Tetrameric Chimera DK4mer is a Tool to Study Mechanisms Of Kinesin-5 Regulation. A Tetrameric Chimera of a Kinesin 1 and a Kinesin 5 is a Fast Microtubule Sliding Motor

Abstract

The homo-tetrameric motor protein Eg5 from X. laevis drives relative sliding of anti-parallel microtubules by the processive action of its two opposing sets of dimeric motor. As shown by Kwok et al. (2006, Nat. Chem. Biol. 2:480) and Kapitein et al. (2008, J. Cell Biol. 182:421), tetrameric motors move slowly (∼20nm/s), but processively on a single microtubule alternating between diffusional and directional episodes, while motors moving between two microtubules move in a highly directional and processive fashion.In order to obtain a tetrameric model system with more clearly defined properties and motile phases, we have constructed a tetrameric chimera by replacing Eg5-motor domain and neck-linker by the homologous regions of D. melanogaster Kinesin 1 (DK4mer).In surface-gliding assays, Dk4mer showed fast motility (553±31nm/s), irrespective of a C-terminal his- or GFP-his-tag. Comparison to DmKHC shows a similar k0.5, ATP of ∼0.06mM, suggesting that the GFP-tagged version is suitable for single-molecule fluorescence studies. Single GFP-tagged DK4mer motors moved processively along the MT at speeds comparable to those seen in surface-gliding assays (499±3nm/s). We observe clearly distinguished directional and diffusional episodes and an overall run length of ∼9μm on average. We further performed relative sliding assays using DK4mer and observe the expected trimodal distribution of velocities at v=0, v=v1 and v=2v1, v1 being 500nm/s, clearly showing that DK4mer is capable of sliding microtubules apart simultaneously using both pairs of motor domains.The DK4mer is thus an excellent model system to study regulatory aspects of Kinesin-5 due to its high speed, its long processivity and its clear separation of diffusive and directional motility and its fast and efficient relative sliding of microtubules.(∗authors contributed equally)