Klp Navigation of Spindle Assembly from Poles

Abstract

We have here investigated the effect of varying neck-linker length on the motile properties of Eg5Kin. As truncated versions of Eg5 contain the native 18 amino acids of the neck linker, we generated six Eg5Kin constructs comprising of 13 to up to the 18 amino acids of the native Eg5 neck linker, possibly providing a physiological context. Using single-molecule fluorescence, we found that all six constructs are active motor molecules capable of processive motility. In a first set of experiments, we found that the neck-linker length influences the run length, but not the velocity of the motor. We thus confirm the findings of Shastry and Hancock (2010, Curr. Biol. 20:939) with a different motor. In addition we used optical-trap assays to investigate the change in the average force the motor constructs generated and found only a small variation. Our data thus suggest that the neck-linker length of Eg5 is at least not the sole determinant for speed and force generation.