Crosstalk between tubulin glutamylation and tyrosination regulates kinesin-3-mediated axonal transport

Tubulin post-translational modifications (PTM) are critical regulators of microtubule function and diversity in neurons. In Caenorhabditis elegans , we investigated how tubulin deglutamylases (CCPP-1 and CCPP-6) and polyglutamylases (TTLL-5 and TTLL-9) affect kinesin-3 KIF1A/UNC-104–mediated axonal transport. Loss of CCPP-1 not only facilitates tubulin polyglutamylation as expected, but it also leads to increased tyrosination in Western blots. Vice versa, loss of TTLL-5 reduces tubulin polyglutamylation as expected, but also leads to reduced tyrosination signals. This crosstalk in tubulin PTM appears to be a critical feature as no tyrosination or detyrosination enzymes are known in C. elegans . Notably, acetylation and detyrosination remain unaffected in the deglutamylation and polyglutamylation mutants. Functionally, reduced glutamylation and tyrosination improved UNC-104 motility. Conversely, increased glutamylation and tyrosination negatively affected the movement of both the UNC-104 motor and its cargo RAB-3. UNC-104 motors visibly accumulate in neuronal cell bodies of ccpp-1 mutants while being significantly reduced in ttll-5 mutants. In ttll-5 mutants, motors tend to cluster along distal axonal regions and these clusters are reduced in ccpp-1 mutants revealing a role of tubulin PTM in axonal motor scaffolding. Employing promoter fusions, we confirmed that all investigated PTM enzymes express in neurons and colocalize with UNC-104. Moreover, co-immunoprecipitation assays revealed that hyperglutamylated tubulin appears in a physical complex with UNC-104, while hypoglutamylated tubulin binds less effectively to the motor. In our model, highly negatively charged polyglutamylated tubulin traps UNC-104 onto microtubules via increased charge-interactions. Tubulin “stickiness” is reduced in polyglutamylase mutants leading to increased motor speeds. Reduced synaptic vesicle transport in ccpp-1 mutants has a negative impact on the nematode’s touch sensing, highlighting C. elegans as a valuable model for investigating tubulin PTM-related neurological disorders.