Accessibility of the unstructured α-tubulin C-terminal tail is controlled by microtubule lattice conformation
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eLife Assessment
This fundamental work reveals that the accessibility of the unstructured C-terminal tails of α- and β-tubulins differs with the state of the microtubule lattice. Their accessibility increases with the expansion of the lattice induced by GTP and certain MAPs, which can then dictate the subsequent interactions between MAPs and microtubules, and post-translational modifications of tubulin tails. The evidence supporting the conclusion is compelling, although the characterisation of the probes does not answer whether they directly affect the lattice or expose the C-terminal tails of tubulin. This work will be of great interest to the cytoskeleton field.
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Abstract
Microtubules are cytoskeletal filaments that self-assemble from the protein tubulin, a heterodimer of α-tubulin and β-tubulin, and are important for cell mechanics, migration, and division. Much work has focused on how the nucleotide state of β-tubulin regulates the structure and dynamics of microtubules. In contrast, less is known about the structure and function of the C-terminal tails (CTTs) of α- and β-tubulin which are thought to freely protrude from the surface of the microtubule. To study the CTTs, we developed three different biosensors that bind the tyrosinated α-tubulin CTT (Y-αCTT) on the microtubule lattice. Surprisingly, live imaging of the probes indicates that the Y-αCTT is not accessible under normal cellular conditions. Lattice binding of the Y-αCTT probes can be increased by three different ways of changing the tubulin conformational state: the drug Taxol, expression of microtubule-associated proteins (MAPs) that recognize or promote an expanded tubulin conformation, or expression of tubulin that cannot hydrolyze GTP. Molecular dynamics simulations indicate that the Y-αCTT undergoes numerous transient interactions with the bodies of α-tubulin and β-tubulin in the lattice, and that the frequency of these interactions is regulated by the tubulin nucleotide state. These findings suggest that accessibility of the Y-αCTT is governed by local nucleotide- and MAP-dependent conformational changes to tubulin subunits within the microtubule lattice.
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eLife Assessment
This fundamental work reveals that the accessibility of the unstructured C-terminal tails of α- and β-tubulins differs with the state of the microtubule lattice. Their accessibility increases with the expansion of the lattice induced by GTP and certain MAPs, which can then dictate the subsequent interactions between MAPs and microtubules, and post-translational modifications of tubulin tails. The evidence supporting the conclusion is compelling, although the characterisation of the probes does not answer whether they directly affect the lattice or expose the C-terminal tails of tubulin. This work will be of great interest to the cytoskeleton field.
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Reviewer #1 (Public review):
Summary:
This is a careful and comprehensive study demonstrating that effector-dependent conformational switching of the MT lattice from compacted to expanded deploys the alpha tubulin C-terminal tails so as to enhance their ability to bind interactors.
Strengths:
The authors use 3 different sensors for the exposure of the alpha CTTs. They show that all 3 sensors report exposure of the alpha CTTs when the lattice is expanded by GMPCPP, or KIF1C, or a hydrolysis-deficient tubulin. They demonstrate that expansion-dependent exposure of the alpha CTTs works in tissue culture cells as well as in vitro.
Weaknesses:
There is no information on the status of the beta tubulin CTTs. The study is done with mixed isotype microtubules, both in cells and in vitro. It remains unclear whether all the alpha tubulins in a …
Reviewer #1 (Public review):
Summary:
This is a careful and comprehensive study demonstrating that effector-dependent conformational switching of the MT lattice from compacted to expanded deploys the alpha tubulin C-terminal tails so as to enhance their ability to bind interactors.
Strengths:
The authors use 3 different sensors for the exposure of the alpha CTTs. They show that all 3 sensors report exposure of the alpha CTTs when the lattice is expanded by GMPCPP, or KIF1C, or a hydrolysis-deficient tubulin. They demonstrate that expansion-dependent exposure of the alpha CTTs works in tissue culture cells as well as in vitro.
Weaknesses:
There is no information on the status of the beta tubulin CTTs. The study is done with mixed isotype microtubules, both in cells and in vitro. It remains unclear whether all the alpha tubulins in a mixed isotype microtubule lattice behave equivalently, or whether the effect is tubulin isotype-dependent. It remains unclear whether local binding of effectors can locally expand the lattice and locally expose the alpha CTTs.
Appraisal:
The authors have gone to considerable lengths to test their hypothesis that microtubule expansion favours deployment of the alpha tubulin C-terminal tail, allowing its interactors, including detyrosinase enzymes, to bind. There is a real prospect that this will change thinking in the field. One very interesting possibility, touched on by the authors, is that the requirement for MAP7 to engage kinesin with the MT might include a direct effect of MAP7 on lattice expansion.
Impact:
The possibility that the interactions of MAPS and motors with a particular MT or region feed forward to determine its future interaction patterns is made much more real. Genuinely exciting.
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Reviewer #2 (Public review):
The unstructured α- and β-tubulin C-terminal tails (CTTs), which differ between tubulin isoforms, extend from the surface of the microtubule, are post-translationally modified, and help regulate the function of MAPs and motors. Their dynamics and extent of interactions with the microtubule lattice are not well understood. Hotta et al. explore this using a set of three distinct probes that bind to the CTTs of tyrosinated (native) α-tubulin. Under normal cellular conditions, these probes associate with microtubules only to a limited extent, but this binding can be enhanced by various manipulations thought to alter the tubulin lattice conformation (expanded or compact). These include small-molecule treatment (Taxol), changes in nucleotide state, and the binding of microtubule-associated proteins and motors. …
Reviewer #2 (Public review):
The unstructured α- and β-tubulin C-terminal tails (CTTs), which differ between tubulin isoforms, extend from the surface of the microtubule, are post-translationally modified, and help regulate the function of MAPs and motors. Their dynamics and extent of interactions with the microtubule lattice are not well understood. Hotta et al. explore this using a set of three distinct probes that bind to the CTTs of tyrosinated (native) α-tubulin. Under normal cellular conditions, these probes associate with microtubules only to a limited extent, but this binding can be enhanced by various manipulations thought to alter the tubulin lattice conformation (expanded or compact). These include small-molecule treatment (Taxol), changes in nucleotide state, and the binding of microtubule-associated proteins and motors. Overall, the authors conclude that microtubule lattice "expanders" promote probe binding, suggesting that the CTT is generally more accessible under these conditions. Consistent with this, detyrosination is enhanced. Mechanistically, molecular dynamics simulations indicate that the CTT may interact with the microtubule lattice at several sites, and that these interactions are affected by the tubulin nucleotide state.
Strengths:
Key strengths of the work include the use of three distinct probes that yield broadly consistent findings, and a wide variety of experimental manipulations (drugs, motors, MAPs) that collectively support the authors' conclusions, alongside a careful quantitative approach.
Weaknesses:
The challenges of studying the dynamics of a short, intrinsically disordered protein region within the complex environment of the cellular microtubule lattice, amid numerous other binders and regulators, should not be understated. While it is very plausible that the probes report on CTT accessibility as proposed, the possibility of confounding factors (e.g., effects on MAP or motor binding) cannot be ruled out. Sensitivity to the expression level clearly introduces additional complications. Likewise, for each individual "expander" or "compactor" manipulation, one must consider indirect consequences (e.g., masking of binding sites) in addition to direct effects on the lattice; however, this risk is mitigated by the collective observations all pointing in the same direction.
The discussion does a good job of placing the findings in context and acknowledging relevant caveats and limitations. Overall, this study introduces an interesting and provocative concept, well supported by experimental data, and provides a strong foundation for future work. This will be a valuable contribution to the field.
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Reviewer #3 (Public review):
Summary:
In this study, the authors investigate how the structural state of the microtubule lattice influences the accessibility of the α-tubulin C-terminal tail (CTT). By developing and applying new biosensors, they reveal that the tyrosinated CTT is largely inaccessible under normal conditions but becomes more accessible upon changes to the tubulin conformational state induced by taxol treatment, MAP expression, or GTP-hydrolysis-deficient tubulin. The combination of live imaging, biochemical assays, and simulations suggests that the lattice conformation regulates the exposure of the CTT, providing a potential mechanism for modulating interactions with microtubule-associated proteins. The work addresses a highly topical question in the microtubule field and proposes a new conceptual link between lattice …
Reviewer #3 (Public review):
Summary:
In this study, the authors investigate how the structural state of the microtubule lattice influences the accessibility of the α-tubulin C-terminal tail (CTT). By developing and applying new biosensors, they reveal that the tyrosinated CTT is largely inaccessible under normal conditions but becomes more accessible upon changes to the tubulin conformational state induced by taxol treatment, MAP expression, or GTP-hydrolysis-deficient tubulin. The combination of live imaging, biochemical assays, and simulations suggests that the lattice conformation regulates the exposure of the CTT, providing a potential mechanism for modulating interactions with microtubule-associated proteins. The work addresses a highly topical question in the microtubule field and proposes a new conceptual link between lattice spacing and tail accessibility for tubulin post-translational modification.
Strengths:
(1) The study targets a highly relevant and emerging topic-the structural plasticity of the microtubule lattice and its regulatory implications.
(2) The biosensor design represents a methodological advance, enabling direct visualization of CTT accessibility in living cells.
(3) Integration of imaging, biochemical assays, and simulations provides a multi-scale perspective on lattice regulation.
(4) The conceptual framework proposed lattice conformation as a determinant of post-translational modification accessibility is novel and potentially impactful for understanding microtubule regulation.
Weaknesses:
There are a number of weaknesses in the paper, many of which can be addressed textually. Some of the supporting evidence is preliminary and would benefit from additional experimental validation and clearer presentation before the conclusions can be considered fully supported.
In particular, the authors should directly test in vitro whether Taxol addition can induce lattice exchange (see comments below).
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