1. Reviewer #3 (Public Review):

    In this study the authors provide a quantitative assessment of the dense microtubule network in dendrites of mammalian neurons. They used 2D and 3D STED as well as Expansion Microscopy to resolve single tubules in the soma and in dendrites. Also, they marked specifically for microtubules specific modifications such as tyrosination and acetylation, which tend to be associated with dynamic and stable tubules respectively.

    I believe the authors achieved very interesting findings, which includes that 1-acetylated microtubules accumulate in the core of the dendritic shaft, surrounded by a shell of tyrosinated microtubules 2- a rigorous quantification of the tyrosination and acetylation levels at the single tube level reveal that the two modifications are anti-correlated and define two distinct microtubule subsets 3-in dendrites the absolute number of acetylated and tyrosinated microtubules is 65-75% and ~20-30% of all microtubules.

    My overall impression is that the choice of methods suits well the study and the image analysis performed is very robust throughout the paper supporting their major findings.

    The fact that they use different methods, both in terms of imaging (2D STED, 3D STED and Expansion) and analysis, and arrive at the same conclusion regarding for example the percentage of dynamic and stable microtubules is very reassuring that they are quantifying relevant numbers in their analysis.

    Overall, I also appreciate how openly they provide their analysis scripts and thorough explanations on the analysis they do and how to use their pipelines, which makes it much easier to check the code and ascertain that it performs as described.

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  2. Reviewer #2 (Public Review):

    In this work the authors take a quantitative approach towards deciphering the spatial distribution of microtubules carrying different tubulin posttranslational modifications (PTMs) in the neuronal cell body and dendrites. They compare two different super-resolution microscopy techniques - STED and expansion microscopy. They also develop a new approach to image the neuronal dendrites perpendicularly in order to further increase the visibility of single microtubules. The team has previously reported that acetylated and tyrosinated microtubules have distinct patterns of organization within the neuron (Tas et al. 2017; Jurriens et al. 2021). The present investigation confirms these findings and takes a step further towards a quantitative segregation of these microtubule subsets. The authors simultaneously provide a thorough pipeline of the process that can be used to address the distribution of other posttranslationally modified microtubules or broadly to quantify absolute microtubule numbers in confined environments such as neurites.

    Altogether, this study demonstrates the power of recently developed super-resolution microscopy techniques for the imaging of single microtubules in neurons. This is especially important in the context of visualizing tubulin PTMs, which require immunostaining for detecting and for which electron microscopy is not a good alternative.

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  3. Reviewer #1 (Public Review):

    In this manuscript, Katrukha et al. use advanced microscopy techniques to quantify the organization of microtubules within neuronal dendrites. This is a challenge due to the tight bundling of microtubules along neuronal processes, and they tackle it using two techniques: STED microscopy and Expansion Microscopy. They thus measure by two independent procedures the density of microtubules along proximal dendrites and the proportion of acetylated and tyrosinated microtubules, showing that dendritic microtubules are either acetylated or tyrosinated, but rarely present both or none of these post-translational modifications.

    The manuscript is a significant methodological advance, with a combination of new sample preparation and image analysis procedures that provide images of dendritic microtubules with enhanced quality. These enhancements don't allow the tracing of a significant number of individual microtubules. They are used for refined intensity-based statistics that provide a worthy insight into the organization of dendritic microtubules. Here the approaches are robust, with clear explanation of the quantitation workflow using open-source tools that are made available. One can regret that these measurements don't address the longitudinal dimension of microtubules, which could help revisiting and answering a couple of important questions (existence of modification domains along single microtubules, microtubule orientation...).

    Nonetheless, the authors bring forward very good imaging and solid quantification workflows that allow them to convincingly answer the main question they ask: what is the radial organization of dendritic microtubules, including their post-translational modification state?

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  4. Evaluation Summary:

    In this manuscript, Katrukha et al. use STED and Expansion Microscopy techniques to map the distribution of different microtubule populations within the dendrites of neurons, a challenging task due to the tight bundling of microtubules along neuronal processes. They are able to show that dendritic microtubules are either acetylated or tyrosinated, but rarely have both or neither of these post-translational modifications. The strength of this paper is the quality of the experiments, the thoroughness of the analyses, and most importantly the transparent and critical discussion of the limitations the authors have encountered. This manuscript is of broad interest to the cytoskeletal and neurobiology fields.

    (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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