Single kinesin-8 microtubule depolymerization activity investigated with optical tweezers
Final Report Abstract
Microtubules are highly dynamic filaments with dramatic structural rearrangements and length changes during the cell cycle. An accurate control of the microtubule length is essential for many cellular processes in particular, during cell division. Motor proteins from the kinesin-8 family depolymerize microtubules by interacting with their ends in a collective and lengthdependent manner. However, it is still unclear how kinesin-8 depolymerizes microtubules. In this project, single molecule approaches will be employed to gain insights into the microtubule end-binding activity of single yeast kinesin, Kip3. First, we tracked the microtubule end-binding activity of Kip3 under varying loads and nucleotide conditions using high-precision optical tweezers. We found that single Kip3 motors spent up to 200 s at the microtubule end and were not stationary there but took several 8-nm forward and backward steps that were suppressed by loads. Interestingly, increased loads, similar to increased motor concentrations, also exponentially decreased the motors’ residence time at the microtubule end. On the microtubule lattice, Kip3 had a different binding behavior suggesting that the observations are distinct for the microtubule end. The force dependence of the end residence time enabled us to estimate what force must act on a single motor to achieve the microtubule depolymerization speed of a motor ensemble. This force is higher than the stall force of a single Kip3 motor, supporting a collective force-dependent depolymerization mechanism. In a second part of this project, we studied the microtubule depolymerization as function of low Kip3 concentration, using interference reflection microscopy (IRM). Surprisingly, we observed that single Kip3 stabilizes microtubules. This implies a complex concentration depend regulation function of Kip3 on microtubules, that was not observed for any other kinesin before. Understanding the mechanics of kinesin-8’s microtubule end activity will provide important insights into cell division with implications for cancer research.
Publications
- Kinesin-8 depolymerizes microtubules with a collective, forcedependent mechanism EMBO|EMBL Symposium Microtubules: From Atoms to Complex Systems 27 - 30 May 2018, EMBL Heidelberg, Germany
Anita Jannasch
- Kinesin-8 depolymerizes microtubules with a force-dependent mechanism. 62nd Annual Biophysical Society Meeting 17 - 21 February 2018, San Francisco, California, USA
Anita Jannasch
- (2019) The kinesin-8 Kip3 depolymerizes microtubules with a collective force-dependent mechanism. (in print, Biophys. J.), biorxiv
M. Bugiel, M. Chugh, T. J Jachowski, E. Schäffer, and A. Jannasch
(See online at https://doi.org/10.1101/844829 https://doi.org/10.1016/j.bpj.2020.02.030)
