The iconic bipolar structure of the mitotic spindle is of extreme importance to proper spindle function. At best, spindle abnormalities result in a delayed mitosis, while worse outcomes include cell death or disease. Recent work has uncovered an important role for the actin-based motor protein myosin-10 in the regulation of spindle structure and function. Here we examine the contribution of the myosin tail homology 4 (MyTH4) domain of the myosin-10 tail to the protein’s spindle functions. The MyTH4 domain is known to mediate binding to microtubules and we verify the suspicion that this domain contributes to myosin-10’s close association with the spindle. More surprisingly, our data demonstrate that some but not all of myosin-10’s spindle functions require microtubule binding. In particular, myosin-10’s contribution to spindle pole integrity requires microtubule binding, whereas its contribution to normal mitotic progression does not. This is demonstrated by the observation that dominant negative expression of the wild-type MyTH4 domain produces multipolar spindles and an increased mitotic index, whereas overexpression of a version of the MyTH4 domain harboring point mutations that abrogate microtubule binding results in only the mitotic index phenotype. Our data suggest that myosin-10 helps to control the metaphase to anaphase transition in cells independent of microtubule binding.

The proper formation and function of mitotic spindles are essential for any organism. Disruption of myosin‐10 (myo10), an actin‐based motor protein, has been shown to perturb mitotic spindle structure and function. Such an important role for a myosin at the mitotic spindle is surprising since spindles are microtubule‐based structures. It thus stands to reason that myo10 either directly or indirectly interacts with spindle microtubules. In support of a direct interaction, a microtubule‐binding domain (MTB) has been identified in the C‐terminal tail of Myo10, suggesting that myo10 can interact with both cytoskeletal polymers. Here we use a dominant negative approach to investigate the function of the MTB in myo10’s spindle function. In brief, mRNA encoding various fragments of the myo10 C‐terminal tail with either intact or altered MTB were injected into Xenopus laevis embryos, and the embryos were subsequently analyzed for protein expression and spindle phenotypes. Results indicate that protein fragments containing intact MTB function as dominant negatives and cause spindle phenotypes similar to myo10 depletion, while fragments that either excluded or contained a mutated MTB did not. This suggests that the MTB of myo10 plays a crucial role in myo10’s function at the mitotic spindle.