Alteration of epithelial structure and function associated with PtdIns(4,5)P2 degradation by a bacterial phosphatase.
Alteration of epithelial structure and function associated with PtdIns(4,5)P2 degradation by a bacterial phosphatase.
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DOI：
10.1085/jgp.200609656
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发表时间：
2007-04
期刊：
影响因子：
--
通讯作者：
Grinstein S
中科院分区：
文献类型：
--
作者：
Mason D;Mallo GV;Terebiznik MR;Payrastre B;Finlay BB;Brumell JH;Rameh L;Grinstein S
PtdIns（4，5）P2在上皮功能中的作用的阐明受到无法选择性地操纵这种磷酸肌醇的细胞内容物的阻碍。在这里，我们报告SigD，一种来源于沙门氏菌的磷酸酶，可以有效地水解PtdIns（4，5）P2，产生PtdIns（5）P。当通过将cDNA显微注射到上皮细胞中形成融合单层来表达时，野生型SigD诱导了显著的形态和功能变化，而磷酸酶缺陷型SigD突变体（C462 S）无法模仿这些变化。通过从磷脂酶Cδ的普列克底物蛋白同源结构域的膜上分离来验证完整SigD注射细胞中PtdIns（4，5）P2的耗尽，所述磷脂酶Cδ通过与绿色荧光蛋白缀合而用作磷酸肌醇的探针。单细胞细胞胞浆pH测量表明，PtdIns（4，5）P2的耗尽显着抑制了上皮细胞的Na+/H+交换活性。同样，阴离子渗透性，使用两种不同的卤化物敏感探针测量，在表达SigD的细胞中被抑制。PtdIns（4，5）P2的耗竭与肌动蛋白细胞骨架及其与质膜的关联的显著改变相关。注射细胞周围的连接复合物逐渐打开，PtdIns（4，5）P2耗尽的细胞最终从单层分离，其经历快速恢复。在肠上皮和肾上皮培养物中也观察到类似的结果。除了对磷酸肌醇的作用外，SigD还显示将肌醇1，3，4，5，6-五磷酸（IP 5）转化为肌醇1，4，5，6-四磷酸（IP 4），后者被认为介导沙门氏菌引起的腹泻。然而，SigD对上皮细胞的影响并没有通过显微注射IP 4来模仿。与此相反，细胞骨架和离子转运的影响，通过水解PtdIns（4，5）P2与膜靶向的5-磷酸酶或封闭肌醇使用高亲和力串联PH结构域的构建体复制。因此，我们认为，开放的紧密连接和抑制Na+/H+交换所造成的PtdIns（4，5）P2水解联合收割机帐户，至少部分，在沙门氏菌引起的腹泻期间观察到的液体损失。
Elucidation of the role of PtdIns(4,5)P2 in epithelial function has been hampered by the inability to selectively manipulate the cellular content of this phosphoinositide. Here we report that SigD, a phosphatase derived from Salmonella, can effectively hydrolyze PtdIns(4,5)P2, generating PtdIns(5)P. When expressed by microinjecting cDNA into epithelial cells forming confluent monolayers, wild-type SigD induced striking morphological and functional changes that were not mimicked by a phosphatase-deficient SigD mutant (C462S). Depletion of PtdIns(4,5)P2 in intact SigD-injected cells was verified by detachment from the membrane of the pleckstrin homology domain of phospholipase Cδ, used as a probe for the phosphoinositide by conjugation to green fluorescent protein. Single-cell measurements of cytosolic pH indicated that the Na+/H+ exchange activity of epithelia was markedly inhibited by depletion of PtdIns(4,5)P2. Similarly, anion permeability, measured using two different halide-sensitive probes, was depressed in cells expressing SigD. Depletion of PtdIns(4,5)P2 was associated with marked alterations in the actin cytoskeleton and its association with the plasma membrane. The junctional complexes surrounding the injected cells gradually opened and the PtdIns(4,5)P2-depleted cells eventually detached from the monolayer, which underwent rapid restitution. Similar observations were made in intestinal and renal epithelial cultures. In addition to its effects on phosphoinositides, SigD has been shown to convert inositol 1,3,4,5,6-pentakisphosphate (IP5) into inositol 1,4,5,6-tetrakisphosphate (IP4), and the latter has been postulated to mediate the diarrhea caused by Salmonella. However, the effects of SigD on epithelial cells were not mimicked by microinjection of IP4. In contrast, the cytoskeletal and ion transport effects were replicated by hydrolyzing PtdIns(4,5)P2 with a membrane-targeted 5-phosphatase or by occluding the inositide using high-avidity tandem PH domain constructs. We therefore suggest that opening of the tight junctions and inhibition of Na+/H+ exchange caused by PtdIns(4,5)P2 hydrolysis combine to account, at least in part, for the fluid loss observed during Salmonella-induced diarrhea.
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DOI：
10.1073/pnas.0403930101
发表时间：
2004-07-13
影响因子：
11.1
作者：
Fuster, D;Moe, OW;Hilgemann, DW
通讯作者：
Hilgemann, DW
影响因子：
5.5
作者：
Bertelsen, LS;Paesold, G;Barrett, KE
通讯作者：
Barrett, KE
Na（+）/H（+）交换的细胞内pH调节需要磷脂酰肌醇4,5-双磷酸。
DOI：
10.1083/jcb.150.1.213
发表时间：
2000-07-10
期刊：
The Journal of cell biology
影响因子：
--
作者：
Aharonovitz O;Zaun HC;Balla T;York JD;Orlowski J;Grinstein S
通讯作者：
Grinstein S
影响因子：
3.6
作者：
Galyov, EE;Wood, MW;Wallis, TS
通讯作者：
Wallis, TS
影响因子：
3.9
作者：
Carpenter, CL
通讯作者：
Carpenter, CL