Molecular mechanisms of cell fate determinant assembly

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PROJECT SUMMARY During embryonic development cells must be correctly allocated to distinct fates needed for organismal growth and reproduction. Germ cells generate eggs and sperm and must be specified to avoid disorders of the reproductive system, including gonadal and ovarian cancers, teratomas and other germ cell tumors, and ultimately infertility. Germ cells often acquire their fate by inheriting cytoplasmic components that are maternally synthesized, membrane-free, gel-like aggregates of proteins and RNAs collectively called germ plasm. The highly conserved proteins, RNAs and organelles within germ plasm are assembled, or nucleated, by other proteins that can be different in sequence across animals, but that share similar evolutionary histories and biophysical properties. The molecular mechanisms by which these nucleators ensure assembly and function of germ plasm remain unclear. Our long-term goal is to understand the molecular mechanisms that drive the assembly and function of cytoplasmic fate determinants. In this proposal, we will elucidate the mechanisms by which the Drosophila nucleator, encoded by the oskar gene, assembles germ plasm. This proposal is significant because it has the potential to uncover generalizable principles of germ plasm assembly, which may be broadly applicable to the formation and function of membrane-less, gel-like cytoplasmic aggregates that regulate cell fates in many different contexts. Our bioinformatic discovery of hundreds of new oskar sequences, combined with X-ray crystallography and biochemical assays, suggested previously unexplored hypotheses for the molecular mechanism of oskar function, which we will test as follows: In Aim 1, we will elucidate the role of the conserved LOTUS domain in germ plasm assembly with in vitro biochemical assays and in vivo transgenic assays of the biological function and biophysical properties of germ plasm, testing hypotheses regarding the importance of dimerization, higher-order aggregate formation, phase separation, and Vasa binding to germ plasm assembly. In Aim 2, we will determine for the first time the specific sequences and structural properties of the Long Oskar Domain that enable the Long Oskar isoform to recruit and anchor mitochondria in the germ plasm. In Aim 3, we will test the novel hypothesis that the conserved OSK domain interacts with specific classes of lipids in the oocyte posterior, to help anchor Oskar to the posterior pole. Since defects in germ cell development can lead to reproductive system pathologies affecting up to 12% of the US population, elucidating the mechanisms that ensure assembly and function of germ line determinants is highly relevant to human health. More generally, germ plasm is a type of ribonucleoprotein multimer (RNP), which are membrane-free, gel-like organelles that regulate translation in both germ line and somatic cells. Understanding germ plasm assembly may thus shed light on the general principles underlying assembly of cytoplasmic RNPs required for the proper function of multiple critical cell types.