Escobar-Aguirre Matias, Zhang Hong, Jamieson-Lucy Allison, Mullins Mary C
Department of Cell and Developmental Biology, University of Pennsylvania Perelman School of Medicine, 1152 BRBII/III, 421 Curie Blvd., Philadelphia, Pennsylvania, United States of America.
PLoS Genet. 2017 Sep 7;13(9):e1006983. doi: 10.1371/journal.pgen.1006983. eCollection 2017 Sep.
Animal-vegetal (AV) polarity of most vertebrate eggs is established during early oogenesis through the formation and disassembly of the Balbiani Body (Bb). The Bb is a structure conserved from insects to humans that appears as a large granule, similar to a mRNP granule composed of mRNA and proteins, that in addition contains mitochondria, ER and Golgi. The components of the Bb, which have amyloid-like properties, include germ cell and axis determinants of the embryo that are anchored to the vegetal cortex upon Bb disassembly. Our lab discovered in zebrafish the only gene known to function in Bb disassembly, microtubule-actin crosslinking factor 1a (macf1a). Macf1 is a conserved, giant multi-domain cytoskeletal linker protein that can interact with microtubules (MTs), actin filaments (AF), and intermediate filaments (IF). In macf1a mutant oocytes the Bb fails to dissociate, the nucleus is acentric, and AV polarity of the oocyte and egg fails to form. The cytoskeleton-dependent mechanism by which Macf1a regulates Bb mRNP granule dissociation was unknown. We found that disruption of AFs phenocopies the macf1a mutant phenotype, while MT disruption does not. We determined that cytokeratins (CK), a type of IF, are enriched in the Bb. We found that Macf1a localizes to the Bb, indicating a direct function in regulating its dissociation. We thus tested if Macf1a functions via its actin binding domain (ABD) and plectin repeat domain (PRD) to integrate cortical actin and Bb CK, respectively, to mediate Bb dissociation at the oocyte cortex. We developed a CRISPR/Cas9 approach to delete the exons encoding these domains from the macf1a endogenous locus, while maintaining the open reading frame. Our analysis shows that Macf1a functions via its ABD to mediate Bb granule dissociation and nuclear positioning, while the PRD is dispensable. We propose that Macf1a does not function via its canonical mechanism of linking two cytoskeletal systems together in dissociating the Bb. Instead our results suggest that Macf1a functions by linking one cytoskeletal system, cortical actin, to another structure, the Bb, where Macf1a is localized. Through this novel linking process, it dissociates the Bb at the oocyte cortex, thus specifying the AV axis of the oocyte and future egg. To our knowledge, this is also the first study to use genome editing to unravel the module-dependent function of a cytoskeletal linker.
大多数脊椎动物卵子的动植物（AV）极性在早期卵子发生过程中通过巴尔比亚尼体（Bb）的形成和分解而确立。Bb是一种从昆虫到人类都保守的结构，表现为一个大颗粒，类似于由mRNA和蛋白质组成的mRNP颗粒，此外还包含线粒体、内质网和高尔基体。具有淀粉样特性的Bb成分包括胚胎的生殖细胞和轴决定因子，它们在Bb分解后锚定在植物性皮质上。我们实验室在斑马鱼中发现了唯一已知在Bb分解中起作用的基因，微管-肌动蛋白交联因子1a（macf1a）。Macf1是一种保守的、巨大的多结构域细胞骨架连接蛋白，可与微管（MTs）、肌动蛋白丝（AF）和中间丝（IF）相互作用。在macf1a突变的卵母细胞中，Bb无法解离，细胞核无中心，卵母细胞和卵子的AV极性无法形成。Macf1a调节Bb mRNP颗粒解离的细胞骨架依赖性机制尚不清楚。我们发现破坏AF会模拟macf1a突变体表型，而破坏MT则不会。我们确定细胞角蛋白（CK），一种IF，在Bb中富集。我们发现Macf1a定位于Bb，表明其在调节Bb解离中具有直接功能。因此，我们测试了Macf1a是否通过其肌动蛋白结合结构域（ABD）和网蛋白重复结构域（PRD）分别整合皮质肌动蛋白和Bb CK，以介导卵母细胞皮质处的Bb解离。我们开发了一种CRISPR/Cas9方法，从macf1a内源基因座中删除编码这些结构域的外显子，同时保持开放阅读框。我们的分析表明，Macf1a通过其ABD介导Bb颗粒解离和核定位，而PRD是可有可无的。我们提出，Macf1a在解离Bb时并非通过其将两个细胞骨架系统连接在一起的经典机制发挥作用。相反，我们的结果表明，Macf1a通过将一个细胞骨架系统，即皮质肌动蛋白，与另一个结构，即Macf1a所在的Bb连接起来发挥作用。通过这个新的连接过程，它在卵母细胞皮质处解离Bb，从而确定卵母细胞和未来卵子的AV轴。据我们所知，这也是第一项使用基因组编辑来揭示细胞骨架连接蛋白模块依赖性功能的研究。
