In vitro reconstitution assays of microtubule amplification and lattice repair by the microtubule-severing enzymes katanin and spastin

Abstract

Microtubules are non-covalent dynamic polymers essential for the life of all eukaryotic cells. Their dynamic behavior is regulated by a large array of cellular effectors. In vitro microtubule assays have been instrumental in dissecting the mechanism of microtubule-associated proteins. In this chapter, we focus on microtubule-severing enzymes katanin and spastin. They are AAA ATPases that generate internal breaks in microtubules by extracting tubulin dimers out of the microtubule lattice. We present protocols for TIRF microscopy-based assays that were instrumental in proving that these enzymes not only sever microtubules but also remodel the microtubule lattice by promoting the exchange of lattice GDP-tubulin with GTP-tubulin from the soluble pool. This activity can modulate microtubule dynamics and support microtubule-dependent microtubule amplification in the absence of a nucleating factor.