Bascom Palmer Eye Institute, Department of Ophthalmology, University of Miami Miller School of Medicine, 1638 NW 10th Avenue, Miami, FL 33136, USA.
Exp Cell Res. 2010 Jan 15;316(2):245-57. doi: 10.1016/j.yexcr.2009.10.008. Epub 2009 Oct 22.
Phagocytosis is an important process for the removal of apoptotic cells or cellular debris. Eat-me signals control the initiation of phagocytosis and hold the key for in-depth understanding of its molecular mechanisms. However, because of difficulties to identify unknown eat-me signals, only a limited number of them have been identified and characterized. Using a newly developed functional cloning strategy of open reading frame (ORF) phage display, we identified nine putative eat-me signals, including tubby-like protein 1 (Tulp1). This further led to the elucidation of tubby as the second eat-me signal in the same protein family. Both proteins stimulated phagocytosis of retinal pigment epithelium (RPE) cells and macrophages. Tubby-conjugated fluorescent microbeads facilitated RPE phagocytosis. Tubby and Tulp1, but not other family members, enhanced the uptake of membrane vesicles by RPE cells in synergy. Retinal membrane vesicles of Tubby mice and Tulp1(-/-) mice showed reduced activities for RPE phagocytosis, which were compensated by purified tubby and Tulp1, respectively. These data reveal a novel activity of tubby and Tulp1, and demonstrate that unbiased identification of eat-me signals by the broadly applicable strategy of ORF phage display can provide detailed insights into phagocyte biology.
吞噬作用对于清除凋亡细胞或细胞碎片是非常重要的。“吃我”信号控制着吞噬作用的起始，为深入理解其分子机制提供了关键信息。然而，由于难以识别未知的“吃我”信号，目前仅鉴定和表征了有限数量的“吃我”信号。我们使用一种新开发的开放阅读框（ORF）噬菌体展示的功能克隆策略，鉴定了 9 个潜在的“吃我”信号，包括类土豆球蛋白 1（Tulp1）。这进一步阐明了类土豆球蛋白作为同一蛋白家族中的第二个“吃我”信号。这两种蛋白都能刺激视网膜色素上皮（RPE）细胞和巨噬细胞的吞噬作用。Tubby 偶联的荧光微球促进了 RPE 的吞噬作用。Tubby 和 Tulp1，但不是其他家族成员，协同增强了 RPE 细胞对膜泡的摄取。Tubby 小鼠和 Tulp1(-/-)小鼠的视网膜膜泡显示出 RPE 吞噬作用活性降低，而纯化的 Tubby 和 Tulp1 分别补偿了这一缺陷。这些数据揭示了 Tubby 和 Tulp1 的新功能，并证明了通过广泛适用的 ORF 噬菌体展示策略对“吃我”信号进行无偏鉴定，可以为吞噬细胞生物学提供详细的见解。
