Organelle positioning in muscles requires cooperation between two KASH proteins and microtubules.

Striated muscle fibers are characterized by their tightly organized cytoplasm. Here, we show that the Drosophila melanogaster KASH proteins Klarsicht (Klar) and MSP-300 cooperate in promoting even myonuclear spacing by mediating a tight link between a newly discovered MSP-300 nuclear ring and a polarized network of astral microtubules (aMTs). In either klar or msp-300(ΔKASH), or in klar and msp-300 double heterozygous mutants, the MSP-300 nuclear ring and the aMTs retracted from the nuclear envelope, abrogating this even nuclear spacing. Anchoring of the myonuclei to the core acto-myosin fibrillar compartment was mediated exclusively by MSP-300. This protein was also essential for promoting even distribution of the mitochondria and ER within the muscle fiber. Larval locomotion is impaired in both msp-300 and klar mutants, and the klar mutants were rescued by muscle-specific expression of Klar. Thus, our results describe a novel mechanism of nuclear spacing in striated muscles controlled by the cooperative activity of MSP-300, Klar, and astral MTs, and demonstrate its physiological significance.