Drosophila katanin-60 Depolymerizes and Severs at Microtubule Defects

Biophys J. 2011 May 18;100(10):2440-9. doi: 10.1016/j.bpj.2011.03.062.

Abstract

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.

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, Non-U.S. Gov't

MeSH terms

  • Adenosine Triphosphatases / chemistry
  • Adenosine Triphosphatases / metabolism*
  • Adenosine Triphosphate / pharmacology
  • Animals
  • Baculoviridae / drug effects
  • Baculoviridae / metabolism
  • Drosophila Proteins / chemistry
  • Drosophila Proteins / metabolism*
  • Drosophila melanogaster / drug effects
  • Drosophila melanogaster / metabolism*
  • Fluorescence
  • Green Fluorescent Proteins / metabolism
  • Guanosine Diphosphate / metabolism
  • Guanosine Triphosphate / analogs & derivatives
  • Guanosine Triphosphate / metabolism
  • Katanin
  • Microtubules / drug effects
  • Microtubules / metabolism*
  • Models, Biological
  • Paclitaxel / pharmacology
  • Photobleaching / drug effects
  • Polymerization* / drug effects
  • Protein Binding / drug effects
  • Protein Structure, Quaternary
  • Protein Structure, Tertiary
  • Protein Transport / drug effects
  • Recombinant Fusion Proteins / metabolism
  • Tubulin / metabolism

Substances

  • Drosophila Proteins
  • Kat60 protein, Drosophila
  • Recombinant Fusion Proteins
  • Tubulin
  • Guanosine Diphosphate
  • Green Fluorescent Proteins
  • 5'-guanylylmethylenebisphosphonate
  • Guanosine Triphosphate
  • Adenosine Triphosphate
  • Adenosine Triphosphatases
  • Katanin
  • Paclitaxel