Department of Cell, Developmental and Regenerative Biology, Icahn School of Medicine at Mount Sinai, New York, NY, United States of America.
PLoS One. 2021 Oct 28;16(10):e0259068. doi: 10.1371/journal.pone.0259068. eCollection 2021.
Wilms tumor-1-interacting protein (Wtip) is a LIM-domain-containing adaptor that links cell junctions with actomyosin complexes and modulates actomyosin contractility and ciliogenesis in Xenopus embryos. The Wtip C-terminus with three LIM domains associates with the actin-binding protein Shroom3 and modulates Shroom3-induced apical constriction in ectoderm cells. By contrast, the N-terminal domain localizes to apical junctions in the ectoderm and basal bodies in skin multiciliated cells, but its interacting partners remain largely unknown. Targeted proximity biotinylation (TPB) using anti-GFP antibody fused to the biotin ligase BirA identified SSX2IP as a candidate protein that binds GFP-WtipN. SSX2IP, also known as Msd1 or ADIP, is a component of cell junctions, centriolar satellite protein and a targeting factor for ciliary membrane proteins. WtipN physically associated with SSX2IP and the two proteins readily formed mixed aggregates in overexpressing cells. By contrast, we observed only partial colocalization of full length Wtip and SSX2IP, suggesting that Wtip adopts a 'closed' conformation in the cell. Furthermore, the double depletion of Wtip and SSX2IP in early embryos uncovered the functional interaction of the two proteins during neural tube closure. Our results suggest that the association of SSX2IP and Wtip is essential for cell junction remodeling and morphogenetic processes that accompany neurulation. We propose that TPB can be a general approach that is applicable to other GFP-tagged proteins.
Wilms 肿瘤 1 相互作用蛋白（Wtip）是一种 LIM 结构域包含的衔接蛋白，它将细胞连接与肌动球蛋白复合物连接起来，并调节 Xenopus 胚胎中的肌动球蛋白收缩和纤毛发生。具有三个 LIM 结构域的 Wtip C 末端与肌动蛋白结合蛋白 Shroom3 结合，并调节 Shroom3 在外胚层细胞中的顶端收缩。相比之下，N 端结构域定位于外胚层的顶端连接和皮肤多纤毛细胞中的基体，但它的相互作用伙伴在很大程度上仍然未知。使用融合了生物素连接酶 BirA 的抗 GFP 抗体进行靶向邻近生物素化（TPB），鉴定出 SSX2IP 作为与 GFP-WtipN 结合的候选蛋白。SSX2IP，也称为 Msd1 或 ADIP，是细胞连接的组成部分、中心粒卫星蛋白和纤毛膜蛋白的靶向因子。WtipN 与 SSX2IP 物理结合，并且这两种蛋白质在过表达细胞中很容易形成混合聚集体。相比之下，我们仅观察到全长 Wtip 和 SSX2IP 的部分共定位，表明 Wtip 在细胞中采用“封闭”构象。此外，在早期胚胎中同时耗尽 Wtip 和 SSX2IP 揭示了这两种蛋白质在神经管闭合过程中的功能相互作用。我们的结果表明，SSX2IP 和 Wtip 的关联对于伴随神经管闭合的细胞连接重塑和形态发生过程是必不可少的。我们提出，TPB 可以是一种适用于其他 GFP 标记蛋白的通用方法。
