The ubiquitiously expressed class 1 myosins are single headed, non-filamentous motor proteins that play a role in various motile processes. Here, we investigated the role of Dictyostelium discoideum Myosin-1C in cytoskeletal organisation and as an instrument to regulate microtubule-based kinesin motility. Myosin-1C molecules have long tail domains. During mitosis, a strong localization of Myosin-1C along microtubules of the mitotic spindle is observed in Dictyostelium discoideum cells. To test for direct interaction, we performed in vitro TIRFM experiments using fluorescently labeled Myosin-1C tail domains and fluorescently labeled microtubules. Our experiments demonstrate a direct association of Myosin-1C with microtubules. Additionally, our experiments showed that Myosin-1C can bind to both microtubules and actin filaments via the tail domain, thus cross-linking microtubules to actin filaments. Moreover, we found that the tail domain of Myosin-1C bound to microtubules have a microtubule stabilizing function. This stabilizing effect is dependent on the Myosin-1C tail concentration. It strongly protects microtubules from cold-induced depolymerisation with an IC50 of about 25nM. A stabilizing effect of Myosin-1C is also suggested from results of studies with Myosin-1C knockout cells. These Myosin-1C deficient cells exhibit reduced microtubule densities at the mitotic spindle as well as reduced size and number of astral microtubules in comparison to wild-type cells. Studying the ternary system of single kinesin molecules, microtubules and Myosin-1C tail domain in motility assays with a TIRF microscope, we observed that the tail domain of Myosin-1C can modulate kinesin motility. With increasing Myosin-1C tail concentration the attachment frequency of kinesin molecules to microtubules decreases, whereas the velocity and run length of individual kinesin molecules along microtubules do not change with increasing Myosin-1C tail concentration.

Our results suggest that Myosin-1C can stabilize (mitotic spindle) microtubules and cross-link them to F-actin. The tail domain of Myosin-1C can also modulate the attachment of kinesin molecules to microtubules, thus possibly regulating kinesin-driven transport.