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Cited by 26Microtubule and mitosis dynamicsCancer Treatment and PharmacologyCellular Mechanics and Interactions

StableMARK-decorated microtubules in cells have expanded lattices

Open access
de Jager, Leanne, Jansen, Klara I., Hoogebeen, Robin, Akhmanova, Anna, Kapitein, Lukas C., Förster, Friedrich, Howes, Stuart C.·2025·Journal of Cell Biology
DOI 10.1083/jcb.202206143PMC PMC11471893

Published version of a preprint

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Abstract

Microtubules are crucial in cells and are regulated by various mechanisms like posttranslational modifications, microtubule-associated proteins, and tubulin isoforms. Recently, the conformation of the microtubule lattice has also emerged as a potential regulatory factor, but it has remained unclear to what extent different lattices co-exist within the cell. Using cryo-electron tomography, we find that, while most microtubules have a compacted lattice (∼41 Å monomer spacing), approximately a quarter of the microtubules displayed more expanded lattice spacings. The addition of the microtubule-stabilizing agent Taxol increased the lattice spacing of all microtubules, consistent with results on reconstituted microtubules. Furthermore, correlative cryo-light and electron microscopy revealed that the stable subset of microtubules labeled by StableMARK, a marker for stable microtubules, predominantly displayed a more expanded lattice spacing (∼41.9 Å), further suggesting a close connection between lattice expansion and microtubule stability. The coexistence of different lattices and their correlation with stability implicate lattice spacing as an important factor in establishing specific microtubule subsets.

AI snapshot

Written from the paper’s full text.

TL;DR

Cryo-electron tomography with in situ Fourier layer-line analysis shows that microtubule lattice spacings vary within U2OS cells, with compacted and expanded lattices coexisting. Taxol treatment hyper-expands cellular microtubules, and StableMARK-positive stable microtubules are predominantly expanded relative to untreated microtubules.

Key findings

  • In untreated U2OS cells, 74% (23/31) of microtubules had a compacted average monomer spacing of 40.8–41.1 Å; the remaining microtubules had expanded spacing of 41.5–42.7 Å.
  • Taxol treatment (1 µM, 16 h) shifted most microtubules to an expanded lattice: 97% (29/30) measured 42.3–43.2 Å, significantly different from untreated cells (P < 0.0001). The average hyper-expansion was 42.6 Å, larger than the in vitro Taxol measurement of ∼42.2 Å (P < 0.01).
  • StableMARK-bound microtubules showed a clear shift toward an expanded distribution centered around 41.5–41.9 Å (P < 0.0001, N = 43), significantly different from untreated microtubules. Nearby undecorated microtubules were compacted and differed from the StableMARK distribution (P < 0.01, N = 12, 10 tomograms).
  • Taxol-treated microtubules averaged 13 protofilaments, with an approximated helical rise of 9.82 Å corresponding to a monomer spacing of 42.5 Å, at ∼25 Å resolution.

Methods

  • U2OS wild-type and StableMARK-expressing Flp-In T-Rex cells were grown on EM grids, vitrified, cryo-FIB milled into ∼150-nm lamellae, and imaged by cryo-ET; in vitro dynamic, Taxol-stabilized, and GMPCPP-bound microtubules served as technical controls.
  • Lattice spacing was measured by layer-line Fourier analysis of masked and aligned microtubule segments using a 1,030-pixel box size (224-nm edge length); subtomogram averaging was performed in Relion for the Taxol dataset.
  • A two-step cryo-CLEM workflow used StableMARK-NeonGreen and fBSA-Au5 fiducials with either an integrated Meteor cryo-FM or a separate CorrSight cryo-FM; lattice spacing distributions were compared with unpaired two-tailed t test–based permutation tests (10,000 iterations).

Limitations

  • Stated: limited cryo-FM resolution means a proportion of StableMARK-bound microtubules may have been identified incorrectly, potentially contributing to the spread in lattice spacings; very low decoration of undecorated microtubules cannot be excluded due to limited cryo-FM sensitivity.
  • Evident: the study is limited to U2OS cells and specific Taxol/StableMARK conditions, so generalization to other cell types or physiological contexts is not established.
  • Evident: lattice measurements are from microtubule shafts, with no microtubule ends observed in the ∼0.8-μm field of view, so tip-specific lattice states are not assessed; the in situ subtomogram average resolution is limited to ∼25 Å.

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

Integrity signals

Registry

Crossref · 10.1083/jcb.202206143

Cites

80 of 80 references verified0 citation errors · 0 not checkable

Statistics

Nothing to recompute.

Claims the paper makes

19 of 22 cited sources checked · 14 supported · 0 flagged · accuracy not yet measured

  • Premisecitation

    StableMARK preferentially binds to a subset of stable microtubules that are often highly acetylated and less dynamic.

    “we used a kinesin-1 rigor construct, StableMARK (Jansen et al., 2023), which preferentially binds to a subset of stable microtubules that are often highly acetylated and less dynamic.”
    Quote in the paperSubstantial claim1 of 1 sources checked
    • cites Jansen et al., 2023 source
  • Findingown data

    The FM-SEM localization errors are lower than previously reported SEM localization errors (SD of 225 nm in x and 341 nm in y).

    “This is lower than previously reported SEM localization errors (SD of 225 nm in x and 341 nm in y), which were based on a workflow similar to our CorrSight workflow (Arnold et al., 2016).”
    Quote in the paperNot a substantial claim1 of 1 sources checked
    • cites Arnold et al., 2016 source
  • Interpretationown data

    The finding that stable cellular microtubules display expanded lattices directly supports the emerging model that microtubule lattice geometry functions as an additional cellular mechanism to guide protein binding specificity, on top of the previously established elements of the tubulin code.

    “Our finding that stable cellular microtubules display expanded lattices now directly supports the emerging model that the microtubule lattice geometry functions as an additional cellular mechanism to guide protein binding specificity, on top of the previously established elements of the tubulin code”
    Quote in the paperSubstantial claim1 of 3 sources checked
    • cites Akhmanova and Kapitein, 2022 source
    • cites Cross, 2019 source
    • cites Iwanski and Kapitein, 2023 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
  • Findingown data

    The Taxol-induced expansion observed in vitro (41.2-42.0 Å) is considerably smaller than the average hyper-expansion of 42.6 Å measured in situ.

    “Remarkably, the Taxol-induced expansion to 41.2–42.0 Å observed in vitro (Alushin et al., 2014; Estevez-Gallego et al., 2020; Kellogg et al., 2017; Rai et al., 2020; Vale et al., 1994) is considerably smaller than the average hyper-expansion of 42.6 Å measured using our in situ approach.”
    Quote in the paperSubstantial claim3 of 3 sources checked
    • cites Alushin et al., 2014 source
    • cites Estevez-Gallego et al., 2020 source
    • cites Kellogg et al., 2017 source
  • Findingown data

    In U2OS cells treated with 1 μM Taxol for 16 h, most microtubules (97%, 29/30) had an expanded lattice spacing of 42.3-43.2 Å.

    “Indeed, in U2OS cells treated with 1 µM Taxol for 16 h, we observed microtubules with typical morphology (Fig. 2 A) but found that most microtubules (97%, 29/30) had an expanded lattice spacing of 42.3–43.2 Å (Fig. 2 B)”
    Quote in the paperSubstantial claim
  • Classified as probably not a substantial claim. 19
  • The quote only partly states this claim. 1
  • May combine two claims. 5

  • Probably not a substantial claim: may not pass S3 (load-bearing). The quote only partly states this claim.

    Study design and conductcitation

    The accuracy of the correlation was measured by leave-one-out calculations as described by Schorb and Briggs (2014).

    “Leave-one-out calculations were performed as described by Schorb and Briggs (2014).”
    • Also stated in: § Statistical analyses · plain
      “The deviation of the predicted bead position from the true bead position was used to calculate the standard deviation of the errors, which was used as an accuracy measurement.”
    Quote in the paperNot a substantial claim1 of 1 sources checked
    • cites Schorb and Briggs (2014) source

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

    Study design and conductown data

    The 13-protofilament assignment of the initial average was confirmed by overlaying and cross-correlating it with the six references (11-16 protofilaments) from the Microtubule RELION-based Pipeline (MiRP).

    “This was confirmed by overlaying and cross-correlating the average with the six references (11–16 PFs) from the Microtubule RELION-based Pipeline (MiRP) (Cook et al., 2020).”
    Quote in the paperNot a substantial claim1 of 1 sources checked
    • cites Cook et al., 2020 source

  • Classified as probably not a substantial claim.

    Premisecitation

    The GTP-bound state is thought to have an expanded lattice spacing of ∼42 Å.

    “The GTP-bound state is thought to have an expanded lattice spacing of ∼42 Å as determined for hydrolysis-deficient mutants and the slowly hydrolysable GTP analogue guanylyl-(α,β)-methylene-diphosphonate (GMPCPP) (Hyman et al., 1995; LaFrance et al., 2022).”
    Quote in the paperSubstantial claim2 of 2 sources checked
    • cites Hyman et al., 1995 source
    • cites LaFrance et al., 2022 source

  • May combine two claims.

    Findingown data

    In vitro, dynamic microtubules have predominantly compacted lattice spacing, while Taxol- and GMPCPP-bound microtubules have expanded lattice spacing.

    “Our analysis recovered the expected lattice spacings, namely predominantly compacted for dynamic microtubules, and expanded for Taxol- and GMPCPP-bound microtubules, verifying our approach (Fig. 1 E).”
    • Also stated in: § Results and discussion · plain
      “A separate in vitro preparation protocol for Taxol and GMPCPP lattices, assembled from tubulin without any fluorophore or biotin labels, similarly yielded expanded microtubules (Fig. S3, D–F) (Estevez-Gallego et al., 2020; Vale et al., 1994).”
    Quote in the paperSubstantial claim2 of 2 sources checked
    • cites Estevez-Gallego et al., 2020 source
    • cites Vale et al., 1994 source

      The Structural Features section (page unavailable) reports a shorter mean axial repeat for GDP microtubules without Taxol (40.74 Å) than for Taxol-treated GDP microtubules (42.02 Å) or GMPCPP microtubules (41.93 Å without Taxol). This supports the compact-versus-expanded comparison, but the paper does not identify the measured untreated GDP microtubules as dynamic or establish that dynamic microtubules predominantly have compacted spacing. (LLM, conf=0.83)

      Experimental — accuracy not yet measured

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

    Premisecitation

    Various microtubule-binding proteins have higher affinities for expanded or compacted lattices in vitro and can induce lattice expansion or compaction, respectively.

    “various microtubule-binding proteins have higher affinities for expanded or compacted lattices in vitro and can also induce lattice expansion or compaction, respectively (Ferro et al., 2022; Guedes-Dias et al., 2019; Peet et al., 2018; Shima et al., 2018; Watanabe et al., 2020; Zhang et al., 2017)”
    Quote in the paperSubstantial claim3 of 3 sources checked
    • cites Ferro et al., 2022 source

      The paper’s structural discussion (indexed as Abstract; page unavailable) describes where MAP7 binds on microtubules, and its motility experiments report MAP7 binding affinity. Neither addresses preferential binding to expanded versus compacted lattices or MAP7-induced lattice expansion or compaction. Searches for “expanded,” “compacted,” and lattice-spacing terms found no passage establishing the attributed point. (LLM, conf=0.74)

      Experimental — accuracy not yet measured
    • cites Guedes-Dias et al., 2019 source

      The Results section reports that KIF1A binds more strongly to GDP-taxol-stabilized than to GMPCPP-stabilized microtubules in vitro. The Discussion describes GDP-like lattices as compacted and GTP-like lattices as expanded. Neither section shows that KIF1A, or another binding protein studied here, induces lattice compaction or expansion. (LLM, conf=0.88)

      Experimental — accuracy not yet measured
    • cites Peet et al., 2018 source

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

    Premisecitation

    Microexpansion joints increase lamellae stability.

    “Microexpansion joints were added during the first step to increase lamellae stability (Wolff et al., 2019).”
    Quote in the paperNot a substantial claim1 of 1 sources checked
    • cites Wolff et al., 2019 source

  • Classified as probably not a substantial claim.

    Premisecitation

    Taxol can alter the lattice spacing of microtubules in vitro.

    “drugs like the anti-cancer drug Taxol (paclitaxel) can alter the lattice spacing of the microtubule in vitro (Kellogg et al., 2017).”
    Quote in the paperSubstantial claim1 of 1 sources checked
    • cites Kellogg et al., 2017 source

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

    Interpretationcitation

    The different lattice spacings observed in the microtubule shaft likely play a role in microtubule stability, MAP binding, and motor protein kinetics.

    “the different lattice spacings that we observe in the shaft of the microtubule likely play a role in microtubule stability, MAP binding, and motor protein kinetics, as has been previously postulated”
    Quote in the paperNot a substantial claim1 of 2 sources checked
    • cites Cross, 2019 source
    • cites Zhang et al., 2018 source

      The Discussion (page not available in our copy) proposes that lattice rearrangements affect binding-partner affinity and microtubule dynamics, supporting the proposed roles in MAP binding and stability. It does not say that lattice spacing affects motor protein kinetics; its discussion of kinesin concerns changes in lattice spacing upon binding, not motor kinetics. (LLM, conf=0.85)

      Experimental — accuracy not yet measured

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

    Premisecitation

    The GDP-bound state makes up the bulk of the microtubule shaft and is generally characterized by a compacted average monomer spacing of ∼41 Å.

    “This GDP-bound state, which makes up the bulk of the microtubule shaft, generally is characterized by a compacted average monomer spacing of ∼41 Å that is distinct from that of the presumed GTP-bound state at the tip (the GTP-cap).”
    Quote in the paperNot a substantial claim1 of 1 sources checked
    • cites Alushin et al., 2014 source

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

    Findingown data

    FM-FIB correlation did not add significant information when tested.

    “An FM-FIB correlation was not included in our workflow as this is a time-intensive procedure and the limited z-resolution of our cryo-FM setup meant this correlation did not add significant information when tested.”
    Quote in the paperNot a substantial claim

  • May combine two claims.

    Findingown data

    Undecorated microtubules near positively identified StableMARK-bound microtubules had compacted lattices, with a distribution significantly different from the StableMARK distribution (P < 0.01, N = 12, 10 tomograms).

    “For undecorated microtubules near the positively identified StableMARK-bound microtubules, we observed compacted lattices (for example, Fig. S7, A–E) and obtained a distribution significantly different from the StableMARK distribution (P value <0.01, N = 12, 10 tomograms, Fig. S7 F)”
    Quote in the paperSubstantial claim

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

    Findingown data

    An initial 3D refinement using a hollow tube as a starting reference yielded an average with 13 protofilaments.

    “An initial 3D refinement, using a hollow tube (Fig. S4 A) as a starting reference, yielded an average with 13 PFs.”
    Quote in the paperNot a substantial claim

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

    Findingown data

    A final 3D refinement using optimized rise and twist values to apply helical symmetry yielded an average with a resolution of ∼25 Å.

    “A final 3D refinement was performed where the optimized rise (−27.7°) and twist (9.82 Å) values were used to apply helical symmetry. This yielded an average with a resolution of ∼25 Å.”
    Quote in the paperNot a substantial claim

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

    Findingown data

    The average scaling factor calculated from 10 lamella sites was 0.584 ± 0.005.

    “This yielded an average scaling factor of 0.584 ± 0.005.”
    Quote in the paperNot a substantial claim

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

    Findingown data

    Attempts to sort subtomograms into different protofilament classes using two, three, or six classes yielded misaligned averages, identical classes, or all particles going to a single, 13-protofilament class.

    “Attempts to sort subtomograms into different PF classes using two, three, or six classes yielded misaligned averages, identical classes, or all particles going to a single, 13 PF class.”
    Quote in the paperNot a substantial claim

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

    Interpretationreasoning

    The lower localization errors are most likely due to the increased number of beads used for calculating the transform (seven to nine instead of six).

    “This is most likely due to the increased number of beads we used for calculating the transform (seven to nine instead of six).”
    Quote in the paperNot a substantial claim

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

    Study design and conductnone given

    Cells were confirmed to be free of mycoplasma.

    “Cells were confirmed to be free of mycoplasma.”
    Quote in the paperNot a substantial claim

  • Classified as probably not a substantial claim.

    Study design and conductown data

    P values for comparison of the different lattice spacing distributions were calculated with an unpaired two-tailed t test-based permutation test with 10,000 iterations.

    “P values for comparison of the different lattice spacing distributions were calculated with an unpaired two-tailed t test–based permutation test with 10,000 iterations.”
    Quote in the paperSubstantial claim

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

    Interpretationreasoning

    This fractional misassignment might contribute to the large spread observed in lattice spacings for the StableMARK-bound microtubule subset.

    “This fractional misassignment might contribute to the large spread observed in lattice spacings for the StableMARK-bound microtubule subset.”
    Quote in the paperNot a substantial claim

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

    Study design and conductreasoning

    A proportion of the StableMARK-bound microtubules may have been identified incorrectly due to the limited resolution of the cryo-FM setup.

    “we cannot exclude that a proportion of the StableMARK-bound microtubules were identified incorrectly due to the limited resolution of our cryo-FM setup.”
    Quote in the paperNot a substantial claim

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

    Study design and conductnone given

    The microtubules in the lamellae of untreated and Taxol-treated cells were chosen at random.

    “The microtubules in the lamellae of untreated and Taxol-treated cells were chosen at random.”
    Quote in the paperNot a substantial claim
21 of 21

  • The paper relies on this without support.

    The microtubule lattice spacing in situ had never been measured directly before this study.

    “However, the microtubule lattice spacing in situ has never been measured directly and its potential to act as a layer of regulation within cells therefore remains uncertain.”

  • The paper relies on this without support.

    The potential of microtubule lattice spacing to act as a layer of regulation within cells remained uncertain before this study.

    “However, the microtubule lattice spacing in situ has never been measured directly and its potential to act as a layer of regulation within cells therefore remains uncertain.”

  • The paper relies on this without support.

    The SEM and FM imaging systems are at a fixed position with respect to each other.

    “FM images could simply be scaled with a predetermined scaling factor, as the SEM and FM imaging systems are at a fixed position with respect to each other.”
3 of 3

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?

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.

  • Particle box width is reported in Å instead of nm

    Minor

    At 2.17 Å per pixel, a 1,030-pixel box is 2,235.1 Å wide, not 224 Å. The paper elsewhere describes the same box as approximately 224 nm wide.

    Reported 224 · recomputed 2235.1

  • StableMARK tomogram count differs between results and figure

    Minor

    The results give 12 tomograms for the 43 StableMARK-bound microtubules, whereas Fig. 4 J gives 25 tomograms for the same 43 microtubules.

  • Helical rise and twist labels are reversed

    Minor

    The final refinement assigns an angle to rise and a length to twist, reversing the assignments used for those parameters immediately beforehand.

3 of 3

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Related

Authors
de Jager, L., Jansen, K. I., Hoogebeen, R., Akhmanova, A., Kapitein, L. C., Förster, F., & Howes, S. C.
Affiliations
  • Bijvoet Centre for Biomolecular Research, Utrecht University 1 Structural Biochemistry, Department of Chemistry, , Utrecht,
  • Utrecht University (NL)
Corresponding author
  • Kapitein, Lukas C.
  • Förster, Friedrich
  • Howes, Stuart C.
ORCID
Venue
Journal of Cell Biology
Publisher
Rockefeller University Press
Published
2024-10-10
Volume
224
Issue
1
ISSN
  • 0021-9525 (ISSN-L)
  • 1540-8140
DOI
10.1083/jcb.202206143
PMC
PMC11471893
OpenAlex
W4403284162
Semantic Scholar
8711822ece3879d82731d62d6f57e2d71715d455
Cited by
25
References
80
Citations by year
2026: 7 · 2025: 7 · 2024: 4 · 2023: 6 · 2022: 1
FWCI
4.03
Type
journal article
Language
en
Open access
Licence
Locations
  • The Journal of Cell Biology (publishedVersion, cc-by)
  • bioRxiv (Cold Spring Harbor Laboratory) (acceptedVersion, cc-by-nc-nd)
  • PubMed Central (publishedVersion, cc-by)
  • PubMed (publishedVersion)
  • Utrecht University Repository (Utrecht University) (submittedVersion, other-oa)
Funders
  • European Research Council (724425, 819219)
  • Dutch Research Council (184.034.014, 022.006.001, OCENW.XL21.XL21.048)
  • Netherlands Electron Microscopy Infrastructure (84.034.014)
Topics
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Cell Biology › Microtubule and mitosis dynamics
  • Health Sciences › Medicine › Oncology › Cancer Treatment and Pharmacology
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Cell Biology › Cellular Mechanics and Interactions
Keywords
microtubule lattice, lattice spacing, microtubule stability, paclitaxel, microtubule-associated proteins, microtubule dynamics, cryo-electron tomography
MeSH terms
Animals, Humans, Microtubule-Associated Proteins, Microtubules, Tubulin, Paclitaxel, Cryoelectron Microscopy, Electron Microscope Tomography

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