Klp61f and ncd function as an accelerator and brake to regulate myonuclear spacing

One of the first genes identified to regulate the spacing of nuclei in the multinucleated myofiber was Kinesin-1. However, the mechanism by which Kinesin-1 or other kinesins regulate myonuclear spacing is not known. Critically, the myofiber lacks centrosomes, and the many myonuclei act as the primary microtubule organizing centers of the cell. Because of this unique re-structuring, we hypothesized that the kinesins that drive centrosomes apart during mitotic spindle elongation may play a similar role in spacing myonuclei. We found that the bipolar Kinesin-5 (Klp61f) and the (-)-end directed Kinesin-14 (ncd) were both necessary for myonuclear spacing at different times, with both being necessary during embryogenesis, but only ncd being necessary in the fully differentiated myofiber. To investigate the shared mechanisms during embryogenesis, we used live-imaging and found that, similar to the mitotic spindle, Klp61f acts as an accelerator for myonuclear movement, whereas ncd acts as a brake contrary to this movement. To investigate these mechanisms and test the hypothesis that this is dependent on microtubule-sliding, we used super-resolution microscopy to visualize and quantify the microtubule network in animals with disrupted Klp61f or ncd. We found that in both cases, there was a decrease in the amount of microtubule overlap between neighboring myonuclei. Furthermore, we found that disrupting ncd led to severe changes in microtubule network organization, supporting our hypotheses that microtubule-sliding is necessary to space myonuclei, and that ncd likely functions through a unique mechanism in the differentiated myofiber to maintain myonuclear spacing. Together, our data supports a model where myonuclear spacing is regulated by a counteracting force generated by different kinesins during embryonic development. Furthermore, one kinesin, ncd, is repurposed in the differentiated myofiber to dynamically crosslink microtubules, a function necessary to anchor nuclei in place. Thus, kinesin motors regulate myonuclear spacing across developmental time by leveraging opposing forces through diverse mechanisms.