Tubulin post-translational modifications orchestrate neuronal growth, regeneration and functions

Abstract

Microtubules, a key cytoskeletal component comprising conserved α/β-tubulins, provide mechanical support for neuronal development and intracellular transport. Tubulins undergo multiple enzymatic modifications after translation, which might regulate the structure of microtubules or their interactions with microtubule-associated proteins. Yet how these post-translational modifications (PTMs) determine microtubule properties in neurons remains to be elucidated. Most studies in the field are conducted via removing putative enzymes, which possibly fulfil various functions and thus deliver misleading information on PTM significance. Transgene overexpression and integration were used to introduce PTM site-specific mutations but tend to disrupt endogenous tubulin expression levels. These technical limitations add layers to the complexity of microtubule regulation.

My PhD project extensively used CRISPR/Cas9 technology to examine how mechanosensory neurons are dynamically regulated by tubulin PTMs in Caenorhabditis elegans. The roles of PTMs could be faithfully revealed by direct manipulation of PTM loci and verified by knocking out corresponding enzymes. The response of C. elegans to gentle mechanical stimuli relies on six touch receptor neurons (TRNs) that extend long sensory neurites along body wall, especially two pairs of lateral ALMs and PLMs. TRNs enjoy substantial quantities of 15-protofilament microtubules comprising two specific tubulin isotypes, α-tubulin MEC-12 and β-tubulin MEC-7.

Through amino acid substitutions of MEC-7 Ser172, we first found that S172 phosphorylation intensely suppresses axonal development. The ALMs of non-phosphorylatable mec-7(S172A) mutant grew an unusually long posterior neurite, while S172E substitution that mimicked permanent phosphorylation led to severe shortening of all neurites. Such neurites were incapable of sensing gentle touch in mec-7(S172E). Phosphorylation greatly introduces microtubule dynamics and therefore hinders cargo transport and regeneration. Conversely, dephosphorylated tubulins constitute hyper-stable but rigid microtubules that induce uncoordinated neurite extension and branching, highlighting the importance of optimal level of β-tubulin phosphorylation. Our in vivo knockout and in vitro kinase assay showed that CDK-1 mediates S172 phosphorylation of MEC-7.

Lys40 acetylation has long been interpreted as protection for microtubules because the loss of acetyltransferase MEC-17 seriously damages microtubules and neuronal growth. We generated endogenous K40Q and K40R mutations in MEC-12 mimicking acetylated and non-acetylated lysine respectively. Acetylation itself has slightly negative effects on neuronal extension, as both mutants overall displayed wild-type morphology except slight decreases in synapse formation and regrowth length in mec-12(K40Q) PLMs and a slightly longer ALM-PN in mec-12(K40R) mutants. MEC-12 tubulins are still noticeably acetylated by the other acetyltransferase ATAT-2 in mec-17(-) mutants, supporting that MEC-17 maintains microtubule structure independently of K40 acetylation.

We conducted a series of genetic modifications to tubulin C-terminal tails and demonstrated for the first time the additive effects of polyglutamylation in vivo and its functional specificity on α-tubulin. Immunostaining of cultured TRNs showed that TTLL-4 and TTLL-5 are major glutamylases in the cell body, while another unknown glutamylase is transported into neurites to modify MEC-12, which then recruits microtubule-depolymerizing kinesin-13 KLP-7 to regulate neurite extension.

Altogether, our work unraveled the nature of multiple tubulin PTMs, including the misunderstood K40 acetylation, polyglutamylation and the functionally conserved phosphorylation. They precisely fine-tune microtubule dynamics and thus guide the development of neurons.