENTH) domain, structurally similar to Armadillo and HEAT repeats, interacts with the transcription factor promyelocytic leukemia Zn(2)+ finger protein (PLZF). J. Cell Biol. 149, 537–546 (2000). source

      The Results section, “General Features of the Structure” (page unavailable), describes the ENTH domain’s helices but does not identify an α0 helix or report membrane insertion or curvature formation. The Introduction says the ENTH domain’s function was unknown; searches for membrane insertion and curvature found no passage addressing the claimed mechanism. (LLM, conf=0.76)

      Experimental — accuracy not yet measured
    • cites [90] Ford, M. G. J. et al. Curvature of clathrin-coated pits driven by epsin. Nature 419, 361–366 (2002). source

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Study design and conductcitation

    Features were quantitated using the SynapsEM family of MATLAB scripts.

    “Features were then quantitated using the SynapsEM (Watanabe et al., 2020) family of MATLAB (MathWorks) scripts”
    Quote in the paperNot a substantial claim1 of 1 sources checked
    • cites Watanabe et al., 2020 source

  • May combine two claims.

    Findingown data

    As fused vesicles flatten, the active zone membrane deforms, generating lateral compression against the periactive zone membrane.

    “Our simulations show that as vesicles flatten out, the active zone membrane starts to deform, generating lateral compression against the periactive zone membrane.”
    Quote in the paperSubstantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Findingown data

    F-actin typically surrounds the active zone like a ring.

    “By 3D gSTED, F-actin is present at presynaptic terminals (Fig. 1a, Supplementary Fig. 1 and 2a for more example micrographs)22,23 and typically surrounds the active zone like a ring.”
    Quote in the paperNot a substantial claim

  • May combine two claims.

    Findingown data

    Latrunculin A treatment decreases EGFP-UtrCH signal intensity and abundance in regions closer to the active zone edge, indicating F-actin disruption at presynapses.

    “After Latrunculin A treatment (Fig. 1a, b), EGFP-UtrCH signals probed by 2D STED decreased in intensity and became less abundant in regions closer to the active zone edge (Fig. 1a, b), suggesting that F-actin is disrupted at presynapses.”
    Quote in the paperNot a substantial claim

  • May combine two claims.

    Findingown data

    Jasplakinolide treatment shifts actin distribution away from the active zone without reducing the abundance of preexisting structures.

    “By contrast, Jasplakinolide treatment shifted actin distribution away from the active zone, likely through enhanced actin polymerization at the periphery, but did not reduce the abundance of preexisting structures (Fig. 1a, b).”
    Quote in the paperNot a substantial claim

  • May combine two claims.

    Findingown data

    The actin ring is in the plane of the active zone; out of this plane, a distinct actin structure overlaps with the reserve pool cluster.

    “Notably, this actin ring is in the plane of the active zone, and when moving out of this plane, a distinct actin structure begins to overlap with the reserve pool cluster, which typically sits directly above or adjacent to the active zone or actin ring (Fig. 1a and Supplementary Fig. 2a).”
    Quote in the paperNot a substantial claim

  • May combine two claims.

    Findingown data

    The lateral membrane pressure from a single vesicle is insufficient to invoke endocytic pit formation; the mechanical energy reaches a steady state with incomplete vesicle flattening (curvature = 0.009 nm−1 at 7 ms).

    “The lateral membrane pressure from a single vesicle is insufficient to invoke the endocytic pit formation; so instead, the mechanical energy of the membrane reaches a steady-state with incomplete vesicle flattening (Supplementary Fig. 5a; curvature = 0.009 nm−1 at 7 ms).”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    F-actin distribution at synapses is either a complete ring encircling the active zone (64%), a partial ring lining 25-75% of the active zone area (27%), or a patch lining 25% or less (9%).

    “F-actin seems to either distribute like a ring that completely encircles the active zone (64% of synapses), like a partial ring (27% of synapses) that lines 25–75% of the active zone area, or like a patch (9% of synapses) that lines 25% or less of the active zone area (Supplementary Fig. 2a, b).”
    Quote in the paperNot a substantial claim

  • May combine two claims.

    Findingown data

    In Latrunculin A-treated neurons, no endocytic pits formed, and ferritin-positive endocytic structures did not accumulate in synaptic terminals by 1 s.

    “In Latrunculin A-treated neurons, no endocytic pits formed, and consequently, ferritin-positive endocytic structures did not accumulate in synaptic terminals by 1 s (Fig. 2c, e).”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    If synaptic vesicle membrane is too soft (below 230 kBT) or active zone membrane too stiff (above 50 kBT), endocytic pits do not form.

    “Furthermore, if synaptic vesicle membrane is too soft (below 230 kBT) or active zone membrane too stiff (above 50 kBT), endocytic pits do not form, further suggesting that relative stiffness is key to mechanical coupling (Fig. 1h).”
    Quote in the paperSubstantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Findingnone given

    The essential model results largely remain insensitive to the specific choice of the bending modulus of the actin-enriched periactive zone (κ3).

    “it is noted that the essential model results largely remain insensitive to the specific choice of κ3.”
    Quote in the paperNot a substantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Findingreasoning

    In the adaptive-timestep simulation, the averaged timestep decreases as the simulation converges with energy minimization and the system reaches the steady state.

    “the averaged timestep decreases (Supplementary Fig. 3c), as the simulation converges with the energy minimization and the system reaching the steady state (Supplementary Fig. 3d).”
    Quote in the paperNot a substantial claim

  • May combine two claims.

    Findingown data

    Actin is organized in a ring-like structure surrounding the active zone, with the endocytic zone also largely free of F-actin.

    “Our work suggests that 1) actin is organized in a ring-like structure surrounding the active zone (Fig. 1a), with the endocytic zone also largely free of F-actin”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Interpretationown data

    The initiation and kinetics of ultrafast endocytosis are unaffected by Jasplakinolide.

    “Ferritin particles were internalized into endocytic vesicles and endosomes by 1 s (Fig. 2c, e), suggesting that the initiation and kinetics of ultrafast endocytosis are unaffected by Jasplakinolide.”
    Quote in the paperSubstantial claim

  • Classified as probably not a substantial claim.

    Interpretationreasoning

    Based on the model, the frequency of ultrafast endocytosis would be expected to differ between synapses under 1.2 mM and 4 mM calcium concentrations.

    “Based on our model, the frequency of ultrafast endocytosis would be expected to differ between synapses under these two calcium concentrations.”
    Quote in the paperSubstantial claim
    • Rests on an assumption: At 4 mM Ca2+, 39% of responding synapses have only one vesicle fusion.

  • May combine two claims.

    Interpretationown data

    A stable actin cortex, but not dynamic actin polymerization, is necessary for ultrafast endocytosis.

    “These data suggest that a stable actin cortex, but not dynamic actin polymerization, is necessary for ultrafast endocytosis.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Interpretationreasoning

    The membrane compression model predicts that the role of actin filaments in ultrafast endocytosis is passive: they are only needed to conserve membrane area, and stable actin polymers are sufficient.

    “The membrane compression model predicts that the role of actin filaments in ultrafast endocytosis is passive - they are only needed to conserve membrane area, and thus stable actin polymers are sufficient.”
    Quote in the paperSubstantial claim
    • The authors state this as an assumption: The actin ring at the periactive zone conserves total membrane area due to the impediment of lipid flow.

  • The quote only partly states this claim.

    Interpretationown data

    There is a disparity between theoretical and experimental results regarding the frequency of ultrafast endocytosis at different calcium concentrations.

    “Thus, there is disparity between theoretical and experimental results.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Interpretationreasoning

    If too many vesicles attempt to fuse in a small presynaptic terminal, flattening out synaptic vesicle membrane incurs a high bending energy penalty, so vesicles would not fully collapse and endocytosis would not initiate.

    “If too many vesicles attempt to fuse in a small presynaptic terminal, flattening out synaptic vesicle membrane would incur a high bending energy penalty. Consequently, the synaptic vesicles would not fully collapse and endocytosis would not initiate.”
    Quote in the paperSubstantial claim

  • Probably not a substantial claim: may not pass S1 (asserted), S4 (based).

    Interpretationreasoning

    Ultrafast exo-endocytosis coupling requires at least two synaptic vesicles to fuse.

    “These simulations predict that ultrafast exo-endocytosis coupling requires at least two synaptic vesicles to fuse.”
    Quote in the paperNot a substantial claim

  • May combine two claims.

    Interpretationreasoning

    Some exocytic events may not trigger successful ultrafast endocytic events because synapses do not always fuse two vesicles either simultaneously or sequentially before the first completely flattens out.

    “Given that synapses do not always fuse two vesicles either simultaneously or sequentially before the first completely flattens out, some exocytic events may not trigger successful ultrafast endocytic events.”
    Quote in the paperSubstantial claim
    • The authors state this as an assumption: Collapsing synaptic vesicles are stiffer than the active zone membrane due to high protein density and enrichment of cholesterol and sphingomyelin lipids.

  • May combine two claims.

    Interpretationreasoning

    Because ultrafast formation of endocytic pits critically depends on the active zone membrane being softer than the vesicle membrane, ultrafast endocytosis may fail under conditions that stiffen the plasma membrane globally.

    “Since the ultrafast formation of endocytic pits critically depends on the active zone membrane being softer than the vesicle membrane (Fig. 1h and Supplementary Fig. 3b), ultrafast endocytosis may fail under such conditions.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Interpretationreasoning

    Two vesicles do not need to fuse simultaneously; one can fuse after the first one has begun to flatten.

    “However, two vesicles do not need to fuse simultaneously as one can fuse after the first one has begun to flatten.”
    Quote in the paperSubstantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Interpretationnone given

    On a longer timescale, synaptic vesicle components will likely mix with the active zone and hence affect the active zone stiffness.

    “On a longer timescale, the synaptic vesicle components will likely mix with the active zone and hence affect the active zone stiffness.”
    Quote in the paperNot a substantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing), S4 (based).

    Interpretationnone given

    Membrane tension may influence the global conservation of membrane area, thus contributing to ultrafast exo-endocytic coupling.

    “Nonetheless, membrane tension may influence the global conservation of membrane area, thus contributing to ultrafast exo-endocytic coupling.”
    Quote in the paperNot a substantial claim

  • Probably not a substantial claim: may not pass S4 (based).

    Interpretationnone given

    The effective stiffness of fused vesicles is likely maintained on a time scale of milliseconds by an inhibition of protein and lipid diffusion due to highly curved membrane at the neck of fused vesicles.

    “Further, the effective stiffness of fused vesicles is likely maintained on a time scale of milliseconds by an inhibition of protein and lipid diffusion due to highly curved membrane at the neck of fused vesicles.”
    Quote in the paperNot a substantial claim
    • The authors state this as an assumption: Collapsing synaptic vesicles are stiffer than the active zone membrane due to high protein density and enrichment of cholesterol and sphingomyelin lipids.

  • May combine two claims.

    Interpretationreasoning

    In synapses with fewer fusions, endocytosis still occurs, but is expected to be slow.

    “Thus, in synapses with fewer fusions, endocytosis still occurs, but is expected to be slow.”
    Quote in the paperNot a substantial claim

  • May combine two claims.

    Interpretationreasoning

    The spatial gradient of membrane tension is expected to relax within about 1-10 ms of vesicle fusion, since neither the active zone nor collapsing vesicles attach to cytoskeleton.

    “However, this spatial gradient is expected to relax within ~1–10 ms of vesicle fusion, since neither the active zone nor collapsing vesicles attach to cytoskeleton.”
    Quote in the paperSubstantial claim
    • Rests on an assumption: Neither the active zone nor collapsing vesicles attach to cytoskeleton.

  • May combine two claims.

    Interpretationreasoning

    Any increase in active zone stiffness from mixing of synaptic vesicle components is expected to be marginal and transient because the active zone area is more than 100 times larger than synaptic vesicle area and synaptic vesicle components are constantly retrieved by endocytosis.

    “However, even if this effect exists, it will be largely diluted because the active zone area is > 100 times larger than synaptic vesicle area. Furthermore, synaptic vesicle components are constantly retrieved by endocytosis. Therefore, such an increase in the active zone stiffness is expected to be marginal and transient.”
    Quote in the paperNot a substantial claim
    • Rests on an assumption: The active zone area is more than 100 times larger than synaptic vesicle area.
    • Rests on an assumption: Synaptic vesicle components are constantly retrieved by endocytosis.

  • Probably not a substantial claim: may not pass S3 (load-bearing), S4 (based). May combine two claims.

    Interpretationnone given

    Excess vesicle proteins are likely recognized by clathrin-adaptors and then internalized via clathrin scaffolds.

    “Still, excess vesicle proteins are likely recognized by clathrin-adaptors and then internalized via clathrin scaffolds.”
    Quote in the paperNot a substantial claim

  • May combine two claims.

    Interpretationown data

    Intact F-actin, but not its dynamic polymerization, is essential for the initiation of ultrafast endocytosis.

    “Our work suggests that 1) actin is organized in a ring-like structure surrounding the active zone (Fig. 1a), with the endocytic zone also largely free of F-actin, and 2) intact F-actin, but not its dynamic polymerization, is essential for the initiation of ultrafast endocytosis (Fig. 2c, d).”
    Quote in the paperSubstantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Premisecitation

    The F1 ATPase uses its beta-sheet as a spring to store energy from ATP, and then rapidly releases it to create high-speed rotary torque of about 100 Hz.

    “For example, the F1 ATPase (the smallest rotary motor) uses its beta-sheet as a spring to store energy from ATP, and then rapidly releases it to create the high-speed rotary torque ( ~100 Hz)91.”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites [91] Wang, H. & Oster, G. Energy transduction in the F1 motor of ATP synthase. Nature 396, 279–282 (1998). source

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Study design and conductnone given

    Samples were blinded before electron microscopy imaging.

    “Samples were blinded before imaging.”
    Quote in the paperNot a substantial claim

  • Probably not a substantial claim: may not pass S2 (checkable), S3 (load-bearing).

    Interpretationreasoning

    Our work provides an example of how mechanics can be exploited to speed up biological functions.

    “Conceptually, our work provides an example of how mechanics can be exploited to speed up biological functions.”
    Quote in the paperNot a substantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Study design and conductcitation

    EM image analysis was performed as previously described in ref. 39.

    “EM image analysis was performed as previously described in ref. 39.”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites ref. 39

      Couldn’t be matched to a paper in Crossref, so it can’t be checked.

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Study design and conductnone given

    All image segmentation was thoroughly checked and edited by a second member of the lab to minimize bias and error and maintain consistency.

    “To minimize bias and error, and to maintain consistency, all image segmentation, still in the form of randomized files, was thoroughly checked and edited by a second member of the lab.”
    Quote in the paperNot a substantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Study design and conductnone given

    For EM experiments using neurons from Doc2a KO and EGTA-AM, a different freeze-substitution protocol was used that generated more consistent results.

    “For EM experiments using neurons from Doc2a KO and EGTA-AM (Fig. 3) a different freeze-substitution protocol was used that generated more consistent results.”
    Quote in the paperNot a substantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Study design and conductnone given

    Sample sizes for each replicate were inferred from previous flash-and-freeze experiments rather than from power analysis.

    “Sample sizes for each replicate were inferred from previous flash-and-freeze experiments as opposed to power analysis.”
    Quote in the paperNot a substantial claim

  • Probably not a substantial claim: may not pass S2 (checkable), S3 (load-bearing).

    Interpretationreasoning

    Despite the overwhelming role of chemical signaling in the nervous system, underpinning the efficacy of this cell-cell communication is a highly organized presynaptic mechanical landscape that is structured to react rapidly to local mechanical changes.

    “Despite the overwhelming role of chemical signaling in the nervous system, underpinning the efficacy of this cell-cell communication is a highly organized presynaptic mechanical landscape that is structured to react rapidly to local mechanical changes.”
    Quote in the paperNot a substantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Premisereasoning

    Due to the high turgor pressure impinging upon yeast plasma membranes, this force generation is essential to invaginate the endocytic membrane.

    “Due to the high turgor pressure impinging upon yeast plasma membranes, this force generation is essential to invaginate the endocytic membrane.”
    Quote in the paperNot a substantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Interpretationnone given

    In the lateral membrane compression model, stable F-actin is proposed to conserve the membrane area.

    “In the lateral membrane compression model, stable F-actin is proposed to conserve the membrane area.”
    Quote in the paperNot a substantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Premisecitation

    Recent work suggests that the actin nucleator formin is required for synaptic vesicle endocytosis at small hippocampal boutons.

    “Additionally, recent work suggests that the actin nucleator formin is required for synaptic vesicle endocytosis at small hippocampal boutons8.”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites [8] Soykan, T. et al. Synaptic vesicle endocytosis occurs on multiple timescales and is mediated by Formin-dependent actin assembly. Neuron 93, 854–866.e4 (2017). source

  • Probably not a substantial claim: may not pass S3 (load-bearing). May combine two claims.

    Study design and conductnone given

    Synaptic clefts were identifiable by their characteristic width, by the apposed membranes following each other closely, and by vesicles near the presynaptic active zone.

    “synaptic clefts were also identifiable by 1) their characteristic width, 2) the apposed membranes following each other closely, and 3) vesicles near the presynaptic active zone.”
    Quote in the paperNot a substantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Study design and conductnone given

    All images were from different synapses.

    “All images were from different synapses.”
    Quote in the paperNot a substantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Premisecitation

    A Venus flytrap leaf preloads its trap and then snaps it at a 100 ms timescale via a buckling instability.

    “For instance, a Venus flytrap leaf preloads its trap and then snaps it at a 100 ms timescale via a buckling instability92.”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites [92] Forterre, Y., Skotheim, J. M., Dumais, J. & Mahadevan, L. How the Venus flytrap snaps. Nature 433, 421–425 (2005). source

  • Probably not a substantial claim: may not pass S3 (load-bearing). May combine two claims.

    Study design and conductown data

    Images were excluded from analysis only after randomization, either because they appeared not to contain a bona fide synapse or because the morphology was too poor for reliable annotation.

    “Only after this randomization were any images excluded from analysis, either because they appeared to not contain a bona fide synapse or the morphology was too poor for reliable annotation.”
    Quote in the paperNot a substantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Study design and conductnone given

    All images from a single experiment were randomized for analysis as a single pool.

    “All the images from a single experiment were randomized for analysis as a single pool.”
    Quote in the paperNot a substantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Premisecitation

    In much softer animal cell membranes, the role of actin polymerization in endocytosis is controversial.

    “However, in the case of much softer animal cell membranes, the role of actin polymerization in endocytosis is controversial55.”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites [55] Kaksonen, M. & Roux, A. Mechanisms of clathrin-mediated endocytosis. Nat. Rev. Mol. Cell Biol. 19, 313–326 (2018). source

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Study design and conductnone given

    An alpha of 0.05 was set for null hypothesis testing.

    “An alpha of 0.05 was set for null hypothesis testing.”
    Quote in the paperNot a substantial claim
65 of 65

  • The authors state this as an assumption.

    The actin ring at the periactive zone conserves total membrane area due to the impediment of lipid flow.

    “First, the actin ring at the periactive zone conserves the total membrane area due to the impediment of lipid flow26–28.”

  • The authors state this as an assumption.

    The membrane of fused vesicles is stiffer than the active zone membrane.

    “Second, the membrane of fused vesicles is stiffer than the active zone membrane (Fig. 1c, d)29,30.”

  • The authors state this as an assumption.

    The endocytic zone has the same bending modulus as the active zone membrane and is included as part of the active zone.

    “The endocytic zone is assumed to have the same bending modulus as the active zone membrane and thus, is included as a part of the active zone (note that no vesicles fuse in this region; Fig. 1c, d).”

  • The authors state this as an assumption.

    When the curvature of resulting membrane pits is sufficiently large (>0.03/nm), endocytosis proceeds by curvature-sensing mechanisms of endocytic proteins.

    “We assumed that when the curvature of the resulting membrane pits is sufficiently large ( >0.03/nm), endocytosis would proceed by well-defined curvature-sensing mechanisms of endocytic proteins45–48, some of which form condensates just outside the active zone17,49.”

  • The paper relies on this without support.

    At 4 mM Ca2+, 39% of responding synapses have only one vesicle fusion.

    “In contrast, at 4 mM Ca2+, which was used in all other experiments described here and in our previous studies, 39% of responding synapses have only one vesicle fusion.”

  • The authors state this as an assumption.

    Collapsing synaptic vesicles are stiffer than the active zone membrane due to high protein density and enrichment of cholesterol and sphingomyelin lipids.

    “The model assumes collapsing synaptic vesicles to be stiffer than the active zone membrane due to high protein density and enrichment of cholesterol and sphingomyelin lipids73.”

  • The paper relies on this without support.

    Neither the active zone nor collapsing vesicles attach to cytoskeleton.

    “However, this spatial gradient is expected to relax within ~1–10 ms of vesicle fusion, since neither the active zone nor collapsing vesicles attach to cytoskeleton.”

  • The paper relies on this without support.

    The active zone area is more than 100 times larger than synaptic vesicle area.

    “However, even if this effect exists, it will be largely diluted because the active zone area is > 100 times larger than synaptic vesicle area.”

  • The paper relies on this without support.

    Synaptic vesicle components are constantly retrieved by endocytosis.

    “Furthermore, synaptic vesicle components are constantly retrieved by endocytosis.”

  • The authors state this as an assumption.

    The spontaneous curvature of the membrane is zero.

    “is the spontaneous curvature of the membrane and assumed to be zero.”

  • The authors state this as an assumption.

    The bending modulus of the stiff post-fusion synaptic vesicle membrane (κ1) ranges from 100 kBT to 400 kBT.

    “the associated bending modulus, κ1, is assumed to range from 100 kBT to 400 kBT (see Supplemental Table 1).”

  • The authors state this as an assumption.

    The bending modulus of the soft membrane comprising the active zone and surrounding endocytic zone (κ2) ranges from 10 kBT to 100 kBT.

    “the associated bending modulus, κ2, is assumed to be range from 10 kBT to 100 kBT (see Supplemental Table 1)40,41.”
12 of 12

Claims other papers make about this one

Each is checked against this paper’s own text: does it say what the citing paper says it does?

  • The molecular switch shifting retrieval toward ultrafast endocytosis is primarily mechanical, driven by a 'membrane compression model' where fused synaptic vesicles exert lateral pressure against an F-actin-enriched stiff periactive zone, inducing rapid membrane buckling and curvature independent of clathrin recruitment.

    “The molecular switch shifting retrieval toward ultrafast endocytosis is primarily mechanical, driven by a ‘membrane compression model’ where fused synaptic vesicles exert lateral pressure against an F-actin-enriched stiff periactive zone, inducing rapid membrane buckling and curvature independent of clathrin recruitment [31,34].”

    From Molecular Physiology of the Neuronal Synapse (2026) · cites this paper as [34]

Self-check

Percentages, sums and reported differences re-computed from the paper’s own numbers.

Each contradiction between two passages listed here was confirmed by an independent check.

  • Different labels specified for the Epsin1-F-actin colocalization assay

    Major

    The Fig. 4a caption describes antibody-labeled Epsin1 and EGFP-UtrCH-labeled F-actin, while the colocalization method specifies Epsin1-EGFP and UtrCH-HaloTag. These are different labeling schemes for the reported colocalization analysis.

  • Conflicting reference for F-actin intensity normalization

    Minor

    The Fig. 4 caption says presynaptic F-actin intensities were normalized to axonal fluorescence, whereas the analysis method says they were normalized to fluorescence in the corresponding cell body.

2 of 2

Cites

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Cited by

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Related

Authors
Ogunmowo, T. H., Jing, H., Raychaudhuri, S., Kusick, G. F., Imoto, Y., Li, S., Itoh, K., Ma, Y., Jafri, H., Dalva, M. B., Chapman, E. R., Ha, T., Watanabe, S., & Liu, J.
Affiliations
  • Johns Hopkins University (US)
  • Thomas Jefferson University (US)
  • Tulane University (US)
  • Howard Hughes Medical Institute (US)
Corresponding author
  • Watanabe, Shigeki
  • Liu, Jian
ORCID
Venue
Nature Communications
Publisher
Springer Science and Business Media LLC
Published
2023-05-20
Volume
14
Issue
1
Pages
2888-2888
ISSN
2041-1723 (ISSN-L)
DOI
10.1038/s41467-023-38595-2
PMC
PMC10199930
OpenAlex
W4377138052
Semantic Scholar
86908888ad8ea2731a1219e86838209ed5fe5dbd
Cited by
51
References
103
Citations by year
2026: 11 · 2025: 18 · 2024: 17 · 2023: 5
FWCI
6.12
Type
journal article
Language
en
Open access
Licence
Locations
  • Nature Communications (publishedVersion, cc-by)
  • PubMed (publishedVersion)
  • PubMed Central (submittedVersion, cc-by)
  • The Medicine Forum (submittedVersion, cc-by)
  • RePEc: Research Papers in Economics (submittedVersion)
  • DOAJ (DOAJ: Directory of Open Access Journals) (submittedVersion, cc-by-sa)
Funders
  • U.S. Department of Health & Human Services | NIH | National Institute of Neurological Disorders and Stroke (NS105810-01, NS111133-01, NS106906, NS097362)
  • National Science Foundation (1727260, 2019241734, 2016217537, 2105837, 2148534)
  • McKnight Endowment Fund for Neuroscience
  • Esther A. and Joseph Klingenstein Fund
  • Vallee Foundation
  • Alfred P. Sloan Foundation
  • U.S. Department of Health & Human Services | NIH | National Institute of General Medical Sciences (T32 GM007445, GM148459-01)
  • Japan Society for the Promotion of Science London
  • U.S. Department of Health & Human Services | NIH | National Institute on Drug Abuse (DA022727)
  • Howard Hughes Medical Institute
  • U.S. Department of Health & Human Services | NIH | National Institute of Mental Health (MH061876)
Topics
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Cell Biology › Cellular transport and secretion
  • Life Sciences › Neuroscience › Cellular and Molecular Neuroscience › Neuroscience and Neuropharmacology Research
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Extracellular vesicles in disease
Keywords
synaptic vesicle exocytosis, clathrin-independent endocytosis, membrane compression, membrane mechanics, actin rings, active zone, epsin1, chemical synapses, hippocampus, vesicle fusion
MeSH terms
Actins, Animals, Cell Membrane, Endocytosis, Exocytosis, Synapses, Synaptic Vesicles, Mice

About these checks

Registry, retraction, Cites and statistics results are computed, not judged, with one exception: a printed reference that no index lists is read once by a model before it counts as a citation error. Claims are extracted by a model and kept only when their quote appears verbatim in the text; each cited source is then checked, with a model’s help, for whether it says what the claim relies on. Those verdicts can be wrong — read the claim’s quote and the cited source before relying on one.

Claims read by deepseek-v4.1-flash under definition version 3 (extract-v3) on 2026-10-12.

Classified by jev-1.13-20260917 (gold-guideline-v0.2) on 2026-10-12.

Cited sources are checked by gpt-6-sol.

Statistics and arithmetic are recomputed, not judged.

Confidence bands are shown only for fields calibrated on our annotated test set. None is calibrated yet, so no band is shown.

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