PgmNr Z649: Microtubule-actin crosslinking factor (Macf1) Function in Oocyte Polarity and Nuclear Positioning.

Authors:
M. Escobar-Aguirre; H. Zhang; R. Fuentes; A. Jamieson-Lucy; M. C. Mullins


Institutes
University of Pennsylvania, Cell and Developmental Biology, Philadelphia, PA.


Abstract:

Cell polarity allows cells to regionalize their intracellular environment to perform a variety of specialized functions. In most vertebrates, polarity of the egg defines the anterior-posterior and dorsal-ventral axes of the embryo. Egg animal-vegetal (AV) polarity is derived from oocyte polarity, which is established early in oogenesis . AV polarity is established through the formation, translocation and disassembly of the Balbiani Body (Bb) at the vegetal pole. The Bb is conserved from insects to humans and is composed of organelles, RNAs and proteins. The Bb components, which include germ cell determinants, anchor to the vegetal cortex upon Bb disassembly in late stage I oocytes. Our lab discovered in zebrafish the only genes known to function in AV polarity formation in vertebrates: bucky ball and macf1. While Bucky ball is required for Bb formation, Macf1 is crucial for its disassembly. Macf1 is a conserved multi-domain cytoskeletal linker protein that can interact with microtubules (MTs), actin filaments (AF) and intermediate filaments (IF). Zebrafish macf1 mutant oocytes display an: 1) enlarged Bb, 2) acentric nucleus, and 3) a failure of Bb disassembly. Our aim is to elucidate the Macf1 dependent cytoskeletal linking mechanism that regulates AV polarity. First, we determined that Macf1 protein localizes perinuclearly, to the Bb, and upon Bb disassembly it distributes to the vegetal cortex. Second, we examined cytoskeleton function. Interestingly, disruption of AF phenocopies the macf1 mutant phenotype. We found that cytokeratins (CK)- a type of intermediate filament- localize around the nucleus, are enriched in the Bb and exhibit a peripheral distribution in the cytoplasm in late stage I oocytes. These findings suggest that Macf1 Actin binding domain (ABD) and Plectin repeat domain (PRD) integrate cortical actin and CK to mediate Bb disassembly. To specifically test if the Macf1 actin binding domain (ABD) and Plectin repeat domain (PRD) function in Bb disassembly and nuclear positioning, we deleted the exons encoding these domains from the macf1 endogenous locus by using Crispr/Cas9 technology. We found that Macf1Del-ABD mutant oocytes display the macf1 null mutant phenotype whereby the nucleus is acentric and the Bb fails to disassemble. Importantly, Macf1Del-ABD protein is localized to the Bb like in wt. Surprisingly, deleting the PRD domain (Macf1Del-PRD) does not affect Bb disassembly or nuclear positioning. In summary, we determined that Macf1 functions via its ABD and actin filaments to mediate Bb disassembly and nuclear positioning, while the PRD is dispensable. To our knowledge, this is the first study to use genome editing to unravel the module-dependent function of a cytoskeletal linker in the context of cell polarity establishment.



ZFIN Genetics Index
1. macf1a