Hansson, K.-A.; Lepperod, M. E.

Skeletal muscle fibers are among the largest cells in biology and rely on the distribution of numerous nuclei to maintain intracellular transport and spatially distributed gene expression across large distances. Although myonuclei are actively positioned, how this organization adapts to changes in nuclear number remains unresolved. Here, we analyze three-dimensional myonuclear positioning across postnatal development (P13-P150) in a mouse model with reduced nuclear number. Across more than 1,000 fibers and 15,000 nuclei, reduced nuclear number leads to increased spatial regularity along the fiber axis, reflected in lower variability of inter-nuclear distances, higher Clark-Evans regularity, and the emergence of a characteristic spacing scale. Density-normalized analyses show that this effect cannot be explained by differences in fiber size or nuclear density alone. Mapping nuclei onto the cylindrical fiber surface further reveals more uniform two-dimensional coverage and increased local orientational order. Null-model analysis demonstrates that this surface organization is largely explained by enhanced longitudinal regularity, indicating that improvements in one-dimensional positioning propagate to higher-dimensional structure. Together, these findings show that myonuclear organization reflects an emergent, adaptive optimization process that enables efficient intracellular organization despite reduced nuclear number.