Microtubule (MT) length and location is tightly controlled in cells. One novel family of MT-associated proteins that regulates MT dynamics is the MT-severing enzymes. In this work, we investigate how katanin (p60), believed to be the first discovered severing enzyme, binds and severs MTs via single molecule total internal reflection fluorescence microscopy. We find that severing activity depends on katanin concentration. We also find that katanin can remove tubulin dimers from the ends of MTs, appearing to depolymerize MTs. Strikingly, katanin localizes and severs at the interface of GMPCPP-tubulin and GDP-tubulin suggesting that it targets to protofilament-shift defects. Finally, we observe that binding duration, mobility, and oligomerization are ATP dependent.

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During axonal transport, an ensemble of molecular motors, including kinesin-1 and kinesin-2, navigate a complex microtubule landscape to deliver cargo to their target destinations within the cell. It has previously been shown in vitro that the neuronal microtubule associated proteins, 3RS-tau and 4RL-tau, reduce kinesin-1 processivity on taxol-stabilized GDP microtubules, but not on microtubules stabilized with GMPCPP (a slowly hydrolyzable GTP analog). Furthermore, kinesin-1 processivity is also reduced on GMPCPP microtubules relative to taxol-stabilized microtubules, suggesting the microtubule lattice modulates interactions with both kinesin-1 and tau (McVicker et al., (2011) J Biol Chem 286:42873). However, the effects of tau and the microtubule lattice structure on kinesin-2 processivity are still unknown. Kinesin-2 is known to have a longer neck-linker than kinesin-1, resulting in reduced coordination between motor domains and decreased processivity on taxol-stabilized microtubules (Shastry et al., (2010) Curr Biol 20:939). We hypothesize that these differences in kinesin-2 function make it less sensitive to alterations in the microtubule lattice than kinesin-1, and allow it to more easily navigate obstacles, such as tau, on the microtubule surface. To directly test this hypothesis, we used single molecule imaging with TIRF microscopy to measure kinesin-2 motility as it stepped along microtubules in different nucleotide states (GDP or GMPCPP) in the absence or presence of 3RS-tau and 4RL-tau. Our results demonstrate that, in contrast to kinesin-1, kinesin-2 processivity is unchanged on taxol-stabilized vs. GMPCPP microtubules and is insensitive to the presence of either 3RStau or 4RL-tau. Thus, while kinesin-2 is less processive than kinesin-1, it may be better optimized to navigate around obstacles on different microtubule lattice structures, allowing the two motors to work together for the efficient delivery of cargo in the complex environment of the neuronal axon.