Wang Sicong, Lin Chien-Wei, Carleton Amber E, Cortez Chari L, Johnson Craig, Taniguchi Linnea E, Sekulovski Nikola, Townshend Ryan F, Basrur Venkatesha, Nesvizhskii Alexey I, Zou Peng, Fu Jianping, Gumucio Deborah L, Duncan Mara C, Taniguchi Kenichiro
Department of Mechanical Engineering, University of Michigan, Ann Arbor, MI 48109, USA.
Department of Cell Biology, Neurobiology and Anatomy, Medical College of Wisconsin, Milwaukee, WI 53226, USA.
Sci Adv. 2021 Apr 23;7(17). doi: 10.1126/sciadv.abd8407. Print 2021 Apr.
Critical early steps in human embryonic development include polarization of the inner cell mass, followed by formation of an expanded lumen that will become the epiblast cavity. Recently described three-dimensional (3D) human pluripotent stem cell-derived cyst (hPSC-cyst) structures can replicate these processes. To gain mechanistic insights into the poorly understood machinery involved in epiblast cavity formation, we interrogated the proteomes of apical and basolateral membrane territories in 3D human hPSC-cysts. APEX2-based proximity bioinylation, followed by quantitative mass spectrometry, revealed a variety of proteins without previous annotation to specific membrane subdomains. Functional experiments validated the requirement for several apically enriched proteins in cyst morphogenesis. In particular, we found a key role for the AP-1 clathrin adaptor complex in expanding the apical membrane domains during lumen establishment. These findings highlight the robust power of this proximity labeling approach for discovering novel regulators of epithelial morphogenesis in 3D stem cell-based models.
人类胚胎发育的关键早期步骤包括内细胞团的极化，随后形成一个将成为上胚层腔的扩大管腔。最近描述的三维（3D）人多能干细胞衍生囊肿（hPSC-囊肿）结构可以复制这些过程。为了深入了解上胚层腔形成过程中鲜为人知的机制，我们对3D人hPSC-囊肿中顶端和基底外侧膜区域的蛋白质组进行了研究。基于APEX2的邻近生物素化，随后进行定量质谱分析，揭示了多种以前未注释到特定膜亚结构域的蛋白质。功能实验验证了几种顶端富集蛋白在囊肿形态发生中的必要性。特别是，我们发现AP-1网格蛋白衔接复合体在管腔形成过程中扩展顶端膜结构域方面起着关键作用。这些发现凸显了这种邻近标记方法在基于3D干细胞模型中发现上皮形态发生新调节因子方面的强大作用。
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