In fruit flies as in humans, skeletal muscle cells are multinucleated with nuclei evenly spaced throughout the cell. The mechanisms by which myonuclei are correctly positioned and the consequences of mispositioning are poorly understood. In Drosophila, myonuclei are initially grouped together in immature muscle cells, prior to moving to the locations that they occupy in fully differentiated muscle. Our group has previously demonstrated that the conserved microtubule (MT) associated protein Ensconsin (Ens) is essential for myonuclear movement in the embryo. Recently, through a yeast two-hybrid screen, we identified Bsg25D (human ortholog: Ninein), a MT-anchoring protein, as an Ens binding partner. Muscle-specific overexpression of Bsg25D causes a complete block in myonuclear movement in the embryo, recapitulating the ens null phenotype. Genetic interaction and rescue experiments combined with colocalization studies suggest that exogenous Bsg25D exerts its effect by sequestering Ens away from MTs where it normally functions. Later in development, Bsg25D-overexpressing larval muscles exhibit severely mispositioned nuclei and dramatic alterations in MT organization; larval motility is reduced, demonstrating a loss of muscle activity. To complement our overexpression studies, we generated a bsg25D null mutant. Even though bsg25D mutant muscles are normal, loss of bsg25D enhances myonuclear positioning defects in ens heterozygotes. Similarly, bsg25D mutant flies have nearly wildtype viability, but loss of bsg25D in an ens-compromised background causes embryonic lethality. We are currently investigating the hypothesis that an Ens-Bsg25D interaction regulates MT dynamics at the myonuclear envelope, a critical MT organizing center in muscle cells (which lack centrosomes). The functions of centrosomal proteins in muscle are unknown, and this is the first evidence for a centrosomal protein participating in myonuclear positioning. As certain muscular dystrophies characteristically feature mispositioned myonuclei, our results will shed light on human disease.