Deutz Lars N, Sarıkaya Sena, Dickinson Daniel J
Department of Molecular Biosciences, The University of Texas at Austin, Austin, Texas.
Department of Molecular Biosciences, The University of Texas at Austin, Austin, Texas.
Biophys J. 2025 Mar 18;124(6):876-890. doi: 10.1016/j.bpj.2023.11.021. Epub 2023 Nov 23.
Embryonic development requires the establishment of cell polarity to enable cell fate segregation and tissue morphogenesis. This process is regulated by Par complex proteins, which partition into polarized membrane domains and direct downstream polarized cell behaviors. The kinase aPKC (along with its cofactor Par6) is a key member of this network and can be recruited to the plasma membrane by either the small GTPase Cdc42 or the scaffolding protein Par3. Although in vitro interactions among these proteins are well established, much is still unknown about the complexes they form during development. Here, to enable the study of membrane-associated complexes ex vivo, we used a maleic acid copolymer to rapidly isolate membrane proteins from single C. elegans zygotes into lipid nanodiscs. We show that native lipid nanodisc formation enables detection of endogenous complexes involving Cdc42, which are undetectable when cells are lysed in detergent. We found that Cdc42 interacts more strongly with aPKC/Par6 during polarity maintenance than polarity establishment, two developmental stages that are separated by only a few minutes. We further show that Cdc42 and Par3 do not bind aPKC/Par6 simultaneously, confirming recent in vitro findings in an ex vivo context. Our findings establish a new tool for studying membrane-associated signaling complexes and reveal an unexpected mode of polarity regulation via Cdc42.
胚胎发育需要建立细胞极性，以实现细胞命运分离和组织形态发生。这一过程由Par复合体蛋白调控，这些蛋白定位于极化的膜结构域，并指导下游的极化细胞行为。激酶aPKC（及其辅因子Par6）是该网络的关键成员，可通过小GTP酶Cdc42或支架蛋白Par3被招募到质膜上。尽管这些蛋白之间的体外相互作用已得到充分证实，但它们在发育过程中形成的复合物仍有许多未知之处。在这里，为了能够在体外研究膜相关复合物，我们使用马来酸共聚物从单个秀丽隐杆线虫受精卵中快速分离膜蛋白到脂质纳米盘中。我们表明，天然脂质纳米盘的形成能够检测到涉及Cdc42的内源性复合物，而当细胞在去污剂中裂解时，这些复合物是检测不到的。我们发现，在极性维持过程中，Cdc42与aPKC/Par6的相互作用比极性建立过程中更强，这两个发育阶段仅相隔几分钟。我们进一步表明，Cdc42和Par3不会同时结合aPKC/Par6，这在体外实验中证实了最近的研究结果。我们的研究结果建立了一种研究膜相关信号复合物的新工具，并揭示了一种通过Cdc42进行极性调节的意外模式。
