Parra Amalia S, Moezzi Cameron A, Johnston Christopher A
Department of Biology, University of New Mexico, Albuquerque, NM, United States.
Front Cell Dev Biol. 2023 Aug 16;11:1220529. doi: 10.3389/fcell.2023.1220529. eCollection 2023.
Asymmetric cell division (ACD) allows stem cells to generate differentiating progeny while simultaneously maintaining their own pluripotent state. ACD involves coupling mitotic spindle orientation with cortical polarity cues to direct unequal segregation of cell fate determinants. In neural stem cells (neuroblasts; NBs), spindles orient along an apical-basal polarity axis through a conserved complex of Partner of Inscuteable (Pins; human LGN) and Mushroom body defect (Mud; human NuMA). While many details of its function are well known, the molecular mechanics that drive assembly of the cortical Pins/Mud complex remain unclear, particularly with respect to the mutually exclusive Pins complex formed with the apical scaffold protein Inscuteable (Insc). Here we identify Hu li tai shao (Hts; human Adducin) as a direct Mud-binding protein, using an aldolase fold within its head domain (Hts) to bind a short Mud coiled-coil domain (Mud) that is adjacent to the Pins-binding domain (Mud). Hts is expressed throughout the larval central brain and apically polarizes in mitotic NBs where it is required for Mud-dependent spindle orientation. analyses reveal that Pins undergoes liquid-liquid phase separation with Mud, but not with Insc, suggesting a potential molecular basis for differential assembly mechanics between these two competing apical protein complexes. Furthermore, we find that Hts binds an intact Pins/Mud complex, reduces the concentration threshold for its phase separation, and alters the liquid-like property of the resulting phase separated droplets. Domain mapping and mutational analyses implicate critical roles for both multivalent interactions (via Mud oligomerization) and protein disorder (via an intrinsically disordered region in Hts; Hts) in phase separation of the Hts/Mud/Pins complex. Our study identifies a new component of the spindle positioning machinery in NBs and suggests that phase separation of specific protein complexes might regulate ordered assembly within the apical domain to ensure proper signaling output.
不对称细胞分裂（ACD）使干细胞能够产生分化的子代细胞，同时维持自身的多能状态。ACD涉及将有丝分裂纺锤体定向与皮质极性线索相耦合，以指导细胞命运决定因子的不均等分离。在神经干细胞（成神经细胞；NBs）中，纺锤体通过保守的Inscuteable伴侣（Pins；人类LGN）和蘑菇体缺陷（Mud；人类NuMA）复合物沿顶-基极性轴定向。虽然其功能的许多细节已为人所知，但驱动皮质Pins/Mud复合物组装的分子机制仍不清楚，特别是关于与顶端支架蛋白Inscuteable（Insc）形成的互斥Pins复合物。在这里，我们鉴定出Hu li tai shao（Hts；人类内收蛋白）是一种直接与Mud结合的蛋白，它利用其头部结构域（Hts）内的醛缩酶折叠来结合与Pins结合结构域（Mud）相邻的短Mud卷曲螺旋结构域（Mud）。Hts在整个幼虫中枢脑中表达，并在有丝分裂的NBs中顶端极化，在那里它是Mud依赖性纺锤体定向所必需的。分析表明，Pins与Mud发生液-液相分离，但与Insc不发生，这表明这两种竞争性顶端蛋白复合物之间差异组装机制的潜在分子基础。此外，我们发现Hts结合完整的Pins/Mud复合物，降低其相分离的浓度阈值，并改变所得相分离液滴的类液体性质。结构域映射和突变分析表明，多价相互作用（通过Mud寡聚化）和蛋白质无序（通过Hts中一个内在无序区域；Hts）在Hts/Mud/Pins复合物的相分离中都起关键作用。我们的研究鉴定出NBs中纺锤体定位机制的一个新成分，并表明特定蛋白复合物的相分离可能调节顶端结构域内的有序组装，以确保正确的信号输出。
