Abstract
ABSTRACTThe assembly and functionality of the mitotic spindle depends on the coordinated activities of microtubule-associated motor proteins of the dynein and kinesin superfamily. Our current understanding of the function of motor proteins is significantly shaped by studies using Xenopus laevis egg extract as its open structure allows complex experimental manipulations hardly feasible in other model systems. Yet, the Kinesin-8 orthologue of human Kif18A has not been described in Xenopus laevis so far. Here, we report the cloning and characterization of Xenopus laevis (Xl) Kif18A. Xenopus Kif18A is expressed during oocyte maturation and its depletion from meiotic egg extract results in severe spindle defects. These defects can be rescued by wild-type Kif18A, but not Kif18A lacking motor activity or the C-terminus. Single-molecule microscopy assays revealed that Xl_Kif18A possesses high processivity, which depends on an additional C-terminal microtubule-binding site. Human tissue culture cells depleted of endogenous Kif18A display mitotic defects, which can be rescued by wild-type, but not tail-less Xl_Kif18A. Thus, Xl_Kif18A is the functional orthologue of human Kif18A whose activity is essential for the correct function of meiotic spindles in Xenopus oocytes.
Highlights
Kinesins are molecular motor proteins that convert the energy released by ATP hydrolysis into mechanical force (Vale and Milligan, 2000)
By combining Xenopus egg extract studies with in-vitro single molecule microscopy assays, we demonstrate that Xl_Kif18A possesses high processivity, which depends on an additional non-motor MT binding site at its C-terminus and which is important for its activity in regulating meiotic spindle function
Xl_Kif18A is expressed during oocyte maturation To characterize Xenopus laevis Kif18A, we PCR amplified the open-reading-frame (ORF) of Kif18A using mRNA purified from mature Xenopus eggs and primers matching the annotated sequence of the start and stop codon
Summary
Kinesins are molecular motor proteins that convert the energy released by ATP hydrolysis into mechanical force (Vale and Milligan, 2000). Because of this characteristic feature, kinesins share a common motor domain of ∼350 amino acids (aa), which couples ATP hydrolysis to conformational changes resulting in altered affinities for microtubules (MTs). The superfamily of kinesins has been classified into 14 different families (Miki et al, 2005). The Kinesin-8 family is unique in that it contains members that integrate two activities: movement towards the plus-ends of MTs and modulation of MT dynamics (Su et al, 2012).
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