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Cited by 0Advanced Electron Microscopy Techniques and ApplicationsEnzyme Structure and FunctionMicrotubule and mitosis dynamics

Ghost Neuron preparation unlocks high-resolution structural mapping of intracellular architecture in axons

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Bodakuntla, Satish, Marelli, Julia, Vasquez-Montes, Victor, Glauser, Simon, Biertümpfel, Christian, Mizuno, Naoko·2026·Nature Communications
DOI 10.1038/s41467-026-77741-4PMC PMC13690744
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Abstract

Cellular cryo-electron microscopy (cryo-EM) offers powerful tools to reveal molecular architecture inside cells, but dense cytoplasm often obscures fine molecular features, especially in narrow neuronal projections. Here, we introduce the Ghost Neuron technique, a preparation combining brief hypotonic treatment with controlled mechanical rupture immediately before freezing, facilitating high-contrast imaging of neuronal ultrastructure within its neurite topology. By removing the plasma membrane and reducing cytoplasmic density, the method allows high-resolution structural analysis while preserving the overall geometry of neurites. The method allows structural information to be mapped back onto neuronal architecture. The preparation also releases endogeneous macromolecular complexes onto EM grids, creating particle-rich regions suitable for high-resolution structural analysis. Using Ghost Neuron , we resolved axonal microtubules at 3.59 Å and 3.9 Å resolution, distinguishing expanded and compact lattice states, and mapped these states as interspersed patches along individual microtubules within axons. We also resolved axonal actin filaments and identified cofilin-decorated actin. These results establish Ghost Neuron as a discovery platform for linking molecular structure to neuronal organization.

AI snapshot

Written from the paper’s full text.

TL;DR

The paper introduces Ghost Neuron, a preparation that combines brief hypotonic treatment with mechanical rupture immediately before vitrification to remove the plasma membrane, reduce cytoplasmic density, and preserve neurite geometry for high-resolution cryo-EM. Using this method, the authors resolve axonal microtubule lattice states and actin/cofilactin structures and map them back onto neuronal architecture.

Key findings

  • Ghost Neuron preparation removes the plasma membrane while preserving overall neurite trajectories and major cytoskeletal architecture, enabling high-contrast cryo-EM and spatial back-mapping of molecular states onto axons.
  • Single-particle analysis resolved axonal microtubules at 3.59 Å (expanded lattice) and 3.9 Å (compact lattice), both with 13-protofilament architecture; 61.4% of classified segments were expanded with an 83.1 Å inter-dimer spacing, and 38.6% were compact with an 81.2 Å repeat. Expanded patches occurred within the mid-shaft of individual microtubules, not only at ends.
  • The method resolved neuronal F-actin at 3.24 Å and cofilin-decorated actin at 5.32 Å; cofilin was identified with a fit correlation coefficient of 0.97 to PDB 6vao. Of 1528 µm total analyzed F-actin, 21.7 µm showed continuous decoration, and decorated filaments appeared predominantly fully decorated or largely undecorated.
  • Hypotonic optimization showed 160 mOsm preserved axon morphology and mitochondrial motility, whereas 80 mOsm caused membrane swellings and impaired motility, and 0 mOsm caused pronounced fragmentation.

Methods

  • Primary thalamus explants from E15.5 CD-1 mice were cultured on EM grids until DIV7–9, exposed to hypotonic media (240, 160, 80, or 0 mOsm) for 5 min, blotted, and vitrified; 160 mOsm was selected as optimal.
  • Live imaging used MemGlow, MitoTracker, fluorescent Epothilone B, and SPY-tubulin; mitochondrial motility was tracked with TrackMate, and osmolarity was measured by freezing-point depression.
  • Cryo-EM data were collected on a Titan Krios G4 at 300 kV with a K3 detector; microtubule SPA used 4172 micrographs, 7787 microtubules, 1296 µm cumulative length, and 158,108 segments, with 78,882 segments classified in CryoSPARC. Actin SPA started from 238,038 particles and yielded final reconstructions for actin and cofilactin.

Limitations

  • Stated: because the plasma membrane is deliberately ruptured, membrane-associated processes such as receptor signaling and ion channel organization cannot be analyzed in their native membrane environment.
  • Stated: selective membrane removal disrupts submembranous structures mechanically coupled to the plasma membrane; the membrane-associated periodic skeleton’s periodic pattern is not preserved, although actin filaments remain axially localized.
  • Stated: membrane rupture causes partial redistribution of soluble and small membranous components, so the method does not provide a complete inventory of all intracellular constituents at their original locations and is best suited for selected or retained assemblies.
  • Evident from the text: the study is a proof-of-principle focused on cytoskeletal targets; rare or transient intermediate microtubule lattice states may fall below the detection threshold, and the authors argue but do not fully exclude that osmotic modulation or rupture could induce conformational changes.

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

Integrity signals

Registry

Crossref · 10.1038/s41467-026-77741-4

Cites

90 of 90 references verified0 citation errors · 0 not checkable

Statistics

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Claims the paper makes

58 of 72 cited sources checked · 33 supported · 0 flagged · accuracy not yet measured

  • Premisecitation

    In situ cryo-EM/cryo-ET remains technically limited to relatively small or thin biological specimens.

    “However, in situ cryo-EM/cryo-ET remains technically limited to relatively small or thin biological specimens, such as bacteria5, yeast6, or peripheral regions of larger eukaryotic cells7.”
    Quote in the paperSubstantial claim3 of 3 sources checked
    • cites [5] Xue, L. et al. Visualizing translation dynamics at atomic detail inside a bacterial cell. Nature 610, 205–211 https://doi.org/10.1038/s41586-022-05255-2 (2022). source
    • cites [6] Wozny, M. R. et al. In situ architecture of the ER-mitochondria encounter structure. Nature 618, 188–192 https://doi.org/10.1038/s41586-023-06050-3 (2023). source
    • cites [7] Medalia, O. et al. Organization of actin networks in intact filopodia. Curr. Biol. 17, 79–84 https://doi.org/10.1016/j.cub.2006.11.022 (2007). source
  • Premisecitation

    Previous studies have shown that crude or partially extracted intracellular mixtures can yield high-resolution SPA reconstructions.

    “Previous studies have shown that crude or partially extracted intracellular mixtures can yield high-resolution SPA reconstructions78–80, highlighting that such fractions can serve as important resources for structural discovery.”
    Quote in the paperSubstantial claim3 of 3 sources checked
    • cites [78] Ho, C. M. et al. Bottom-up structural proteomics: cryoEM of protein complexes enriched from the cellular milieu. Nat. Methods 17, 79–85 https://doi.org/10.1038/s41592-019-0637-y (2020). source
    • cites [79] Skalidis, I. et al. Cryo-EM and artificial intelligence visualize endogenous protein community members. Structure 30, 575–589 e576 https://doi.org/10.1016/j.str.2022.01.001 (2022). source
    • cites [80] May, M. B., Lopez-Perez, G. S. & Davis, J. H. Capturing ribosomal structures in cellular extracts with cryoPRISM: A purification-free cryoEM approach reveals novel structural states. Proc. Natl. Acad. Sci. USA 123, e2521210123. https://doi.org/10.1073/pnas.2521210123 (2026). source
  • Premisecitation

    Thalamus neural explants produced long neurites extending from brain tissue, allowing unambiguous assignment to axonal origins at this developmental stage.

    “We used thalamus neural explants, which produced long neurites extending from brain tissue, allowing unambiguous assignment to axonal origins at this developmental stage25,33,34.”
    Quote in the paperSubstantial claim2 of 3 sources checked
    • cites [25] Nedozralova, H. et al. In situ cryo-electron tomography reveals local cellular machineries for axon branch development. J. Cell Biol. 221 https://doi.org/10.1083/jcb.202106086 (2022). source

      The Materials and methods section describes E15.5 mouse thalamus tissue explants, and the Results and discussion section identifies axons and branches in those cultures. Neither passage says that long neurites extended from the tissue or that their axonal origin could be assigned unambiguously at that developmental stage. Page numbers are unavailable in our copy. (LLM, conf=0.83)

      Experimental — accuracy not yet measured
    • cites [33] Molnar, Z. & Blakemore, C. Development of signals influencing the growth and termination of thalamocortical axons in organotypic culture. Exp. Neurol. 156, 363–393 https://doi.org/10.1006/exnr.1999.7032 (1999). source
    • cites [34] Bodakuntla, S. et al. In situ structural mechanism of epothilone-B-induced CNS axon regeneration. Nature 648, 477–487 https://doi.org/10.1038/s41586-025-09654-z (2025). source

      The Fig. 1 caption in “Cryo-EM platform for axon injury model” says embryonic thalamus explants extended axons from the tissue and describes their neurite extensions as axon-dominant. The Methods confirm that the explants came from E15.5 embryos. The source does not establish that every long neurite could be assigned unambiguously to an axonal origin; “axon-dominant” is a narrower claim. (LLM, conf=0.82)

      Experimental — accuracy not yet measured
  • Findingcitation

    Measured axial repeat distances correspond closely to canonical compact and expanded lattice parameters reported in vitro and in intact cells.

    “The measured axial repeat distances correspond closely to canonical compact and expanded lattice parameters reported in vitro45–47 and in intact cells48.”
    Quote in the paperSubstantial claim4 of 4 sources checked
    • cites [45] Alushin, G. M. et al. High-resolution microtubule structures reveal the structural transitions in αβ-tubulin upon GTP hydrolysis. Cell 157, 1117–1129 https://doi.org/10.1016/j.cell.2014.03.053 (2014). source
    • cites [46] Zhang, R., Alushin, G. M., Brown, A. & Nogales, E. Mechanistic Origin of Microtubule Dynamic Instability and Its Modulation by EB Proteins. Cell 162, 849–859 https://doi.org/10.1016/j.cell.2015.07.012 (2015). source
    • cites [47] Estevez-Gallego, J. et al. Hydrolysis-deficient mosaic microtubules as faithful mimics of the GTP cap. Nat. Commun. 16, 2396. https://doi.org/10.1038/s41467-025-57555-6 (2025). source
    • cites [48] de Jager, L. et al. StableMARK-decorated microtubules in cells have expanded lattices. J. Cell Biol. 224 https://doi.org/10.1083/jcb.202206143 (2025). source
  • Findingown data

    Measured axial spacings match the well-established expanded (83 Å) and compact (81 Å) lattice spacing described for in vitro reconstituted microtubules.

    “The measured axial spacings match the well-established expanded (83 Å) and compact (81 Å) lattice spacing described for in vitro reconstituted microtubules45–47.”
    Quote in the paperSubstantial claim3 of 3 sources checked
    • cites [45] Alushin, G. M. et al. High-resolution microtubule structures reveal the structural transitions in αβ-tubulin upon GTP hydrolysis. Cell 157, 1117–1129 https://doi.org/10.1016/j.cell.2014.03.053 (2014). source
    • cites [46] Zhang, R., Alushin, G. M., Brown, A. & Nogales, E. Mechanistic Origin of Microtubule Dynamic Instability and Its Modulation by EB Proteins. Cell 162, 849–859 https://doi.org/10.1016/j.cell.2015.07.012 (2015). source

      The Results section on the compacted EB3-GTPγS lattice (page unavailable) describes an extended GMPCPP-K microtubule lattice and compacted EB3-GTPγS and GDP-K lattices, supporting the distinction between expanded and compact states. The passages in our copy do not give the claimed 83 Å and 81 Å axial spacings, so those values cannot be verified from this source. (LLM, conf=0.82)

      Experimental — accuracy not yet measured
    • cites [47] Estevez-Gallego, J. et al. Hydrolysis-deficient mosaic microtubules as faithful mimics of the GTP cap. Nat. Commun. 16, 2396. https://doi.org/10.1038/s41467-025-57555-6 (2025). source
  • Classified as probably not a substantial claim. 29
  • May combine two claims. 63

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

    Premisecitation

    Ghost cell formation was originally described as a degenerative process in red blood cells that lost hemoglobin and later adapted as a specialized preparation to visualize membrane architecture.

    “Similarly, ghost cell formation was originally described as a degenerative process in red blood cells (RBCs) that have lost hemoglobin and later adapted as a specialized preparation to visualize membrane architecture28,29.”
    Quote in the paperNot a substantial claim1 of 2 sources checked
    • cites [28] Hoffman, J. F. Evidence that asymmetry of the membrane/cytoskeletal complex in human red blood cell ghosts is responsible for their biconcave shape. Proc. Natl. Acad. Sci. USA 115, 1641–1645 https://doi.org/10.1073/pnas.1721463115 (2018). source

      Materials and Methods (page unavailable) describes prepared red blood cell ghosts containing less than 2% hemoglobin. The Significance section and the discussion (pages unavailable) use those ghosts to study and image the membrane/cytoskeletal basis of red blood cell shape. The paper does not say ghost formation was originally described as a degenerative process or trace a later adaptation from that description. (LLM, conf=0.86)

      Experimental — accuracy not yet measured
    • cites [29] Steck, T. L. & Kant, J. A. Preparation of impermeable ghosts and inside-out vesicles from human erythrocyte membranes. Methods Enzymol. 31, 172–180 https://doi.org/10.1016/0076-6879(74)31019-1 (1974). source

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

    Premisecitation

    For larger and thicker samples, cryo-FIB milling combined with CLEM has been developed to access regions deep within cells.

    “In contrast, for larger and thicker samples, cryo-focused ion beam (cryo-FIB) milling combined with correlative light and electron microscopy (CLEM) has been developed to access regions deep within cells8.”
    Quote in the paperNot a substantial claim1 of 1 sources checked
    • cites [8] Barad, B. A., Medina, M., Fuentes, D., Wiseman, R. L. & Grotjahn, D. A. Quantifying organellar ultrastructure in cryo-electron tomography using a surface morphometrics pipeline. J. Cell Biol. 222 https://doi.org/10.1083/jcb.202204093 (2023). source

      The Results (Figure 1 caption; page unavailable) describe using cryo-fluorescence microscopy to select cells for cryo-FIB milling into thin lamellae, supporting the combined CLEM–cryo-FIB approach. The passages do not say that this approach was developed specifically for larger or thicker samples or to access regions deep within cells. (LLM, conf=0.84)

      Experimental — accuracy not yet measured

  • Classified as probably not a substantial claim.

    Premisecitation

    Cell unroofing mechanically removes the dorsal surface of adherent cells, exposing the cytoplasmic face of the plasma membrane attached to a solid substrate.

    “Cell unroofing mechanically removes the dorsal surface of adherent cells, thereby exposing the cytoplasmic face of the plasma membrane that remains attached to a solid substrate26,27.”
    Quote in the paperSubstantial claim2 of 2 sources checked
    • cites [26] Vassilopoulos, S., Gibaud, S., Jimenez, A., Caillol, G. & Leterrier, C. Ultrastructure of the axonal periodic scaffold reveals a braid-like organization of actin rings. Nat. Commun. 10, 5803. https://doi.org/10.1038/s41467-019-13835-6 (2019). source
    • cites [27] Sun, W. W. et al. Cryo-electron tomography pipeline for plasma membranes. Nat. Commun. 16, 855. https://doi.org/10.1038/s41467-025-56045-z (2025). source

      The Results figure caption (Fig. 1; page unavailable) describes pressurized fluid washing away the apical portions of cells to leave basal plasma membranes on an EM grid, supporting the claim for that preparation. But the same caption also describes unroofing cells in an inverted orientation by washing away their basal portions to isolate apical membranes. Thus, removal of the dorsal surface is not a general description of every unroofing preparation in this paper. (LLM, conf=0.85)

      Experimental — accuracy not yet measured

  • Classified as probably not a substantial claim. May combine two claims.

    Premisecitation

    Several studies have imaged neuronal processes by cryo-EM and cryo-ET, showing neurites are accessible to cryogenic imaging but persistent challenges remain at molecular resolution due to poor contrast and cytoplasmic denseness.

    “Several studies have imaged neuronal processes by cryo-EM and cryo-ET19–25, showing that neurites are accessible to cryogenic imaging, while also revealing persistent challenges in obtaining information at a molecular resolution due to poor contrast and cytoplasmic denseness.”
    Quote in the paperSubstantial claim6 of 7 sources checked
    • cites [19] Hoffmann, P. C. et al. Electron cryo-tomography reveals the subcellular architecture of growing axons in human brain organoids. Elife 10 https://doi.org/10.7554/eLife.70269 (2021). source

      The Introduction and Discussion describe cryo-ET imaging of neuronal processes, and the Results report detailed cryo-ET images of growing axons. The Image processing and analysis section notes that signal-to-noise ratio and proximity to other microtubules can prevent some subtomogram analyses, but the paper does not attribute a persistent molecular-resolution challenge to poor contrast and cytoplasmic denseness generally. No page numbers are available in our copy. (LLM, conf=0.84)

      Experimental — accuracy not yet measured
    • cites [20] Petrovic, A., Do, T. T. & Fernandez-Busnadiego, R. New insights into the molecular architecture of neurons by cryo-electron tomography. Curr. Opin. Neurobiol. 90, 102939. https://doi.org/10.1016/j.conb.2024.102939 (2025). source
    • cites [21] Foster, H. E., Ventura Santos, C. & Carter, A. P. A cryo-ET survey of microtubules and intracellular compartments in mammalian axons. J. Cell Biol. 221 https://doi.org/10.1083/jcb.202103154 (2022). source
    • cites [22] Atherton, J., Stouffer, M., Francis, F. & Moores, C. A. Microtubule architecture in vitro and in cells revealed by cryo-electron tomography. Acta Crystallogr D. Struct. Biol. 74, 572–584 https://doi.org/10.1107/S2059798318001948 (2018). source

      In “Ultrastructure of MTs in neurons” (page not provided), the paper reports that parts of neuronal processes are well preserved in cryo-EM images and tomographic sections, supporting their accessibility to cryogenic imaging. It identifies excessive soma thickness as an imaging limitation, but does not attribute difficulty obtaining molecular-resolution information in neurites to poor contrast or cytoplasmic denseness. (LLM, conf=0.85)

      Experimental — accuracy not yet measured
    • cites [23] Atherton, J., Stouffer, M., Francis, F. & Moores, C. A. Visualising the cytoskeletal machinery in neuronal growth cones using cryo-electron tomography. J. Cell Sci. 135 https://doi.org/10.1242/jcs.259234 (2022). source

      The Results section, “Cryo-ET of primary hippocampal neuron growth cones” (page unavailable), reports imaging vitrified growth cones and clearly visualising the neuron interior, supporting the claim that neuronal processes are accessible to cryogenic imaging. The passages do not attribute persistent difficulty obtaining molecular-resolution information to poor contrast or cytoplasmic denseness; searches for those limitations did not find such a statement. (LLM, conf=0.82)

      Experimental — accuracy not yet measured
    • cites [24] Zamponi, E., Meehl, J. B. & Voeltz, G. K. The ER ladder is a unique morphological feature of developing mammalian axons. Dev. Cell 57, 1369–1382 e1366 https://doi.org/10.1016/j.devcel.2022.05.002 (2022). source

      The Results (page unavailable) describe cryo-ET imaging of developing axons and visualization of their structures at nanometer resolution, supporting the claim that neuronal processes are accessible to cryogenic imaging. The paper does not attribute difficulty obtaining molecular-resolution information to poor contrast or cytoplasmic denseness; its comment about poor resolution concerns conventional TEM instead. (LLM, conf=0.84)

      Experimental — accuracy not yet measured
    • cites [25] Nedozralova, H. et al. In situ cryo-electron tomography reveals local cellular machineries for axon branch development. J. Cell Biol. 221 https://doi.org/10.1083/jcb.202106086 (2022). source

      The Results and discussion (Structural analysis of branching axons; page not provided) reports cryo-ET reconstructions of axon branches and shafts, supporting that neuronal processes are accessible to cryogenic imaging. It also notes a tomographic limitation, the “missing wedge,” but does not attribute difficulty obtaining molecular-resolution information to poor contrast or cytoplasmic denseness. (LLM, conf=0.85)

      Experimental — accuracy not yet measured

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

    Premisecitation

    Expanded lattice states have been detected in intact mammalian U2OS cells by cryo-ET, supporting lattice heterogeneity within a cell.

    “In addition, the expanded lattice states have been detected in intact mammalian U2OS cells by cryo-ET48, supporting the lattice heterogeneity within a cell.”
    Quote in the paperNot a substantial claim1 of 1 sources checked
    • cites [48] de Jager, L. et al. StableMARK-decorated microtubules in cells have expanded lattices. J. Cell Biol. 224 https://doi.org/10.1083/jcb.202206143 (2025). source

  • May combine two claims.

    Premisecitation

    Alternative perturbations such as mechanical stretching, RhoA-ROCK activation, or modification of lipid composition can induce significant disruption ranging from membrane-cytoskeleton decoupling, contractility-induced retraction to altered lipid composition and gene expression.

    “Alternative perturbations such as mechanical stretching73, RhoA-ROCK activation74–76, or the modification of lipid composition77 can induce significant disruption ranging from membrane-cytoskeleton decoupling, contractility-induced retraction to altered lipid composition and gene expression.”
    Quote in the paperSubstantial claim3 of 5 sources checked
    • cites [73] Sheetz, M. P. & Dai, J. Modulation of membrane dynamics and cell motility by membrane tension. Trends Cell Biol. 6, 85–89 https://doi.org/10.1016/0962-8924(96)80993-7 (1996). source
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      Split into 2 atoms — 0 confirmed, 1 partially supported, 0 disputed, 0 misattributed, 1 unverifiable. Expand the claim to see per-atom verdicts.

      Experimental — accuracy not yet measured
    • cites [76] Nambiar, R., McConnell, R. E. & Tyska, M. J. Control of cell membrane tension by myosin-I. Proc. Natl. Acad. Sci. USA 106, 11972–11977 https://doi.org/10.1073/pnas.0901641106 (2009). source

      Split into 2 atoms — 0 confirmed, 1 partially supported, 0 disputed, 0 misattributed, 1 unverifiable. Expand the claim to see per-atom verdicts.

      Experimental — accuracy not yet measured
    • cites [77] Khatibzadeh, N., Gupta, S., Farrell, B., Brownell, W. E. & Anvari, B. Effects of cholesterol on nano-mechanical properties of the living cell plasma membrane. Soft Matter 8, 8350–8360 https://doi.org/10.1039/C2SM25263E (2012). source

  • May combine two claims.

    Premisecitation

    Similar expanded lattice spacing was observed in a previous reconstruction of Epothilone B-stabilized microtubules at 3.19 Å resolution in regenerating axons, where membrane rupture also occurred during vitrification due to expanded membranes at regeneration sites, indicating membrane disruption itself does not distort lattice geometry.

    “Similar expanded lattice spacing was observed in our previous reconstruction of Epothilone B-stabilized microtubules at 3.19 Å resolution in regenerating axons34, where membrane rupture also occurred during vitrification due to expanded membranes at regeneration sites, indicating that membrane disruption itself does not distort lattice geometry.”
    Quote in the paperNot a substantial claim1 of 1 sources checked
    • cites [34] Bodakuntla, S. et al. In situ structural mechanism of epothilone-B-induced CNS axon regeneration. Nature 648, 477–487 https://doi.org/10.1038/s41586-025-09654-z (2025). source

      Split into 2 atoms — 1 confirmed, 1 partially supported, 0 disputed, 0 misattributed, 0 unverifiable. Expand the claim to see per-atom verdicts.

      Experimental — accuracy not yet measured

  • Classified as probably not a substantial claim. May combine two claims.

    Premisecitation

    Recent work reported that axonal microtubules in human iPSC-derived neurons are GDP-bound yet adopt an expanded, GTP-like lattice, indicating that lattice spacing is not determined solely by nucleotide state.

    “Recent work reported that axonal microtubules in human iPSC-derived neurons are GDP-bound yet adopt an expanded, GTP-like lattice, indicating that lattice spacing is not determined solely by nucleotide state52.”
    Quote in the paperSubstantial claim1 of 1 sources checked
    • cites [52] Zehr, E. A., Sun, S., Sarbanes, S. L. & Roll-Mecak, A. Microtubules in the axon are GDP bound but adopt a stable GTP-like expanded state. Nat. Struct. Mol. Biol. 33, 631–640 https://doi.org/10.1038/s41594-026-01787-7 (2026). source

  • Classified as probably not a substantial claim. May combine two claims.

    Premisecitation

    Two models have been proposed to explain microtubule stability in axons: one suggests centrally located microtubules are generally more stable while peripheral ones are more dynamic, and the other suggests intra-microtubule heterogeneity with alternating domains of differing stability.

    “Two models have been proposed to explain microtubule stability in axons: one suggests that centrally located microtubules are generally more stable while peripheral ones are more dynamic53–55, and the other model suggests intra-microtubule heterogeneity, in which individual microtubules contain alternating domains with differing stability56,57.”
    Quote in the paperSubstantial claim3 of 5 sources checked
    • cites [53] Conde, C. & Caceres, A. Microtubule assembly, organization and dynamics in axons and dendrites. Nat. Rev. Neurosci. 10, 319–332 https://doi.org/10.1038/nrn2631 (2009). source
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      The Discussion (page unavailable) reports stable microtubules enriched in the core of dendritic shafts and dynamic microtubules in a surrounding shell. It also notes that different parts of one microtubule can have different modifications, but does not describe alternating domains of stability. The claim applies these patterns to axons, whereas this paper's spatial-mapping result concerns dendrites. (LLM, conf=0.86)

      Experimental — accuracy not yet measured
    • cites [55] Tas, R. P. et al. Differentiation between oppositely oriented microtubules controls polarized neuronal transport. Neuron 96, 1264–1271 e1265 https://doi.org/10.1016/j.neuron.2017.11.018 (2017). source

      Split into 2 atoms — 0 confirmed, 1 partially supported, 0 disputed, 0 misattributed, 1 unverifiable. Expand the claim to see per-atom verdicts.

      Experimental — accuracy not yet measured
    • cites [56] Baas, P. W. & Black, M. M. Individual microtubules in the axon consist of domains that differ in both composition and stability. J. Cell Biol. 111, 495–509 (1990). source

      Split into 2 atoms — 1 confirmed, 1 partially supported, 0 disputed, 0 misattributed, 0 unverifiable. Expand the claim to see per-atom verdicts.

      Experimental — accuracy not yet measured
    • cites [57] Baas, P. W., Slaughter, T., Brown, A. & Black, M. M. Microtubule dynamics in axons and dendrites. J. Neurosci. Res 30, 134–153 https://doi.org/10.1002/jnr.490300115 (1991). source

  • May combine two claims.

    Interpretationcitation

    Such regional variation is consistent with previous studies and suggests that lattice conformation can be locally influenced by lattice-binding microtubule-associated proteins, post-translational modifications, mechanical stress, or repair processes.

    “Such regional variation is consistent with previous studies58and suggest that lattice conformation can be locally influenced by lattice-binding microtuble-associated proteins (MAPs)59–61, post-translational modifications, mechanical stress50,51, or repair processes62–66.”
    Quote in the paperNot a substantial claim10 of 10 sources checked
    • cites [58] Paquette, A. L. et al. Competition for microtubule lattice spacing between a microtubule expander and compactor. Curr. Biol. 35, 4442–4452 e4444 https://doi.org/10.1016/j.cub.2025.07.080 (2025). source
    • cites [59] Kellogg, E. H. et al. Near-atomic model of microtubule-tau interactions. Science 360, 1242–1246 https://doi.org/10.1126/science.aat1780 (2018). source

      Split into 2 atoms — 0 confirmed, 2 partially supported, 0 disputed, 0 misattributed, 0 unverifiable. Expand the claim to see per-atom verdicts.

      Experimental — accuracy not yet measured
    • cites [60] Kirimtay, K. et al. Tau and MAP6 establish labile and stable domains on microtubules. iScience 28, 111785. https://doi.org/10.1016/j.isci.2025.111785 (2025). source
    • cites [61] Nakata, T., Niwa, S., Okada, Y., Perez, F. & Hirokawa, N. Preferential binding of a kinesin-1 motor to GTP-tubulin-rich microtubules underlies polarized vesicle transport. J. Cell Biol. 194, 245–255 https://doi.org/10.1083/jcb.201104034 (2011). source
    • cites [50] Manka, S. W. & Moores, C. A. The role of tubulin-tubulin lattice contacts in the mechanism of microtubule dynamic instability. Nat. Struct. Mol. Biol. 25, 607–615 https://doi.org/10.1038/s41594-018-0087-8 (2018). source

      Split into 2 atoms — 1 confirmed, 1 partially supported, 0 disputed, 0 misattributed, 0 unverifiable. Expand the claim to see per-atom verdicts.

      Experimental — accuracy not yet measured
    • cites [51] Manka, S. W. & Moores, C. A. Microtubule structure by cryo-EM: snapshots of dynamic instability. Essays Biochem 62, 737–751 https://doi.org/10.1042/EBC20180031 (2018). source

      Figure 3 and the ‘Tubulin conformational landscape’ section describe differences in lattice conformation across nucleotide states and binding conditions. The ‘Beyond the lattice’ section describes structural heterogeneity at microtubule ends, but the copy does not establish regional variation in lattice conformation along a microtubule. Citation 58 is attached to the comparison with previous studies; the proposed local influences have separate citations. (LLM, conf=0.79)

      Experimental — accuracy not yet measured
    • cites [62] Triclin, S. et al. Self-repair protects microtubules from destruction by molecular motors. Nat. Mater. 20, 883–891 https://doi.org/10.1038/s41563-020-00905-0 (2021). source

      Split into 2 atoms — 0 confirmed, 2 partially supported, 0 disputed, 0 misattributed, 0 unverifiable. Expand the claim to see per-atom verdicts.

      Experimental — accuracy not yet measured
    • cites [63] Schaedel, L. et al. Microtubules self-repair in response to mechanical stress. Nat. Mater. 14, 1156–1163 https://doi.org/10.1038/nmat4396 (2015). source
    • cites [64] Aumeier, C. et al. Self-repair promotes microtubule rescue. Nat. Cell Biol. 18, 1054–1064 https://doi.org/10.1038/ncb3406 (2016). source
    • cites [65] Vemu, A. et al. Severing enzymes amplify microtubule arrays through lattice GTP-tubulin incorporation. Science 361 https://doi.org/10.1126/science.aau1504 (2018). source

      Split into 2 atoms — 0 confirmed, 2 partially supported, 0 disputed, 0 misattributed, 0 unverifiable. Expand the claim to see per-atom verdicts.

      Experimental — accuracy not yet measured

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

    Premisecitation

    Surfactant-based extraction methods have provided access to cytoskeletal organization, including neuronal processes.

    “Related surfactant-based extraction methods have provided informative access to cytoskeletal organization, including neuronal processes30–32.”
    Quote in the paperNot a substantial claim3 of 3 sources checked
    • cites [30] Korobova, F. & Svitkina, T. Molecular architecture of synaptic actin cytoskeleton in hippocampal neurons reveals a mechanism of dendritic spine morphogenesis. Mol. Biol. Cell 21, 165–176 https://doi.org/10.1091/mbc.e09-07-0596 (2010). source
    • cites [31] Jones, S. L., Korobova, F. & Svitkina, T. Axon initial segment cytoskeleton comprises a multiprotein submembranous coat containing sparse actin filaments. J. Cell Biol. 205, 67–81 https://doi.org/10.1083/jcb.201401045 (2014). source
    • cites [32] Efimova, N. et al. betaIII spectrin is necessary for formation of the constricted neck of dendritic spines and regulation of synaptic activity in neurons. J. Neurosci. 37, 6442–6459 https://doi.org/10.1523/JNEUROSCI.3520-16.2017 (2017). source

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

    Premisecitation

    The classical view describes newly polymerized plus ends, where GTP-tubulin is incorporated and subsequently hydrolyzed as microtubules mature.

    “This classical view describes newly polymerized plus ends, where GTP-tubulin is incorporated and subsequently hydrolyzed as the microtubules mature45,50,51.”
    Quote in the paperNot a substantial claim3 of 3 sources checked
    • cites [45] Alushin, G. M. et al. High-resolution microtubule structures reveal the structural transitions in αβ-tubulin upon GTP hydrolysis. Cell 157, 1117–1129 https://doi.org/10.1016/j.cell.2014.03.053 (2014). source
    • cites [50] Manka, S. W. & Moores, C. A. The role of tubulin-tubulin lattice contacts in the mechanism of microtubule dynamic instability. Nat. Struct. Mol. Biol. 25, 607–615 https://doi.org/10.1038/s41594-018-0087-8 (2018). source
    • cites [51] Manka, S. W. & Moores, C. A. Microtubule structure by cryo-EM: snapshots of dynamic instability. Essays Biochem 62, 737–751 https://doi.org/10.1042/EBC20180031 (2018). source

  • Classified as probably not a substantial claim.

    Premisecitation

    Microtubule lattice spacing has classically been linked to nucleotide state, with expanded lattices associated with GTP-like states and compact lattices associated with GDP-tubulin.

    “Microtubule lattice spacing has classically been linked to nucleotide state, with expanded lattices associated with GTP-like states and compact lattices associated with GDP-tubulin45,50,51.”
    Quote in the paperSubstantial claim3 of 3 sources checked
    • cites [45] Alushin, G. M. et al. High-resolution microtubule structures reveal the structural transitions in αβ-tubulin upon GTP hydrolysis. Cell 157, 1117–1129 https://doi.org/10.1016/j.cell.2014.03.053 (2014). source
    • cites [50] Manka, S. W. & Moores, C. A. The role of tubulin-tubulin lattice contacts in the mechanism of microtubule dynamic instability. Nat. Struct. Mol. Biol. 25, 607–615 https://doi.org/10.1038/s41594-018-0087-8 (2018). source
    • cites [51] Manka, S. W. & Moores, C. A. Microtubule structure by cryo-EM: snapshots of dynamic instability. Essays Biochem 62, 737–751 https://doi.org/10.1042/EBC20180031 (2018). source

  • May combine two claims.

    Findingown data

    Axonal actin filaments were resolved and cofilin-decorated actin was identified.

    “We also resolved axonal actin filaments and identified cofilin-decorated actin.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingreasoning

    The Ghost Neuron preparation removes the plasma membrane and reduces cytoplasmic density while preserving overall neurite geometry, allowing high-resolution structural analysis.

    “By removing the plasma membrane and reducing cytoplasmic density, the method allows high-resolution structural analysis while preserving the overall geometry of neurites.”
    Quote in the paperSubstantial claim
    • Rests on an assumption: Hypotonic treatment increases membrane tension, which is necessary for membrane rupture and high-contrast imaging of intracellular components by cryo-EM.

  • May combine two claims.

    Findingown data

    Using Ghost Neuron, axonal microtubules were resolved at 3.59 Å and 3.9 Å resolution, distinguishing expanded and compact lattice states.

    “Using Ghost Neuron, we resolved axonal microtubules at 3.59 Å and 3.9 Å resolution, distinguishing expanded and compact lattice states”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingreasoning

    As a result of Ghost Neuron preparation, the outer membrane barrier is removed and cytoplasmic density is reduced, producing thin, high-contrast traces of neurites suitable for cryo-EM/ET and downstream high-resolution structural analysis.

    “As a result, the outer membrane barrier is removed and the cytoplasmic density is reduced, producing thin, high-contrast traces of neurites suitable for cryo-EM and cryo-ET imaging, and downstream high-resolution structural analysis.”
    Quote in the paperSubstantial claim
    • Rests on an assumption: Hypotonic treatment increases membrane tension, which is necessary for membrane rupture and high-contrast imaging of intracellular components by cryo-EM.

  • May combine two claims.

    Findingown data

    Actin filaments decorated by cofilin adopt an expanded conformation (C-form), which has also been observed upon full binding of cofilin.

    “We also observed that the actin filaments decorated by cofilin adopt an expanded conformation (C-form), which has also been observed upon full binding of cofilin.”
    Quote in the paperNot a substantial claim

  • May combine two claims.

    Findingown data

    We mapped compact and expanded microtubule lattice conformations along individual axons, providing new insights into the conformational variability of microtubules.

    “We also mapped compact and expanded microtubule lattice conformations along individual axons, which provides new insights into the conformational variability of microtubules.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    Classification revealed two major microtubule lattice conformations at resolutions of 3.59 Å and 3.9 Å, both displaying canonical 13-protofilament architecture.

    “Classification revealed two major lattice conformations at resolutions of 3.59 Å and 3.9 Å, respectively, both displaying the canonical 13-protofilament architecture (Fig. 7).”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    When hypotonic treatment was followed by filter paper blotting without vitrification, robust microtubule labeling was observed under 160 and 80 mOsm conditions, while samples treated with 240 mOsm media showed no labeling.

    “When hypotonic treatment was followed by filter paper blotting, but without vitrification, robust microtubule labeling was observed under 160 and 80 mOsm conditions, while samples treated with 240 mOsm media showed no labeling”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    By back-plotting classified segments onto cryo-EM montages, microtubule conformational states and cytoskeletal architectures were mapped along individual neurites, revealing patches of expanded lattice within shafts of axonal microtubules.

    “By back-plotting classified segments onto cryo-EM montages, we mapped microtubule conformational states and cytoskeletal architectures along individual neurites, revealing patches of expanded lattice within the shafts of axonal microtubules.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    We resolved F-actin at molecular resolution and identified a sub-population of fully cofilin-decorated filaments.

    “We resolved F-actin at molecular resolution and identified a sub-population of fully cofilin-decorated filaments.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    High-magnification montages revealed membranes remained mostly intact at 320 mOsm and 240 mOsm, whereas treatment at 160 mOsm and 80 mOsm partially disrupted the plasma membrane.

    “High-magnification montages (HMMs) revealed that membranes remained mostly intact at 320 mOsm (control, Fig. 4A) and 240 mOsm (Fig. S2A), whereas the treatment at 160 mOsm and 80 mOsm partially disrupted the plasma membrane (Fig. 4B and Fig. S2B).”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingreasoning

    Preserved geometry allows structural information to be mapped back onto neuronal topology, while dispersed material on the grid provides an additional source for single-particle analysis of endogenous assemblies.

    “Importantly, the preserved geometry allows structural information to be mapped back onto neuronal topology, while the dispersed material on the grid provides an additional source for single-particle analysis of endogenous assemblies.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingnone given

    During Ghost Neuron preparation, the plasma membrane remains intact during hypotonic incubation; rupture occurs only upon blotting immediately before freezing.

    “During the incubation, the plasma membrane remains intact; rupture occurs only upon blotting immediately before freezing.”
    Quote in the paperSubstantial claim
    • Rests on an assumption: Hypotonic treatment increases membrane tension, which is necessary for membrane rupture and high-contrast imaging of intracellular components by cryo-EM.

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

    Findingnone given

    The osmolarity of normal neural explant media was approximately 320 mOsm at DIV8.

    “The osmolarity of normal neural explant media was approximately 320 mOsm at 8 days in vitro (DIV8).”
    Quote in the paperNot a substantial claim

  • May combine two claims.

    Findingown data

    Larger organelles and the microtubule cytoskeleton followed the original trajectory of the neurite, allowing visualization of spatial organization and potential interactions with associated components.

    “In contrast, larger organelles and the microtubule cytoskeleton followed the original trajectory of the neurite, allowing the visualization of its spatial organization and potential interactions with associated components (Fig. S5A and S5B).”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    From the decorated subset, 2328 asymmetric particles were collected and a structure at 5.32 Å resolution was achieved.

    “While we collected only 2328 asymmetric particles from this subset, we achieved a structure at 5.32 Å resolution (Fig. 6C, D).”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    Using Ghost Neuron, high-resolution structures of axonal microtubules were obtained, resolving coexisting expanded and compact lattice conformations within axons, as well as actin filaments with a sub-population of fully cofilin-decorated actin filaments.

    “Using the Ghost Neuron approach, we obtained high-resolution structures of axonal microtubules, resolving coexisting expanded and compact lattice conformations within axons, as well as actin filaments with a sub-population of fully cofilin-decorated actin filaments.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    Approximately 61% of classified microtubule segments adopted the expanded lattice conformation with an inter-dimer spacing of 83.1 Å, while 39% displayed a compact lattice with an 81.2 Å repeat.

    “Approximately 61% of the classified microtubule segments adopted the expanded lattice conformation with an inter-dimer spacing of 83.1 Å, while 39% displayed a compact lattice with an 81.2 Å repeat (Fig. 7D).”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    The Ghost Neuron approach selectively removes the plasma membrane from neurites while preserving overall neurite geometry.

    “Here, we introduce the Ghost Neuron approach, which selectively removes the plasma membrane from neurites while preserving overall neurite geometry.”
    • Also stated in: § Establishment of the Ghost Neuron approach · plain
      “This combined procedure selectively ruptures the plasma membrane while leaving the underlying cytoskeletal architecture and overall neurite geometry largely intact.”
    Quote in the paperSubstantial claim
    • Rests on an assumption: Hypotonic treatment increases membrane tension, which is necessary for membrane rupture and high-contrast imaging of intracellular components by cryo-EM.

  • May combine two claims.

    Findingreasoning

    The Ghost Neuron method facilitates structural analysis at defined positions along neurites and allows individual molecular states to be mapped back onto specific neuronal locations.

    “The method facilitates structural analysis at defined positions along neurites and allows individual molecular states to be mapped back onto specific neuronal locations.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    In all conditions, no axonal breakage was observed after 10 min of exposure, suggesting short-term hypotonic exposure does not cause gross axonal morphological disruption under the conditions tested.

    “In all conditions, no axonal breakage was observed after 10 min of exposure, suggesting that short-term hypotonic exposure does not cause gross axonal morphological disruption under the conditions tested35–37.”
    Quote in the paperSubstantial claim0 of 3 sources checked
    • cites [35] Bray, D., Money, N. P., Harold, F. M. & Bamburg, J. R. Responses of growth cones to changes in osmolality of the surrounding medium. J. Cell Sci. 98, 507–515 https://doi.org/10.1242/jcs.98.4.507 (1991). source
    • cites [36] Wan, X., Harris, J. A. & Morris, C. E. Responses of neurons to extreme osmomechanical stress. J. Membr. Biol. 145, 21–31 https://doi.org/10.1007/BF00233304 (1995). source
    • cites [37] Pontes, B., Monzo, P. & Gauthier, N. C. Membrane tension: A challenging but universal physical parameter in cell biology. Semin Cell Dev. Biol. 71, 30–41 https://doi.org/10.1016/j.semcdb.2017.08.030 (2017). source

  • May combine two claims.

    Findingown data

    Neurons remained morphologically intact when exposed to 160 mOsm for an extended duration up to 24 h, showing hypotonic tolerance of neurons at 160 mOsm.

    “Neurons remained morphologically intact when exposed to 160 mOsm for an extended duration of up to 24 h (Fig. S1F and S1G), showing hypotonic tolerance of neurons at 160 mOsm.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    Secondary structure assignment and backbone tracing revealed cofilin as the decorating protein, consistent with the available structure of an in vitro prepared cofilin-actin complex (PDB 6vao) with a fit correlation coefficient of 0.97.

    “Secondary structure assignment and backbone tracing revealed cofilin as the decorating protein, consistent with the available structure of an in vitro prepared cofilin-actin complex (PDB 6vao) (Fig. 6E) with a fit correlation coefficient of 0.97.”
    Quote in the paperSubstantial claim0 of 1 sources checked
    • cites PDB 6vao

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

  • May combine two claims.

    Findingnone given

    The Ghost Neuron approach selectively removes the plasma membrane immediately prior to vitrification, resulting in reduced cytoplasmic density and local thinning of the specimen while maintaining architectural continuity of neurite trajectories.

    “We established the Ghost Neuron approach to address this constraint by selectively removing the plasma membrane immediately prior to vitrification, resulting in reduced cytoplasmic density and locally thinning of the specimen, while maintaining the architectural continuity of neurite trajectories.”
    Quote in the paperSubstantial claim
    • Rests on an assumption: Hypotonic treatment increases membrane tension, which is necessary for membrane rupture and high-contrast imaging of intracellular components by cryo-EM.

  • Classified as probably not a substantial claim.

    Findingown data

    4172 micrographs containing 7787 microtubules were used for analysis, corresponding to a cumulative microtubule length of 1296 μm.

    “4172 micrographs containing 7787 microtubules were used for analysis, corresponding to a cumulative microtubule length of 1296 μm.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingreasoning

    After Ghost Neuron preparation, actin filaments remain axially localized along neurites, but the periodic pattern of the MPS is not preserved.

    “After Ghost Neuron preparation, actin filaments remain axially localized along neurites, but the periodic pattern of the MPS is not preserved.”
    Quote in the paperNot a substantial claim

  • May combine two claims.

    Findingown data

    The C1 reconstructions achieved resolutions of 3.9 Å for the compact lattice and 3.59 Å for the expanded lattice.

    “The resulting C1 reconstructions achieved resolutions of 3.9 Å for the compact lattice, and 3.59 Å for the expanded lattice (Fig. S8D).”
    Quote in the paperSubstantial claim

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

    Findingown data

    Extensive 2D classification yielded 6835 undecorated actin filaments and 147 decorated filaments.

    “Extensive 2D classification yielded 6835 undecorated actin filaments and 147 decorated filaments.”
    Quote in the paperNot a substantial claim

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

    Findingown data

    Decorated filaments were identified in 17 micrographs.

    “Decorated filaments were identified in 17 micrographs.”
    Quote in the paperNot a substantial claim

  • May combine two claims.

    Findingown data

    Of the 78,882 classified microtubule segments, 48,459 (61.4% of classified segments, 30.6% of initial segments) were assigned the expanded conformation, and 30,423 (38.6% of classified segments, 19.2% of initial segments) the compact conformation.

    “Of the 78,882 classified segments, 48,459 (61.4% of classified segments, 30.6% of initial segments) were assigned the expanded conformation, and 30,423 (38.6% of classified segments, 19.2% of initial segments) the compact conformation.”
    Quote in the paperNot a substantial claim

  • May combine two claims.

    Findingown data

    Posterior probabilities clustered near 0 or 1 rather than populating intermediate values, supporting distinct separation of the two classified lattice states, though it does not exclude rare or transient intermediate states below the current detection threshold.

    “Posterior probabilities clustered near 0 or 1 than populating intermediate values (Fig. S8B), supporting distinct separation of the two classified lattice states, though it does not exclude rare or transient intermediate states below our current detection threshold.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    The final C1 3D reconstruction without application of pseudo-helical symmetry revealed a clear distinction in the S9-S10 loop that differentiates between α and β tubulin subunits, indicating the success of the seam detection and alignment.

    “The final C1 3D reconstruction without application of pseudo-helical symmetry revealed a clear distinction in the S9-S10 loop that differentiates between α and β tubulin subunits, indicating the success of the seam detection and alignment.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    Initial helical refinement yielded reconstructions at 4.12 Å resolution for actin and 6.58 Å for decorated filaments; local refinement improved the resolutions to 3.88 Å and 5.88 Å, respectively.

    “Initial helical refinement yielded reconstructions at 4.12 Å resolution for actin (rise = 27.51 Å, twist = 166.65°) and 6.58 Å for decorated filaments (rise = 27.38 Å, twist = 162.33°). Local refinement improved the resolutions to 3.88 Å and 5.88 Å, respectively.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingreasoning

    Membrane rupture leads to partial redistribution of soluble and small membranous components, and it is not possible to fully determine which species are retained versus released in any given preparation.

    “Finally, membrane rupture leads to partial redistribution of soluble and small membranous components, and it is not possible to fully determine which species are retained versus released in any given preparation.”
    Quote in the paperSubstantial claim

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

    Findingnone given

    The peptide bonds of P274 in α-tubulin and P272 in β-tubulin showed cis configuration in both cases.

    “As a note, the peptide bonds of P274 in α-tubulin and P272 in β-tubulin showed cis configuration in both cases.”
    Quote in the paperNot a substantial claim

  • May combine two claims.

    Findingown data

    Final local refinement yielded reconstructions at 3.24 Å for actin and 5.32 Å for decorated.

    “Final local refinement yielded reconstructions at 3.24 Å for actin and 5.32 Å for decorated.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingnone given

    Cultured neurons prepared on EM grids can be imaged directly by light microscopy either during live imaging or after vitrification, and the resulting fluorescence data can be correlated to map regions of interest for further imaging by cryo-EM.

    “Cultured neurons prepared on EM grids can be imaged directly by light microscopy either during live imaging or after vitrification, and the resulting fluorescence data can be correlated to map the regions of interest for further imaging by cryo-EM.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Premisereasoning

    Although neurites are within the geometric thickness limits of cryo-EM and cryo-ET, their dense cytoplasm and abundant membrane-bound organelles reduce image contrast, limiting alignment accuracy and achievable resolution.

    “Although neurites are within the geometric thickness limits of cryo-EM and cryo-ET, their dense cytoplasm and the abundance of membrane-bound organelles substantially reduce image contrast, limiting alignment accuracy and ultimately achievable resolution.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Interpretationown data

    The absence of actin filaments within neuronal microtubule lumen suggests this phenomenon may be a cell type-specific mechanism.

    “We have not observed actin filaments within the lumen of neuronal microtubules, suggesting this phenomenon may be a cell type-specific mechanism.”
    Quote in the paperNot a substantial claim

  • May combine two claims.

    Interpretationown data

    160 mOsm was selected as the optimal compromise, permitting controlled membrane removal while preserving neurite geometry and intracellular dynamics as assessed by mitochondrial motility.

    “We therefore selected 160 mOsm as the optimal compromise, permitting controlled membrane removal while preserving neurite geometry and intracellular dynamics as assessed by mitochondrial motility.”
    Quote in the paperSubstantial claim
    • Rests on an assumption: Hypotonic treatment increases membrane tension, which is necessary for membrane rupture and high-contrast imaging of intracellular components by cryo-EM.

  • May combine two claims.

    Interpretationown data

    Our structural analysis is consistent with intra-microtubule heterogeneity, revealing local differences in lattice conformation within single axonal microtubules.

    “While these models are not mutually exclusive, our structural analysis is consistent with intra-microtubule heterogeneity, revealing local differences in lattice conformation within single axonal microtubules.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Premisenone given

    Surfactant-based extraction methods are largely limited by chemical fixation and metal coating and are incompatible with high-resolution cryo-EM in a frozen-hydrated state.

    “However, these methods are largely limited by the usage of chemical fixation and metal coating and, therefore, incompatible with high-resolution cryo-EM in a frozen-hydrated state.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Interpretationown data

    Our observation of the dominant expanded microtubule conformation is consistent with the view that lattice spacing is not determined solely by nucleotide state, while detection of compact segments suggests local lattice heterogeneity is detectable in our dataset.

    “Our observation of the dominant expanded microtubule conformation is consistent with this view, while the detection of compact segments suggests that local lattice heterogeneity is detectable in our dataset.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Study design and conductown data

    Lattice classifications were reproducible across independent runs and multiple neurites.

    “Lattice classifications were reproducible across independent runs (Fig. S8) and multiple neurites (Fig. S7).”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Interpretationown data

    Short-term hypotonic treatment provides a practical strategy to transiently weaken membranes while maintaining intracellular organization for cryo-EM applications and keeping cells in a vital state.

    “Short-term hypotonic treatment provides a practical strategy to transiently weaken membranes while maintaining intracellular organization for cryo-EM applications and keeping cells in a vital state.”
    Quote in the paperSubstantial claim
    • Rests on an assumption: Hypotonic treatment increases membrane tension, which is necessary for membrane rupture and high-contrast imaging of intracellular components by cryo-EM.

  • May combine two claims.

    Interpretationreasoning

    The Ghost Neuron approach provides two complementary structural readouts within a single specimen: a continuous cytoskeletal scaffold for topological mapping and a dispersed pool of endogenous assemblies amenable to high-resolution SPA.

    “Thus, the Ghost Neuron approach provides two complementary structural readouts within a single specimen: a continuous cytoskeletal scaffold for topological mapping and a dispersed pool of endogenous assemblies amenable to high-resolution SPA.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Study design and conductown data

    Independent classification runs yielded highly consistent class assignments (>90% consistency), indicating robust separation of the two states.

    “Independent classification runs yielded highly consistent class assignments ( > 90% consistency) (Fig. S8), indicating robust separation of the two states.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Study design and conductown data

    Decorated filaments were initially recognizable by increased diameter, and this visual distinction was validated by 2D classification separating decorated from undecorated particles.

    “These filaments were initially recognizable in the images by their characteristic increased diameter (Fig. 6C and Fig. S6), and this visual distinction was subsequently validated by 2D classification of the filament segments, which separated decorated from undecorated particles.”
    Quote in the paperSubstantial claim

  • Probably not a substantial claim: may not pass S4 (based). May combine two claims.

    Premisenone given

    Classical unroofing or detergent-based extraction methods typically require chemical fixation or metal coating prior to imaging to preserve subcellular architecture and therefore are incompatible with molecular-resolution cryo-EM of hydrated specimens.

    “In contrast, classical unroofing or detergent-based extraction methods typically require chemical fixation or metal coating prior to imaging to preserve subcellular architecture and therefore, are incompatible with molecular-resolution cryo-EM of hydrated specimens.”
    Quote in the paperNot a substantial claim

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

    Interpretationnone given

    Differences in neuronal origin, developmental stage, sampled axonal region, or analysis strategy may influence the extent by which compact lattice segments are detected.

    “Differences in neuronal origin, developmental stage, sampled axonal region, or analysis strategy may influence the extent by which compact lattice segments are detected.”
    Quote in the paperNot a substantial claim

  • May combine two claims.

    Study design and conductown data

    Axonal identity of neurites was validated by measuring the angular distribution of microtubules relative to neurite direction, which follows a plus-end-out alignment with angles less than 90°.

    “We validated axonal identity of the neurites by measuring the angular distribution of microtubules relative to neurite direction which follows a plus-end-out alignment with angles of less than 90˚ (Fig. S7B).”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Interpretationown data

    We interpret the expanded and compact segments identified here as local structural states of axonal microtubules, though molecular determinants and functional consequences remain to be established.

    “Thus, we interpret the expanded and compact segments identified here as local structural states of axonal microtubules, though molecular determinants and functional consequences remain to be established.”
    Quote in the paperSubstantial claim

  • Probably not a substantial claim: may not pass S2 (checkable).

    Interpretationown data

    These observations highlight the strength of the Ghost Neuron approach in uncovering molecular architectures within neurons.

    “Altogether, these observations highlight the strength of the Ghost Neuron approach in uncovering molecular architectures within neurons.”
    Quote in the paperNot a substantial claim

  • May combine two claims.

    Interpretationown data

    These observations indicate the presence of heterogeneous lattice conformations along individual microtubules, which might contribute to regional specialization of axonal microtubules, although molecular origin and functional consequence remain to be determined.

    “These observations indicate the presence of heterogeneous lattice conformations along individual microtubules, which might contribute to the regional specialization of axonal microtubules (Fig. 8 and Fig. S7C), although the molecular origin and functional consequence remain to be determined.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Interpretationown data

    Secondary structure assignment and backbone tracing of the decorated actin filaments revealed cofilin as the binding partner, consistent with the available structure of a cofilin-actin complex (PDB 6vao) with a correlation coefficient of the fit of 0.97.

    “Secondary structure assignment and backbone tracing of the decorated actin filaments revealed cofilin as the binding partner, consistent with the available structure a cofilin-actin complex (PDB 6vao) with a correlation coefficient of the fit of 0.97.”
    Quote in the paperSubstantial claim0 of 1 sources checked
    • cites PDB 6vao

      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

    The F-actin reconstruction was deposited under EMD-76718 with coordinates PDB 12rv (β-actin).

    “The F-actin reconstruction was deposited under EMD-76718 with coordinates PDB 12rv [β-actin].”
    Quote in the paperNot a substantial claim

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

    Study design and conductown data

    The cryo-EM reconstructions of microtubules were deposited in the Electron Microscopy database (EMDB) under accession codes EMD-76716 (compact) and EMD-76717 (expanded).

    “The cryo-EM reconstructions of microtubules were deposited in the Electron Microscopy database (EMDB) under accession codes EMD-76716 [compact, https://www.ebi.ac.uk/pdbe/entry/emdb/EMD-76716] and EMD-76717 [expanded, https://www.ebi.ac.uk/pdbe/entry/emdb/EMD-76717].”
    Quote in the paperNot a substantial claim

  • Classified as probably not a substantial claim.

    Interpretationreasoning

    By improving contrast and allowing access to otherwise difficult-to-visualize compartments, the Ghost Neuron platform expands the capabilities of cellular structural biology.

    “By improving contrast and allowing access to otherwise difficult-to-visualize compartments, the Ghost Neuron platform expands the capabilities of cellular structural biology.”
    Quote in the paperSubstantial claim

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

    Premisecitation

    Mus musculus cytoplasmic β-actin (Uniprot P60710) is the major neuronal actin isoform together with γ-actin, and they only differ by four amino acids at the N-terminus.

    “The protein sequence was adjusted to Mus musculus cytoplasmic β-actin (Uniprot P60710) during the refinement process, which is the major neuronal actin isoform together with γ-actin, and they only differ by four amino acids at the N-terminus.”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites Uniprot P60710

      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

    Source data are provided with this paper.

    “Source data are provided with this paper.”
    Quote in the paperNot a substantial claim

  • May combine two claims.

    Study design and conductown data

    The final model for the expanded lattice had a MolProbity score of 1.65 with a correlation coefficient CC(mask) of 0.77 and an FSCmodel (masked) of 0.5 at 3.93 Å and of 0.143 at 3.53 Å.

    “The final model for the expanded lattice had a MolProbity score of 1.65 with a correlation coefficient CC(mask) of 0.77 and an FSCmodel (masked) of 0.5 at 3.93 Å and of 0.143 at 3.53 Å.”
    Quote in the paperSubstantial claim

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

    Interpretationnone given

    The Ghost Neuron approach can be applied not only to primary neuronal tissues, but also to various neuronal cell types including iPSC-derived neurons, as well as those derived from genetically modified models such as knockout mice, disease models, and neurons expressing fluorescent markers.

    “The Ghost Neuron approach can be applied not only to primary neuronal tissues, but also to various neuronal cell types including iPSC-derived neurons, as well as those derived from genetically modified models such as knockout mice, disease models, and neurons expressing fluorescent markers.”
    Quote in the paperNot a substantial claim

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

    Study design and conductnone given

    The cofilactin reconstruction (cofilin-decorated F-actin) was deposited under EMD-71056.

    “The cofilactin reconstruction (cofilin-decorated F-actin) was deposited under EMD-71056.”
    Quote in the paperNot a substantial claim

  • May combine two claims.

    Study design and conductown data

    94.8-96.6% of particles were assigned to the same class in each of the other five runs, indicating high reproducibility of classification.

    “94.8–96.6% of particles were assigned to the same class in each of the other five runs, indicating high reproducibility of classification (Fig. S8).”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Study design and conductown data

    The final actin model had a MolProbity score of 1.32 with a correlation coefficient CC(mask) of 0.87 and an FSC(model, masked) of 0.5 at 3.48 Å and of 0.143 at 3.19 Å.

    “The final model had a MolProbity score of 1.32 with a correlation coefficient CC(mask) of 0.87 and an FSC(model, masked) of 0.5 at 3.48 Å and of 0.143 at 3.19 Å.”
    Quote in the paperNot a substantial claim

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

    Study design and conductnone given

    All structures for which spatial mapping is shown in this study were derived from regions with corresponding montages.

    “All structures for which spatial mapping is shown in this study were derived from regions with corresponding montages.”
    Quote in the paperNot a substantial claim

  • May combine two claims.

    Interpretationreasoning

    Although the 1.9 Å difference between the two lattice repeats is smaller than the nominal resolution of the maps, the measurement reflects a systematic shift in the axial repeat measured across equivalent tubulin heterodimers within classified reconstructions.

    “Although the 1.9 Å difference between the two lattice repeats is smaller than the nominal resolution of the maps, this measurement does not rely on resolving two isolated densities separated by 1.9 Å but, it reflects a systematic shift in the axial repeat measured across equivalent tubulin heterodimers within classified reconstructions.”
    Quote in the paperSubstantial claim

  • Classified as probably not a substantial claim.

    Study design and conductown data

    The inter-dimer spacing reported for each lattice state is a relative distance measured from atomic models fit into two independently classified and refined reconstructions, each representing an average over individually aligned segments.

    “The inter-dimer spacing reported for each lattice state (Fig. 7D) is a relative distance measured from atomic models fit into two independently classified and refined reconstructions, each representing an average over individually aligned segments (48,459 for the expanded class, 30,423 for the compact class).”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Study design and conductown data

    The final model for the compact lattice had a MolProbity score of 1.75 with a correlation coefficient CC(mask) of 0.78 and an FSCmodel (masked) of 0.5 at 4.30 Å and of 0.143 at 3.79 Å.

    “The final model for the compact lattice had a MolProbity score of 1.75 with a correlation coefficient CC(mask) of 0.78 and an FSCmodel (masked) of 0.5 at 4.30 Å and of 0.143 at 3.79 Å.”
    Quote in the paperSubstantial claim

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

    Study design and conductnone given

    The corresponding microtubule coordinates were deposited in the Protein Data Bank (PDB) under PDB 12rt (compact) and PDB 12ru (expanded).

    “The corresponding microtubule coordinates were deposited in the Protein Data Bank (PDB) under PDB 12rt [compact] and PDB 12ru [expanded].”
    Quote in the paperNot a substantial claim

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

    Study design and conductnone given

    All experiments and observations were conducted and analyzed using at least three biologically independent replicates.

    “All experiments and observations were conducted and analyzed using at least three biologically independent replicates.”
    Quote in the paperNot a substantial claim
84 of 84

  • The paper relies on this without support.

    Hypotonic treatment increases membrane tension, which is necessary for membrane rupture and high-contrast imaging of intracellular components by cryo-EM.

    “Incubation of neuronal cultures in hypotonic media was used to increase membrane tension, a step necessary to perform membrane rupture and high-contrast imaging of intracellular components by cryo-EM.”

  • The paper relies on this without support.

    The Ghost Neuron preparation (hypotonic treatment and blotting) preserves the native intracellular architecture sufficiently for structural mapping.

    “To mimic cryo-EM specimen preparation, the grid medium was replaced with hypotonic media containing MemGlow 560 (1:5000 dilution) and 500 nM fluorescently labeled Epothilone-B for at least 10 min, and the grids were imaged by Differential Interference Contrast (DIC) and fluorescence methods.”

  • The paper relies on this without support.

    Fluorescently labeled Epothilone-B39 specifically binds microtubules in membrane-compromised axons.

    “To visualize microtubules in membrane-compromised axons, we used 500 nM fluorescently labeled Epothilone-B39.”

  • The paper relies on this without support.

    MemGlow and MitoTracker dyes do not affect mitochondrial transport or morphology.

    “To visualize the plasma membrane and mitochondria, we used 2 nM MemGlow 488 (Cytoskeleton #MG01) or 2 nM MitoTracker Red CMXRos (Thermo Fisher Scientific #M7512) for 1 min, respectively.”
4 of 4

Self-check

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

  • Fig. 3 ANOVA degrees of freedom do not match the stated sample size

    Minor

    The caption specifies five groups-the control and four hypotonic conditions-with three data points per group. A one-way ANOVA on those 15 observations has 15 − 5 = 10 residual degrees of freedom, not 15.

    Reported 15 · recomputed 10

  • Fig. 2 ANOVA degrees of freedom do not match the stated sample size

    Minor

    The caption specifies four groups-the control and three hypotonic conditions-with three data points per group. A one-way ANOVA on those 12 observations has 12 − 4 = 8 residual degrees of freedom, not 12.

    Reported 12 · recomputed 8

2 of 2

Cites

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Related

Authors
Bodakuntla, S., Marelli, J., Vasquez-Montes, V., Glauser, S., Biertümpfel, C., & Mizuno, N.
Affiliations
  • National Institutes of Health (US)
  • ETH Zurich (CH)
Corresponding author
Mizuno, Naoko
ORCID
Venue
Nature Communications
Publisher
Springer Science and Business Media LLC
Published
2026-09-23
Volume
17
Issue
1
ISSN
2041-1723 (ISSN-L)
DOI
10.1038/s41467-026-77741-4
PMC
PMC13690744
OpenAlex
W7214077015
Cited by
0
References
90
FWCI
0.00
Type
journal article
Language
en
Open access
Licence
Locations
  • Nature Communications (publishedVersion, cc-by)
  • PubMed (publishedVersion)
  • PubMed Central (publishedVersion, cc-by)
  • Research Explorer (The University of Manchester) (publishedVersion)
  • Repository for Publications and Research Data (ETH Zurich) (publishedVersion, cc-by)
  • DOAJ (DOAJ: Directory of Open Access Journals) (submittedVersion)
  • ETH Zürich Research Collection (cc-by)
Funders
U.S. Department of Health & Human Services | NIH | National Heart, Lung, and Blood Institute (1ZIAHL006264)
Topics
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Structural Biology › Advanced Electron Microscopy Techniques and Applications
  • Physical Sciences › Materials Science › Materials Chemistry › Enzyme Structure and Function
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Cell Biology › Microtubule and mitosis dynamics
Keywords
axon ultrastructure, hypotonic treatment, mechanical rupture, F-actin, cofilin, cryo-EM sample preparation
MeSH terms
Animals, Axons, Actin Cytoskeleton, Microtubules, Neurons, Neurites, Cryoelectron Microscopy

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 2026-11-02.

Classified by jev-1.13-20260917 (gold-guideline-v0.2) on 2026-11-02.

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Statistics and arithmetic are recomputed, not judged.

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