Efa 6 protects axons and regulates their growth and branching by inhibiting 1 microtubule polymerisation at the cortex 2 3

32 Cortical collapse factors affect microtubule (MT) dynamics at the plasma membrane. They 33 play important roles in neurons, as suggested by inhibition of axon growth and regeneration 34 through the Arf activator Efa6 in C. elegans, and by neurodevelopmental disorders linked to 35 the mammalian kinesin Kif21A. How cortical collapse factors influence axon growth is little 36 understood. Here we studied them, focussing on the function of Drosophila Efa6 in 37 experimentally and genetically amenable fly neurons. First, we show that Drosophila Efa6 can 38 inhibit MTs directly without interacting molecules via an N-terminal 18 amino acid motif (MT 39 elimination domain/MTED) that binds tubulin and inhibits microtubule growth in vitro and cells. 40 If N-terminal MTED-containing fragments are in the cytoplasm they abolish entire microtubule 41 networks of mouse fibroblasts and whole axons of fly neurons. Full-length Efa6 is membrane42 attached, hence primarily blocks MTs in the periphery of fibroblasts, and explorative MTs that 43 Qu et al. – The role of Efa6 in axon maintenance 2 have left axonal bundles in neurons. Accordingly, loss of Efa6 causes an increase of 44 explorative MTs: in growth cones they enhance axon growth, in axon shafts they cause 45 excessive branching, as well as atrophy through perturbations of MT bundles. Efa6 over46 expression causes the opposite phenotypes. Taken together, our work conceptually links 47 molecular and sub-cellular functions of cortical collapse factors to axon growth regulation and 48 reveals new roles in axon branching and in the prevention of axonal atrophy. Furthermore, the 49 MTED delivers a promising tool that can be used to inhibit MTs in a compartmentalised 50 fashion when fusing it to specifically localising protein domains. 51