Cell polarity is fundamental to the biology of most cells and is characterized by the asymmetric distribution of factors at the cell cortex (the region just beneath the plasma membrane) and in the cytoplasm. A conserved set of proteins, the PAR (PARtitioning defective) proteins regulate polarity in most polarized animal cells. While we know a great deal about how they generate cortical asymmetries, little is known about how cortical PAR proteins segregate diffusive proteins in the cytoplasm. In the C. elegans zygote, the PAR proteins localize to two complementary domains at the cell cortex from which they orchestrate cellular polarities. The posterior kinase PAR-1 drives the segregation of the essential protein MEX-5 to the anterior cytoplasm. In turn, MEX-5 drives the segregation of germ line determinants (collectively, the germ plasm) to the anterior cytoplasm. The conserved mitotic kinase PLK-1 is recruited by MEX-5 to the anterior cytoplasm and is important for germ plasm segregation. We recently found that PLK-1 phosphorylates one germ plasm protein, POS-1, to inhibit its retention in the anterior, thereby driving its segregation to the posterior. In the proposed work, we will identify additional substrates of PLK-1 that mediate germ plasm segregation. In addition, we will determine the role of the endoplasmic reticulum and mitochondrial networks in scaffolding cytoplasmic asymmetries. Because the core polarity regulators are conserved, knowledge of the principles and mechanisms that establish asymmetries in the C. elegans zygote will provide a foundation for understanding how asymmetries are generated in human cells.