Editing of endogenous tubulins reveals varying effects of tubulin posttranslational modifications on axonal growth and regeneration
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eLife assessment
This important study addresses a fundamental question: how do post-translational modifications of tubulin regulate the function of the microtubule cytoskeleton in vivo? The authors generate a large panel of tubulin mutants designed to lack specific modifications and describe their effects using endogenous editing and touch receptor neurons in C. elegans as an in vivo model. While the work presents an impressive amount of data, it is in part incomplete, since the presence and absence of specific tubulin modifications and their effects on microtubules are not demonstrated in all cases.
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Abstract
Tubulin posttranslational modifications (PTMs) modulate the dynamic properties of microtubules and their interactions with other proteins. However, the effects of tubulin PTMs were often revealed indirectly through the deletion of modifying enzymes or the overexpression of tubulin mutants. In this study, we directly edited the endogenous tubulin loci to install PTM-mimicking or -disabling mutations and studied their effects on microtubule stability, neurite outgrowth, axonal regeneration, cargo transport, and sensory functions in the touch receptor neurons of Caenorhabditis elegans . We found that the status of β-tubulin S172 phosphorylation and K252 acetylation strongly affected microtubule dynamics, neurite growth, and regeneration, whereas α-tubulin K40 acetylation had little influence. Polyglutamylation and detyrosination in the tubulin C-terminal tail had more subtle effects on microtubule stability likely by modulating the interaction with kinesin-13. Overall, our study systematically assessed and compared several tubulin PTMs for their impacts on neuronal differentiation and regeneration and established an in vivo platform to test the function of tubulin PTMs in neurons.
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eLife assessment
This important study addresses a fundamental question: how do post-translational modifications of tubulin regulate the function of the microtubule cytoskeleton in vivo? The authors generate a large panel of tubulin mutants designed to lack specific modifications and describe their effects using endogenous editing and touch receptor neurons in C. elegans as an in vivo model. While the work presents an impressive amount of data, it is in part incomplete, since the presence and absence of specific tubulin modifications and their effects on microtubules are not demonstrated in all cases.
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Reviewer #1 (Public Review):
The manuscript by Lu et al aims to study the effects of tubulin post-translational modification in C. elegans touch receptor neurons. Authors use gene editing to engineer various predicted PTM mutations in a-tubulin MEC-12 and b-tubulin MEC-7. Authors generate and analyze an impressive battery of mutants in predicted phosphorylation site and acetylation site of b-tubulin MEC-7, K40 acetylation site in a-tubulin MEC-12, enzymatic site of the a-tubulin acetyltransferase MEC-17, and PTM sites in the MEC-12 and MEC-7 C-tails (glutamylation, detyrosination, delta-tubulin). This represents a lot of work, and will appeal to a readership interested in C. elegans touch receptor neurons. The major concern/criticism of this manuscript is whether the introduced mutation(s) directly affects a specific PTM or whether the …
Reviewer #1 (Public Review):
The manuscript by Lu et al aims to study the effects of tubulin post-translational modification in C. elegans touch receptor neurons. Authors use gene editing to engineer various predicted PTM mutations in a-tubulin MEC-12 and b-tubulin MEC-7. Authors generate and analyze an impressive battery of mutants in predicted phosphorylation site and acetylation site of b-tubulin MEC-7, K40 acetylation site in a-tubulin MEC-12, enzymatic site of the a-tubulin acetyltransferase MEC-17, and PTM sites in the MEC-12 and MEC-7 C-tails (glutamylation, detyrosination, delta-tubulin). This represents a lot of work, and will appeal to a readership interested in C. elegans touch receptor neurons. The major concern/criticism of this manuscript is whether the introduced mutation(s) directly affects a specific PTM or whether the mutation affects gene expression, protein expression/stability/localization, etc. As such, this work does convincingly demonstrate, as stated in the title, that "Editing of endogenous tubulins reveals varying effects of tubulin posttranslational modifications on axonal growth and regeneration."
For example, the authors manipulate the C-terminal tail of MEC-12 and MEC-7, to test the idea that polyglutamylation may be an important PTM. These mutants displayed subtle phenotypes. The authors show that branch point GT335 and polyglutamyation polyE recognizing antibodies stain cultured embryonic touch receptor neurons (TRNs), but did not examine staining in C. elegans TRNs in situ. To my knowledge, these antibodies have not been shown to stain the TRNs in any published papers, raising the question of how these "glutamylation" mutations are affecting mec-12 and -7. The rationale for using cultured embryonic TRNs and the relevance of the data and its interpretation are not clear.
The final paragraph of the discussion is factually incorrect. The C. elegans homologs of the CCP carboxypeptidases are called CCPP-1 and CCPP-6. There are several publications on their functions in C. elegans.
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Reviewer #2 (Public Review):
Summary:
The tubulin subunits that make up microtubules can be posttranslationally modified and these PTMs are proposed to regulate microtubule dynamics and the proteins that can interact with microtubules in many contexts. However, most studies investigating the roles of tubulin PTMs have been conducted in vitro either with purified components or in cultured cells. Lu et al. use CRISPR/Cas9 genome editing to mutate tubulin genes in C. elegans, testing the role of specific tubulin residues on neuronal development. This study is a real tour de force, tackling multiple proposed tubulin modifications and following the resulting phenotypes with respect to neurite outgrowth in vivo. There is a ton of data that experts in the field will likely reference for years to come as this is one of the most comprehensive in …Reviewer #2 (Public Review):
Summary:
The tubulin subunits that make up microtubules can be posttranslationally modified and these PTMs are proposed to regulate microtubule dynamics and the proteins that can interact with microtubules in many contexts. However, most studies investigating the roles of tubulin PTMs have been conducted in vitro either with purified components or in cultured cells. Lu et al. use CRISPR/Cas9 genome editing to mutate tubulin genes in C. elegans, testing the role of specific tubulin residues on neuronal development. This study is a real tour de force, tackling multiple proposed tubulin modifications and following the resulting phenotypes with respect to neurite outgrowth in vivo. There is a ton of data that experts in the field will likely reference for years to come as this is one of the most comprehensive in vivo analyses of tubulin PTMs in vivo.This paper will be very important to the field, however would be strengthened if: 1) the authors demonstrated that the mutations they introduced had the intended consequences on microtubule PTMs, 2) the authors explored how the various tubulin mutations directly affect microtubules, and 3) the findings are made generally more accessible to non C. elegans neurobiologists.
(1) The authors introduce several mutations to perturb tubulin PTMs, However, it is unclear to what extent the engineered mutations affect tubulin in the intended way i.e. are the authors sure that the PTMs they want to perturb are actually present in C. elegans. Many of the antibodies used did not appear to be specific and antibody staining was not always impacted in the mutant cases as expected. For example, is there any evidence that S172 is phosphorylated in C. elegans, e.g. from available phosphor-proteomic data? Given the significant amount of staining left in the S172A mutant, the antibody seems non-specific in this context and therefore not a reliable readout of whether MTs are actually phosphorylated at this residue. As another example, there is no evidence presented that K252 is acetylated in C. elegans. At the very least, the authors should consider demonstrating the conservation of these residues and the surrounding residues with other organisms where studies have demonstrated PTMs exist.
(2) Given that the authors have the mutants in hand, it would be incredibly valuable to assess the impact of these mutations on microtubules directly in all cases. MT phenotypes are inferred from neurite outgrowth phenotypes in several cases, the authors should look directly at microtubules and/or microtubule dynamics via EBP-2 when possible OR show evidence that the only way to derive the neurite phenotypes shown is through the inferred microtubule phenotypes. For example, the effect of the acetylation or detyrosination mutants on MTs was not assessed.
(3) There is a ton of data here that will be important for experts working in this field to dig into, however, for the more general cell biologist, some of the data are quite inaccessible. More cartoons and better labeling will be helpful as will consistent comparisons to control worms in each experiment.
(4) In addition, I am left unconvinced of the negative data demonstrating that MBK does not phosphorylate tubulin. First, the data described in lines 207-211 does not appear to be presented anywhere. Second, RNAi is notoriously finicky in neurons, thus necessitating tissue-specific degradation using either the ZF/ZIF-1 or AID/TIR1 systems which both work extremely well in C. elegans. Third, there appears to be increasing S172 phosphorylation in Figure 3 Supplement 2 with added MBK-2, but there is no anti-tubulin blot to show equal loading, so this experiment is hard to interpret.
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