Institute for Molecular Bioscience and Australia Research Council, Centre of Excellence in Bioinformatics, The University of Queensland, St Lucia, Brisbane, Australia.
PLoS One. 2010 Oct 29;5(10):e13763. doi: 10.1371/journal.pone.0013763.
Macropinocytosis is an actin-driven endocytic process, whereby membrane ruffles fold back onto the plasma membrane to form large (>0.2 µm in diameter) endocytic organelles called macropinosomes. Relative to other endocytic pathways, little is known about the molecular mechanisms involved in macropinocytosis. Recently, members of the Sorting Nexin (SNX) family have been localized to the cell surface and early macropinosomes, and implicated in macropinosome formation. SNX-PX-BAR proteins form a subset of the SNX family and their lipid-binding (PX) and membrane-curvature sensing (BAR) domain architecture further implicates their functional involvement in macropinosome formation.
METHODOLOGY/PRINCIPAL FINDINGS: We exploited the tractability of macropinosomes through image-based screening and systematic overexpression of SNX-PX-BAR proteins to quantitate their effect on macropinosome formation. SNX1 (40.9+/-3.19 macropinosomes), SNX5 (36.99+/-4.48 macropinosomes), SNX9 (37.55+/-2.4 macropinosomes), SNX18 (88.2+/-8 macropinosomes), SNX33 (65.25+/-6.95 macropinosomes) all exhibited statistically significant (p<0.05) increases in average macropinosome numbers per 100 transfected cells as compared to control cells (24.44+/-1.81 macropinosomes). SNX1, SNX5, SNX9, and SNX18 were also found to associate with early-stage macropinosomes within 5 minutes following organelle formation. The modulation of intracellular PI(3,4,5)P(3) levels through overexpression of PTEN or a lipid phosphatase-deficient mutant PTEN(G129E) was also observed to significantly reduce or elevate macropinosome formation respectively; coexpression of PTEN(G129E) with SNX9 or SNX18 synergistically elevated macropinosome formation to 119.4+/-7.13 and 91.4+/-6.37 macropinosomes respectively (p<0.05).
CONCLUSIONS/SIGNIFICANCE: SNX1, SNX5, SNX9, SNX18, and SNX33 were all found to elevate macropinosome formation and (with the exception of SNX33) associate with early-stage macropinosomes. Moreover the effects of SNX9 and SNX18 overexpression in elevating macropinocytosis is likely to be synergistic with the increase in PI(3,4,5)P(3) levels, which is known to accumulate on the cell surface and early-stage macropinocytic cups. Together these findings represent the first systematic functional study into the impact of the SNX-PX-BAR family on macropinocytosis.
巨胞饮作用是一种肌动蛋白驱动的内吞过程，在此过程中，膜皱襞向内折叠回到质膜上，形成大的（直径大于 0.2 µm）内吞细胞器，称为巨胞饮体。与其他内吞途径相比，人们对巨胞饮作用涉及的分子机制知之甚少。最近，分选连接蛋白（Sorting Nexin，SNX）家族的成员被定位到细胞表面和早期巨胞饮体上，并与巨胞饮体的形成有关。SNX-PX-BAR 蛋白形成了 SNX 家族的一个子集，其脂质结合（PX）和膜曲率感应（BAR）结构域架构进一步表明它们在巨胞饮体的形成中具有功能上的参与。
方法/主要发现：我们利用巨胞饮体的可追踪性，通过基于图像的筛选和系统地过表达 SNX-PX-BAR 蛋白，定量分析它们对巨胞饮体形成的影响。SNX1（40.9+/-3.19 个巨胞饮体）、SNX5（36.99+/-4.48 个巨胞饮体）、SNX9（37.55+/-2.4 个巨胞饮体）、SNX18（88.2+/-8 个巨胞饮体）、SNX33（65.25+/-6.95 个巨胞饮体）在每 100 个转染细胞中平均巨胞饮体数量方面均表现出统计学上的显著增加（p<0.05），与对照细胞（24.44+/-1.81 个巨胞饮体）相比。还发现 SNX1、SNX5、SNX9 和 SNX18 在细胞器形成后 5 分钟内与早期巨胞饮体相关联。通过过表达 PTEN 或脂质磷酸酶缺陷型突变体 PTEN(G129E)来调节细胞内 PI(3,4,5)P(3)水平，也观察到分别显著减少或增加巨胞饮体的形成；与 SNX9 或 SNX18 共表达 PTEN(G129E)协同作用，将巨胞饮体的形成分别提高到 119.4+/-7.13 和 91.4+/-6.37 个巨胞饮体（p<0.05）。
结论/意义：SNX1、SNX5、SNX9、SNX18 和 SNX33 均被发现能增加巨胞饮体的形成，并且（除 SNX33 外）与早期巨胞饮体相关联。此外，SNX9 和 SNX18 过表达在增加巨胞饮作用中的作用可能与 PI(3,4,5)P(3)水平的增加协同作用，PI(3,4,5)P(3)水平已知在细胞表面和早期巨胞饮杯中积累。这些发现共同代表了对 SNX-PX-BAR 家族对巨胞饮作用影响的首次系统的功能研究。
