Padilla, J. R. | bioRxiv Science

bioRxiv2026

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.

cell biology↗

bioRxiv2024

Dystrophin interacts with Msp300 to Regulate Myonuclear Positioning and Microtubule Organization

During Drosophila myognesis, myonuclei are actively moved during embryogenesis, and their spacing is maintained through an anchoring mechanism in the fully differentiated myofiber. While we have identified microtubule associated proteins, motors, and nuclear envelope proteins that regulate myonuclear spacing, the developmental time during which each gene functions has not been tested. Here we have identified a Dystrophin as required only for the maintenance of myonuclear spacing. Furthermore, we demonstrate that Dystrophin genetically interacts with the KASH-domain protein Msp300 to maintain myonuclear spacing. Mechanistically, both Dystrophin and Msp300 regulate microtubule organization. Specifically, in animals with disrupted expression of both Dystrophin and Msp300, microtubule colocalization with sarcomeres is reduced. Taken altogether, these data indicate that the peripheral membrane protein Dystrophin, and the outer nuclear membrane protein Msp300, together regulate the organization of the microtubule network which then acts as an anchor to restrict myonuclear movement in contractile myofibers. These data are consistent with growing evidence that myonuclear movement and myonuclear spacing are critical to muscle development, muscle function, and muscle repair and provide a mechanism to connect disparate muscle diseases. Summary StatementHere we show that Dystrophin is required to maintain the spacing of nuclei in differentiated myofibers. Furthermore, Dystrophin achieves this function via a genetic interaction with Msp300 which regulates microtubule organization.

cell biology↗