Sequential activities of phosphoinositide 3-kinase, PKB/Aakt, and Rab7 during macropinosome formation in Dictyostelium.
Sequential activities of phosphoinositide 3-kinase, PKB/Aakt, and Rab7 during macropinosome formation in Dictyostelium.
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DOI：
10.1091/mbc.12.9.2813
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发表时间：
2001-09
影响因子：
3.3
通讯作者：
A. Rupper;Kyung Lee;David A. Knecht;J. Cardelli
中科院分区：
文献类型：
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作者：
A. Rupper;Kyung Lee;David A. Knecht;J. Cardelli
巨噬细胞吞噬在树突状细胞对抗原的内化过程中起着重要作用，也是许多细菌病原体的进入途径；然而，调控巨噬细胞形成或成熟的分子机制却知之甚少。与树突状细胞一样，网柄扁平苔藓也活跃在巨噬细胞吞噬过程中，各种蛋白质已被确定参与了这一过程。正如这里所描述的，对缺失突变的显微镜分析表明，I类磷脂酰肌醇3-激酶PIK1和PIK2以及下游效应蛋白激酶B(PKB/Akt)在调节巨噬细胞吞噬完成过程中起重要作用。尽管在这些细胞系中形成了富含肌动蛋白的膜突起，但它们退缩而不形成大胞体。表达绿色荧光蛋白(GFP)的细胞融合到PKB的Pleckstrin同源域(PH)(GFP-PHPKB)的成像结果表明，D3磷脂酰肌醇在形成的大胞杯中富含，并与新形成的大胞体结合1分钟。融合蛋白由融合到F-肌动蛋白结合区的GFP组成，与GFP-PHPKB在结合形成大胞体的时间上重叠。虽然在表达显性阴性Rab7的细胞中巨噬细胞吞噬减少，但显微镜成像研究表明，GFP-Rab7仅在F-肌动蛋白和D3磷脂酰肌醇水平下降的大约时间与形成的大胞体结合。这些结果支持一个模型，在该模型中，F-肌动蛋白调节蛋白和囊泡运输蛋白协调调节大胞体的形成和成熟。
Macropinocytosis plays an important role in the internalization of antigens by dendritic cells and is the route of entry for many bacterial pathogens; however, little is known about the molecular mechanisms that regulate the formation or maturation of macropinosomes. Like dendritic cells, Dictyostelium amoebae are active in macropinocytosis, and various proteins have been identified that contribute to this process. As described here, microscopic analysis of null mutants have revealed that the class I phosphoinositide 3-kinases, PIK1 and PIK2, and the downstream effector protein kinase B (PKB/Akt) are important in regulating completion of macropinocytosis. Although actin-rich membrane protrusions form in these cell lines, they recede without forming macropinosomes. Imaging of cells expressing green fluorescent protein (GFP) fused to the pleckstrin homology domain (PH) of PKB (GFP-PHPKB) indicates that D3 phosphoinositides are enriched in the forming macropinocytic cup and remain associated with newly formed macropinosomes for <1 minute. A fusion protein, consisting of GFP fused to an F-actin binding domain, overlaps with GFP-PHPKB in the timing of association with forming macropinosomes. Although macropinocytosis is reduced in cells expressing dominant negative Rab7, microscopic imaging studies reveal that GFP-Rab7 associates only with formed macropinosomes at approximately the time that F-actin and D3 phosphoinositide levels decrease. These results support a model in which F-actin modulating proteins and vesicle trafficking proteins coordinately regulate the formation and maturation of macropinosomes.