Clasps Depolymerize Microtubules in a Nucleotide-Dependent Manner

Abstract

CLASPs constitute a family of evolutionarily-conserved TOG-domain proteins that regulate microtubules in fundamental cellular processes including cell division, cell migration and neuronal development. CLASPs stabilize dynamically growing microtubules by suppressing catastrophe and promoting rescue, the switch-like transitions between growth and shrinkage. However, the molecular mechanisms underlying CLASP's activity are not fully understood. Previous work found that another TOG-domain protein, XMAP215/chTOG, promotes microtubule polymerization in the presence of soluble tubulin but depolymerizes microtubules in the absence of tubulin. Here, we report the surprising finding that human CLASP1 depolymerizes stabilized microtubules in the presence of GTP and GDP but not in the presence of GMPCPP nor in the no nucleotide condition. Thus, CLASP1 depolymerizes stable microtubules in a nucleotide-dependent manner. Further analyses revealed that other members of the human CLASP family (CLASP2α and CLASP2γ) also possess nucleotide-dependent depolymerase activity, as does a minimal TOG2-domain construct. Next, we demonstrate that CLASP1 depolymerizes both plus and minus microtubule ends in a nucleotide-dependent manner; this is unexpected, given that the exchangeable nucleotide site in tubulin is presumed to be buried within the polymer at the minus end. by performing single molecule dwell time analysis, we find that GTP reduces the dwell time of TOG2 at microtubule tips compared to the no nucleotide condition. Finally, we report that the depolymerase activity of CLASP1 exhibits significantly greater sensitivity to GTP than that of XMAP215/chTOG, indicating that the two TOG-domain proteins employ distinct mechanisms to depolymerize microtubules. The unanticipated finding that CLASPs possesses nucleotide-sensitive depolymerase activity provides critical mechanistic insights into the activity of an important family of microtubule regulatory proteins. This work will further our understanding of the mechanisms employed by CLASPs in regulating microtubule network dynamics and organization in cells.