Beemiller Peter, Hoppe Adam D, Swanson Joel A
Cellular and Molecular Biology Graduate Program, University of Michigan Medical School, Ann Arbor, Michigan, USA.
PLoS Biol. 2006 Jun;4(6):e162. doi: 10.1371/journal.pbio.0040162. Epub 2006 May 9.
Fcgamma receptor (FcgammaR)-mediated phagocytosis of IgG-coated particles is regulated by 3'-phosphoinositides (3'PIs) and several classes of small GTPases, including ARF6 from the ADP Ribosylation Factor subfamily. The insensitivity of phagocytosis to brefeldin A (BFA), an inhibitor of certain ARF guanine nucleotide exchange factors (GEFs), previously indicated that ARF1 did not participate in phagocytosis. In this study, we show that ARF1 was activated during FcgammaR-mediated phagocytosis and that blocking normal ARF1 cycling inhibited phagosome closure. We examined the distributions and activation patterns of ARF6 and ARF1 during FcgammaR-mediated phagocytosis using fluorescence resonance energy transfer (FRET) stoichiometric microscopy of macrophages expressing CFP- or YFP-chimeras of ARF1, ARF6, and a GTP-ARF-binding protein domain. Both GTPases were activated by BFA-insensitive factors at sites of phagocytosis. ARF6 activation was restricted to the leading edge of the phagocytic cup, while ARF1 activation was delayed and delocalized over the phagosome. Phagocytic cups formed after inhibition of PI 3-kinase (PI-3K) contained persistently activated ARF6 and minimally activated ARF1. This indicates that a PI-3K-dependent signal transition defines the sequence of ARF GTPase activation during phagocytosis and that ARF6 and ARF1 coordinate different functions at the forming phagosome.
Fcγ受体（FcγR）介导的IgG包被颗粒的吞噬作用受3'-磷酸肌醇（3'PIs）和几类小GTP酶调节，包括ADP核糖基化因子亚家族的ARF6。吞噬作用对布雷菲德菌素A（BFA）不敏感，BFA是某些ARF鸟嘌呤核苷酸交换因子（GEFs）的抑制剂，这表明ARF1不参与吞噬作用。在本研究中，我们发现ARF1在FcγR介导的吞噬作用过程中被激活，并且阻断正常的ARF1循环会抑制吞噬体的封闭。我们使用表达ARF1、ARF6和GTP-ARF结合蛋白结构域的CFP或YFP嵌合体的巨噬细胞的荧光共振能量转移（FRET）化学计量显微镜，研究了FcγR介导的吞噬作用过程中ARF6和ARF1的分布及激活模式。两种GTP酶在吞噬作用位点均被对BFA不敏感的因子激活。ARF6的激活局限于吞噬杯的前缘，而ARF1的激活延迟且在吞噬体上分布不均。抑制PI 3激酶（PI-3K）后形成的吞噬杯含有持续激活的ARF6和极少激活的ARF1。这表明PI-3K依赖性信号转换定义了吞噬作用过程中ARF GTP酶激活的顺序，并且ARF6和ARF1在形成中的吞噬体上协调不同的功能。
