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

Abstract

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 nematodes touch sensing, highlighting C. elegans as a valuable model for investigating tubulin PTM-related neurological disorders.