Tubulin polyglutamylation is a post‐translational modification of the microtubule cytoskeleton, which is generated by a variety of enzymes with different specificities. The “tubulin code” hypothesis predicts that modifications generated by specific enzymes selectively control microtubule functions. Our recent finding that excessive accumulation of polyglutamylation in neurons causes their degeneration and perturbs axonal transport provides an opportunity for testing this hypothesis. By developing novel mouse models and a new glutamylation‐specific antibody, we demonstrate here that the glutamylases TTLL1 and TTLL7 generate unique and distinct glutamylation patterns on neuronal microtubules. We find that under physiological conditions, TTLL1 polyglutamylates α‐tubulin, while TTLL7 modifies β‐tubulin. TTLL1, but not TTLL7, catalyses the excessive hyperglutamylation found in mice lacking the deglutamylase CCP1. Consequently, deletion of TTLL1, but not of TTLL7, prevents degeneration of Purkinje cells and of myelinated axons in peripheral nerves in these mice. Moreover, loss of TTLL1 leads to increased mitochondria motility in neurons, while loss of TTLL7 has no such effect. By revealing how specific patterns of tubulin glutamylation, generated by distinct enzymes, translate into specific physiological and pathological readouts, we demonstrate the relevance of the tubulin code for homeostasis.

The microtubule (MT) cytoskeleton forms complex macromolecular assemblies with a plethora of MT-associated proteins (MAPs) that play fundamental roles in cell architecture, division and motility. Determining how an individual MAP modulates MT behaviour is an important step in understanding the physiological roles of various MT assemblies. To characterise how the ever-increasing number of MAPs control MT properties and functions, we developed an approach allowing for medium-throughput analyses of MAPs in cell-free conditions using lysates of mammalian cells. Our pipeline allows for quantitative as well as ultrastructural analyses of MT-MAP assemblies. Analysing about fifty bona-fide and potential mammalian MAPs, we uncovered previously unknown activities that lead to distinct and unique MT behaviours, such as MT coiling or hook formation, or liquid-liquid phase separation along MT lattice that initiates MT branching. We have thus established a powerful tool for a thorough characterisation of a wide range of MAPs and MAP variants, thus opening avenues for the determination of mechanisms underlying their physiological roles and pathological implications.