Shank2 contributes to the apical retention and intracellular redistribution of NaPiIIa in OK cells.
Shank2 contributes to the apical retention and intracellular redistribution of NaPiIIa in OK cells.
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Shank2 有助于 OK 细胞中 NaPiIIa 的顶端保留和细胞内重新分布。
DOI：
10.1152/ajpcell.00189.2012
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发表时间：
2013
期刊：
影响因子：
--
通讯作者：
Doctor,RBrian
中科院分区：
文献类型：
--
作者：
Dobrinskikh,Evgenia;Lanzano,Luca;Rachelson,Joanna;Cranston,DeeAnn;Moldovan,Radu;Lei,Tim;Gratton,Enrico;Doctor,RBrian
在肾近曲小管（PT）细胞中，钠-磷酸盐协同转运蛋白IIa（NaPIIa）通常集中在顶端膜内，在那里它重吸收约70%的管腔磷酸盐（Pi）。NaPiIIa活性通过调节其在顶膜内的丰度而被剧烈调节。在低Pi条件下，NaPiIIa保留在顶端膜内。在高Pi条件下，NaPiIIa从顶膜中回收并运输至溶酶体进行降解。本研究探讨了Shank 2在调节NaPiIIa分布中的作用。在负鼠肾细胞中，PT细胞模型，在低Pi培养基中维持的细胞中敲低Shank 2导致NaPIIa丰度显著降低。在转移到高Pi培养基中后，活细胞成像显示mRFP-Shank 2 E和GFP-NaPiIIa经历内吞作用并一起通过亚顶端结构域运输。荧光交叉相关光谱表明，GFP-NaPIIIa和mRFP-Shank 2具有不可区分的扩散系数，并在时间同步迁移通过亚顶端域。光栅图像互相关光谱表明，这两种蛋白的过程中，在时间-空间同步通过亚顶端域。在低Pi条件下的细胞的微绒毛和在高Pi条件下的细胞的亚顶端域中，Cer-NaPiIIa和EYFP-Shank 2 E的荧光寿命成像显微镜-福斯特共振能量转移分析发现这些荧光体彼此驻留在10 nm内。证明了功能的复杂性，在低Pi条件下维持的细胞中，Shank 2在NaPilla的顶端保留中起着重要作用，而在高Pi条件下，Shank 2仍然与NaPilla相关联，并护送NaPilla通过细胞内部。
In renal proximal tubule (PT) cells, sodium-phosphate cotransporter IIa (NaPiIIa) is normally concentrated within the apical membrane where it reabsorbs ∼70% of luminal phosphate (Pi). NaPiIIa activity is acutely regulated by moderating its abundance within the apical membrane. Under low-Pi conditions, NaPiIIa is retained within the apical membrane. Under high-Pi conditions, NaPiIIa is retrieved from the apical membrane and trafficked to the lysosomes for degradation. The present study investigates the role of Shank2 in regulating the distribution of NaPiIIa. In opossum kidney cells, a PT cell model, knockdown of Shank2 in cells maintained in low-Pi media resulted in a marked decrease in NaPiIIa abundance. After being transferred into high-Pi media, live-cell imaging showed that mRFP-Shank2E and GFP-NaPiIIa underwent endocytosis and trafficked together through the subapical domain. Fluorescence cross-correlation spectroscopy demonstrated that GFP-NaPiIIa and mRFP-Shank2 have indistinguishable diffusion coefficients and migrated through the subapical domain in temporal synchrony. Raster image cross-correlation spectroscopy demonstrated these two proteins course through the subapical domain in temporal-spatial synchrony. In the microvilli of cells under low-Pi conditions and in the subapical domain of cells under high-Pi conditions, fluorescence lifetime imaging microscopy-Forster resonance energy transfer analysis of Cer-NaPiIIa and EYFP-Shank2E found these fluors reside within 10 nm of each other. Demonstrating a complexity of functions, in cells maintained under low-Pi conditions, Shank2 plays an essential role in the apical retention of NaPiIIa while under high-Pi conditions Shank2 remains associated with NaPiIIa and escorts NaPiIIa through the cell interior.