Folker, E. | Search bioRxiv

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↗

bioRxiv2025

The LINC complex and microtubule motors regulate the number and position of nuclei in the subperineurial glial cells of the Drosophila blood-brain barrier

Multinucleated cells, or syncytia, provide a unique system in which to understand the mechanisms of cellular organization. The two most dramatic features of syncytial cells are the number of nuclei and the positioning of nuclei within a shared cytoplasm. While the mechanisms that regulate these features have been studied in some syncytial cells, most syncytial cells are uncharacterized. Furthermore, whether the formation of the syncytia and the organization of the syncytia are linked is not known. We have characterized the subperineurial glial cells (SPG) which form the most restrictive layer of the Drosophila blood-brain barrier. We have found that disruption of the Linker of Nucleoskeleton and Cytoskeleton (LINC) complex, Kinesin, or cytoplasmic Dynein specifically in SPG cells affected both SPG cell development and general brain development. Specifically, the brains were smaller in each case and the SPG cells were smaller when the LINC complex or cytoplasmic Dynein were disrupted. The number of nuclei per cell was increased when Kinesin was disrupted, decreased when cytoplasmic Dynein was disrupted, and abnormal numbers of nuclei were found when the LINC complex was disrupted. Finally, the positions of nuclei relative to their nearest neighbor was decreased when the expression of each gene was disrupted and nuclei were closer to the cell edge when either Kinesin or cytoplasmic Dynein were disrupted. Finally, the evenness of nuclear spacing was reduced when LINC complex or Kinesin expression was disrupted. Together, these data illustrate that formation of SPG cells and the organization of SPG cells are dependent on microtubule motors and the LINC complex.

cell biology↗