Department of Biotechnology, Graduate School of Agricultural and Life Sciences, The University of Tokyo, Bunkyo-ku, Tokyo 113-8657, Japan.
Department of Cell Biology, Graduate School of Medical and Dental Science, Tokyo Medical and Dental University, Bunkyo-ku, Tokyo 113-8510, Japan.
J Cell Sci. 2017 Sep 15;130(18):3158-3172. doi: 10.1242/jcs.194126. Epub 2017 Aug 4.
Macropinocytosis involves the uptake of large volumes of fluid, which is regulated by various small GTPases. The protein GflB is a guanine nucleotide exchange factor (GEF) of Rap1, and is involved in chemotaxis. Here, we studied the role of GflB in macropinocytosis, phagocytosis and cytokinesis. In plate culture of vegetative cells, compared with the parental strain AX2, -knockout (KO) cells were flatter and more polarized, whereas GflB-overproducing cells were rounder. The -KO cells exhibited impaired crown formation and retraction, particularly retraction, resulting in more crowns (macropinocytic cups) per cell and longer crown lifetimes. Accordingly, -KO cells showed defects in macropinocytosis and also in phagocytosis and cytokinesis. F-actin levels were elevated in -KO cells. GflB localized to the actin cortex most prominently at crowns and phagocytic cups. The villin headpiece domain (VHP)-like N-terminal domain of GflB directly interacted with F-actin Furthermore, a domain enriched in basic amino acids interacted with specific membrane cortex structures such as the cleavage furrow. In conclusion, GflB acts as a key local regulator of actin-driven membrane protrusion possibly by modulating Rap1 signaling pathways.
巨胞饮作用涉及大量液体的摄取，这受各种小 GTP 酶的调节。蛋白 GflB 是 Rap1 的鸟嘌呤核苷酸交换因子（GEF），参与趋化作用。在这里，我们研究了 GflB 在巨胞饮作用、吞噬作用和胞质分裂中的作用。在营养细胞的平板培养中，与亲本菌株 AX2 相比，-敲除（KO）细胞更平坦且极化，而 GflB 过表达细胞更圆。-KO 细胞表现出冠形成和回缩受损，特别是回缩，导致每个细胞的冠（巨胞饮杯）更多，冠寿命更长。因此，-KO 细胞在巨胞饮作用以及吞噬作用和胞质分裂中都存在缺陷。-KO 细胞中的 F-肌动蛋白水平升高。GflB 主要定位于冠和吞噬杯中肌动蛋白皮质。GflB 的 villin 头部结构域（VHP）样 N 端结构域直接与 F-肌动蛋白相互作用。此外，富含碱性氨基酸的结构域与特定的膜皮质结构（如分裂沟）相互作用。总之，GflB 作为肌动蛋白驱动的膜突起的关键局部调节剂，可能通过调节 Rap1 信号通路发挥作用。
