Genome Editing Approach to Uncover Microtubule-Actin Crosslinking Factor (Macf1) Essential Domains in Establishing Oocyte Polarity and Nuclear Positioning

The totipotent egg of most vertebrates is polarized in a so called animal-vegetal (AV) axis that is crucial for early embryonic development. AV polarity is established during early oogenesis through the formation and disassembly of the Balbiani Body (Bb) at the vegetal pole. The Bb is a non-membrane bound large mRNP granule, conserved from insects to humans and composed of mitochondria, RNAs and proteins. The Bb components, which include germ cell determinants, anchor to the vegetal cortex upon Bb dissociation in late stage I oocytes. Importantly, Bb dissociation at the oocyte cortex defines the future vegetal pole of the egg. Our lab discovered in zebrafish the only genes known to function in AV polarity formation in vertebrates: bucky ball and macf1. On one hand, Bucky ball is required for Bb formation, and is thought to act by the formation of amyloid-like fibers that capture Bb components. On the other hand, Macf1 is crucial for Bb dissociation. Macf1 is a conserved and giant multi-domain cytoskeletal linker protein that can interact with microtubules (MTs), actin filaments (AF) and intermediate filaments (IF). Macf1 is the only factor known to regulate Bb dissociation, however the Macf1 and cytoskeleton-dependent mechanism by which Macf1 regulates Bb mRNP granule dissociation and, thus, defines AV polarity in the egg is unknown. Here, we unravel Macf1 function via interrogating, for the first time, individual macf1-encoded domains from its endogenous locus to determine their requirement in Bb dissociation and ultimately in egg polarity establishment. Our results show that the Macf1 actin binding domain is essential for Bb dissociation, whereas the Macf1 plakin repeat domain, which interacts with IF, is dispensable for Macf1 function in this context. The method presented here is applicable to other cytolinkers involved in human diseases