Mechanisms Establishing Oocyte Polarity

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 DESCRIPTION (provided by applicant): Cell polarity is essential to formation of epithelial tissues, organ formation, and to embryonic axis formation in many animals. In most vertebrates, polarity of the egg determines anterior-posterior polarity of the embryo, and is essential to establishing the embryonic dorsal-ventral axis. Egg polarity originates during early stages of oogenesis when distinct animal-vegetal domains are established in the oocyte. The first polarized structure in vertebrate oocytes is the Balbiani body (Bb), a structure conserved from insects to vertebrates. The Bb in frogs and fish is composed of ribonucleoproteins, germ plasm, an aggregate of mitochondria, and RNAs destined to the vegetal pole of the oocyte and egg, and to the future germ cells of the embryo. The Bb is a transient structure that moves to the oocyte cortex, where it dissociates delivering its contents and determining the vegetal pole. Although fundamental to forming the major axes of most vertebrate embryos, vertebrate oocyte polarity has been little studied due to the inaccessibility of these early oocyte stages within the ovary and the difficulty of genetic analysis in adult females. Through a maternal-effect mutant screen in the zebrafish, two genes were discovered that establish oocyte polarity, bucky ball (buc) and macf1 (microtubule actin crosslinking factor 1), the first and only genes known to function in vertebrate oocyte polarity. Females mutant for buc fail to form the Bb during oogenesis, and do not establish oocyte polarity. In macf1 mutant oocytes the Bb forms but fails to disassemble, disrupting oocyte polarity, and the nucleus is asymmetrically positioned. Macf1 is an extraordinarily large cytoskeletal linker protein of the spectraplakin family. It contains an actin binding domain, a Microtubule binding domain (MTBD), Plakin repeat domains that can interact with Cytokeratin filaments (CK), and a series of spectrin repeats. It is hypothesized and will be tested that Macf1 has dual functions in the oocyte linking Bb contents to the actin cortex via CK in Bb disassembly, and positioning the nucleus by linking perinuclear CK to cytoplasmic MTs. The Bb is a large membrane-free organelle or RNA granule, similar to stress granules, P bodies, and P granules. A key property of these granules is their ability to undergo phase transitions into granule form or dissolution into non-granule form, and the dependence of the phase transition on intrinsically disordered proteins (IDP). Buc, the only protein known to be required for Bb formation, is an IDP. Here Bb granule dynamics and properties are investigated, including the nature of the IDP Buc. These proteins provide the first functional entry points to understanding how the Bb is regulated in its function to establish oocyte polarity. Importantly, inappropriate protein granule aggregates in cells is a major cause of disease, including neurodegenerative diseases of the synucleinopathies. These studies provide an in vivo paradigm for granule formation, function, regulation, and disassembly, which is significantly lacking in this field.