The importance of lattice defects in katanin-mediated microtubule severing in vitro.
The microtubule-severing enzyme katanin uses ATP hydrolysis to disrupt noncovalent bonds between tubulin dimers within the microtubule lattice. Although its microtubule severing activity is likely important for fundamental processes including mitosis and axonal outgrowth, its mechanism of action is poorly understood. To better understand this activity, an in vitro assay was developed to enable the real-time observation of katanin-mediated severing of individual, mechanically unconstrained microtubules. To interpret the experimental observations, a number of theoretical models were developed and compared quantitatively to the experimental data via Monte Carlo simulation. Models that assumed that katanin acts on a uniform microtubule lattice were incompatible with the in vitro data, whereas a model that assumed that katanin acts preferentially on spatially infrequent microtubule lattice defects was found to correctly predict the experimentally observed breaking rates, number and spatial frequency of severing events, final levels of severing, and sensitivity to katanin concentration over the range 6-300 nM. As a result of our analysis, we propose that defects in the microtubule lattice, which are known to exist but previously not known to have any biological function, serve as sites for katanin activity.
Davis
Liza J
LJ
Department of Chemical Engineering and Materials Science, University of Minnesota, Minneapolis, Minnesota 55455, USA.
Odde
David J
DJ
Block
Steven M
SM
Gross
Steven P
SP
D000251:Adenosine Triphosphatases / Q000378:metabolism*; D000255:Adenosine Triphosphate / Q000378:metabolism; D000818:Animals; D055592:Biophysical Phenomena; D001703:Biophysics; D019281:Dimerization; D006868:Hydrolysis; D066298:In Vitro Techniques; D000074201:Katanin; D007700:Kinetics; D018715:Microscopy, Video; D008870:Microtubules / Q000737:chemistry* / Q000378:metabolism* / Q000648:ultrastructure; D008954:Models, Biological; D008958:Models, Molecular; D009010:Monte Carlo Method; D012617:Sea Urchins; D014404:Tubulin / Q000737:chemistry* / Q000378:metabolism* / Q000648:ultrastructure
D014404:Tubulin; D000255:Adenosine Triphosphate; D000251:Adenosine Triphosphatases; D000074201:Katanin