SSNA1 stabilizes dynamic microtubules and detects microtubule damage
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Evaluation Summary:
In this manuscript, Lawrence et al. investigate the direct effects of the microtubule-associated protein, SSNA1, on microtubule dynamics and damage using purified proteins and TIRF microscopy. The authors conclude that SSNA1 is a microtubule stabilizing protein and a sensor of microtubule damage. This paper is of high interest to scientists within the field of microtubule mechanics and of broad interest to scientists studying cilia, cell division and neuronal development.
(This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. Reviewer #3 agreed to share their name with the authors.)
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Abstract
Sjögren’s syndrome nuclear autoantigen-1 (SSNA1/NA14) is a microtubule-associated protein with important functions in cilia, dividing cells, and developing neurons. However, the direct effects of SSNA1 on microtubules are not known. We employed in vitro reconstitution with purified proteins and TIRF microscopy to investigate the activity of human SSNA1 on dynamic microtubule ends and lattices. Our results show that SSNA1 modulates all parameters of microtubule dynamic instability—slowing down the rates of growth, shrinkage, and catastrophe, and promoting rescue. We find that SSNA1 forms stretches along growing microtubule ends and binds cooperatively to the microtubule lattice. Furthermore, SSNA1 is enriched on microtubule damage sites, occurring both naturally, as well as induced by the microtubule severing enzyme spastin. Finally, SSNA1 binding protects microtubules against spastin’s severing activity. Taken together, our results demonstrate that SSNA1 is both a potent microtubule-stabilizing protein and a novel sensor of microtubule damage; activities that likely underlie SSNA1’s functions on microtubule structures in cells.
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Reviewer #3 (Public Review):
By means of in vitro reconstitution, the authors find that the microtubule associated protein Sjögren's Syndrome Nuclear Autoantigen 1 (SSNA1), know to form fibrils binding longitudinally along microtubules, modulates microtubule instability by reducing dynamicity and inducing rescues, prevents catastrophes in absence of free tubulin or in presence of the tubulin-sequestering protein stathmin, inhibits microtubule severing of spastin and detects spastin-induced damage sites. SSNA1, thus, is revealed as a very potent microtubule stabilizing factor.
The reconstitution of microtubule dynamics is sound and well performed, and the parameters of dynamicity are thoroughly analyzed. The observed intensity of SSNA1 fluorescence demonstrates that the proteins do not bind uniformly along microtubules. Consequently, the …
Reviewer #3 (Public Review):
By means of in vitro reconstitution, the authors find that the microtubule associated protein Sjögren's Syndrome Nuclear Autoantigen 1 (SSNA1), know to form fibrils binding longitudinally along microtubules, modulates microtubule instability by reducing dynamicity and inducing rescues, prevents catastrophes in absence of free tubulin or in presence of the tubulin-sequestering protein stathmin, inhibits microtubule severing of spastin and detects spastin-induced damage sites. SSNA1, thus, is revealed as a very potent microtubule stabilizing factor.
The reconstitution of microtubule dynamics is sound and well performed, and the parameters of dynamicity are thoroughly analyzed. The observed intensity of SSNA1 fluorescence demonstrates that the proteins do not bind uniformly along microtubules. Consequently, the rates of microtubule dynamics are not affected globally. Instead the observed rates are affected at different times for individual microtubules and, importantly, directly correlate with locally accumulating SSNA1. The authors thus validly conclude that nucleotide state recognition is not the primary mechanism of SSNA1 localization and activity. Clues towards the mechanism of SSNA1 activity are provided by the observation that SSNA1 detects spastin-induced damage sites, indicating that SSNA1 binds to partial, open microtubule structures and then stabilizes them, which is consistent with cryo-electron-tomograms available in the literature. To me it is not clear, however, if SSNA1 localize-to and act-on distinct sites of microtubule damage exclusively, or if these sites rather serve as positions of initiation or nucleation of cooperative SSNA1 binding, which the kymographs and movies seem to suggest.
The presented observations nicely explain how the microtubule severing enzyme spastin, which directly interacts with SSNA1 and thus recruits it to the very sites of immediate damage, promotes regrowth of microtubules and increases their number and mass in vivo. The manuscript would benefit from further investigation-into and quantifications-of the "progressive accumulation" of SSNA1 on the dynamic microtubules, which, thus far are presented only by way of representational example.
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Reviewer #2 (Public Review):
The manuscript provides some long awaited follow-up work to a controversial publication implicating SSNA1/NA14 in microtubule branching (Basnet et al. NCB 2018). The authors have strong expertise in in-vitro microtubule dynamic behaviour. While the experiments are technically strong, the authors use unphysiological amounts of the SSNA1, making interpretations about biological function hard.
The authors take a rigorous approach to analyze details of microtubule dynamic behaviour presented in Figure 1. While I recognize the enormous amount of work that went into Figure 1, in my opinion these experiments shows that SSNA1 has no effect on microtubule dynamics at physiological concentration (sub 100 nM). That finding is i) very publishable and ii) should not take away from SSNA1 as an important molecule, but …
Reviewer #2 (Public Review):
The manuscript provides some long awaited follow-up work to a controversial publication implicating SSNA1/NA14 in microtubule branching (Basnet et al. NCB 2018). The authors have strong expertise in in-vitro microtubule dynamic behaviour. While the experiments are technically strong, the authors use unphysiological amounts of the SSNA1, making interpretations about biological function hard.
The authors take a rigorous approach to analyze details of microtubule dynamic behaviour presented in Figure 1. While I recognize the enormous amount of work that went into Figure 1, in my opinion these experiments shows that SSNA1 has no effect on microtubule dynamics at physiological concentration (sub 100 nM). That finding is i) very publishable and ii) should not take away from SSNA1 as an important molecule, but rather open up alternative ways of thinking about the protein.
I believe similar conclusions should be applied to microtubule slow-down in Figure 2 and the stabilization against tubulin loss by dilution/sequestration in Figure 3. If 5 uM (the only concentration shown) are required to achieve above effects, these observations are likely not relevant to SSNA1's biological function.
Taking into account SSNA1's cellular localization at centrosomes, midbodies, and branch points etc., I am not sure a major effect on microtubule dynamics other than nucleation should be expected.
The authors pursue an alternative and very interesting avenue in Figure 4, by examining the interplay between spastin and SSNA1 with regards to microtubules. Here, (1 uM) SSNA1 has protective effects against severing by spastin.
The discussion could use a direct contrast to differences in findings between the current work and the branched nucleation. It is not stated in the manuscript, though presumably no branching has been observed in several thousands of microtubule growth events? I would find a lot of value in such a potential statement.
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Reviewer #1 (Public Review):
In their manuscript, Lawrence et al. investigate the direct effects of the microtubule-associated protein, SSNA1, on microtubule (MT) dynamics and damage using purified proteins and TIRF microscopy. Prior work on this protein showed that SSNA1 self-assembles into higher-order filaments and binds longitudinally along stabilised MTs, inducing MT branching and nucleation. In this study, they find that SSNA1 promotes templated MT nucleation, consistent with prior results, but further define the effect of SSNA1 on MT dynamics. SSNA1 overall dampens MT dynamics by reducing both growth and shrinkage rates, suppressing catastrophe frequency, and increasing rescues. The authors also quantify SSNA1 on GMPCPP over a timecourse both at single-molecule and multi-molecule concentrations. On dynamic MTs, SSNA1 recognizes …
Reviewer #1 (Public Review):
In their manuscript, Lawrence et al. investigate the direct effects of the microtubule-associated protein, SSNA1, on microtubule (MT) dynamics and damage using purified proteins and TIRF microscopy. Prior work on this protein showed that SSNA1 self-assembles into higher-order filaments and binds longitudinally along stabilised MTs, inducing MT branching and nucleation. In this study, they find that SSNA1 promotes templated MT nucleation, consistent with prior results, but further define the effect of SSNA1 on MT dynamics. SSNA1 overall dampens MT dynamics by reducing both growth and shrinkage rates, suppressing catastrophe frequency, and increasing rescues. The authors also quantify SSNA1 on GMPCPP over a timecourse both at single-molecule and multi-molecule concentrations. On dynamic MTs, SSNA1 recognizes the growing end and promotes end curvature, but it did not recognize the curves of taxol-stabilised MTs, leading the authors to conclude that it likely induces curvature, rather than recognizes it. Perhaps this is the mechanism by which SSNA1 prevents catastrophe, a role which the authors demonstrate for SSNA1 after both tubulin dilution or stathmin sequestration of tubulin. The most interesting part of this study is found in Figure 4, where the authors show that SSNA1 prevents MT severing by spastin and also localizes to sites of lattice damage. The authors conclude that SSNA1 is a MT stabilizing protein and a sensor of MT damage. The results on MT dynamics do not provide much insight into the mechanism of this protein, which isn't even found to colocalize with MTs in vivo (SSNA1 instead accumulates at branchpoints in neurons). The role of SSNA1 in lattice damage recognition is the highlight of this paper, and also correlates well with its in vivo localization pattern, indicating this could be a true function of this protein. This damage recognition ability could potentially be the first step that leads to SSNA1-induced MT nucleation and branching from an existing MT.
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Evaluation Summary:
In this manuscript, Lawrence et al. investigate the direct effects of the microtubule-associated protein, SSNA1, on microtubule dynamics and damage using purified proteins and TIRF microscopy. The authors conclude that SSNA1 is a microtubule stabilizing protein and a sensor of microtubule damage. This paper is of high interest to scientists within the field of microtubule mechanics and of broad interest to scientists studying cilia, cell division and neuronal development.
(This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. Reviewer #3 agreed to share their name with the authors.)
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