The cell nucleus is positioned at specific places within the cytoplasm and this position is important for different cellular, developmental and physiological processes. Nuclear positioning depends on connections between nuclear envelope proteins and the cytoskeleton. In migrating cells, we found that the nucleus is positioned away from the front of the cell and this event is important for cell migration. We performed an RNAi screen for nuclear positioning and found new nuclear envelope proteins involved in nuclear positioning. In fully developed myofibers, nuclei are specifically positioned at the periphery of the myofiber, while during development and regeneration, as well as in multiple muscle pathologies, the nucleus is centrally positioned. We found new mechanisms drive nuclear movement during myofiber formation. We also showed that nuclear position is important for muscle function. However why nuclear positioning is important for myofiber activity remains an open question.We now propose to use unique systems to monitor cell migration and myofiber formation in combination with biochemistry, cell biology, high- and super-resolution microscopy approaches to:1) Identify novel molecular mechanisms that mediate nuclear positioning during cell migration and myofiber formation.3) Determine a role for nuclear positioning in myofiber function as well as the significance of altered nuclear positioning in different forms of muscle pathology.The proposed work will establish new mechanisms for nuclear positioning. Importantly, by identifying mechanisms and understanding the role of nuclear positioning in myofiber function, we will lay the foundations for future studies to ameliorate or treat muscle disorders as well as other conditions where nucleus positioning may prove to play a role such as cancer.