In vivo tracking of phosphoinositides in Drosophila photoreceptors.
In vivo tracking of phosphoinositides in Drosophila photoreceptors.
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DOI：
10.1242/jcs.180364
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发表时间：
2015-12-01
影响因子：
4
通讯作者：
Sengupta S
中科院分区：
文献类型：
--
作者：
Hardie RC;Liu CH;Randall AS;Sengupta S
为了监测磷脂酶 C (PLC) 介导的果蝇光转导过程中磷酸肌醇的周转，荧光标记的脂质探针在光感受器中表达，并在离解细胞和完整活果蝇的眼睛中成像。在测试的 6 个探针中，TbR332H（Tubby 蛋白 pleckstrin 同源结构域的突变体）被认为是磷脂酰肌醇 (4,5)-二磷酸 [PtdIns(4,5)P2] 的最佳报告基因，以及军团菌 SidM 中磷脂酰肌醇 4-磷酸 (PtdIns4P) 的 P4M 结构域。使用精确校准的照明，我们发现野生型果蝇中只有约 50% 的 PtdIns(4,5)P2 和极少的 PtdIns4P 被全日光强度耗尽，但在缺乏 Ca2+ 通透性瞬时受体电位 (TRP) 通道或蛋白激酶 C (PKC) 的突变体中，两者都被约 100 倍的调光强度严重耗尽。 PtdIns4P (t½ ∼12 s) 的再合成比 PtdIns(4,5)P2 (t½ ∼40 s) 更快，但在 DAG 激酶 (rdgA) 或 PtdIns 转移蛋白 (rdgB) 突变体中，两者均大大减慢。结果表明，尽管 PLC 水解率很高，但需要 Ca2+ 和 PKC 依赖性 PLC 抑制才能使光感受器维持磷酸肌醇水平，并表明 PtdIns4P 磷酸化为 PtdIns(4,5)P2 是循环的限速步骤。重点文章：一系列基因靶向脂质探针被用来追踪和量化完整完整的活果蝇光感受器中磷酸肌醇的周转，其精确度是其他系统无法超越的。
In order to monitor phosphoinositide turnover during phospholipase C (PLC)-mediated Drosophila phototransduction, fluorescently tagged lipid probes were expressed in photoreceptors and imaged both in dissociated cells, and in eyes of intact living flies. Of six probes tested, TbR332H (a mutant of the Tubby protein pleckstrin homology domain) was judged the best reporter for phosphatidylinositol (4,5)-bisphosphate [PtdIns(4,5)P2], and the P4M domain from Legionella SidM for phosphatidylinositol 4-phosphate (PtdIns4P). Using accurately calibrated illumination, we found that only ∼50% of PtdIns(4,5)P2 and very little PtdIns4P were depleted by full daylight intensities in wild-type flies, but both were severely depleted by ∼100-fold dimmer intensities in mutants lacking Ca2+-permeable transient receptor potential (TRP) channels or protein kinase C (PKC). Resynthesis of PtdIns4P (t½ ∼12 s) was faster than PtdIns(4,5)P2 (t½ ∼40 s), but both were greatly slowed in mutants of DAG kinase (rdgA) or PtdIns transfer protein (rdgB). The results indicate that Ca2+- and PKC-dependent inhibition of PLC is required for enabling photoreceptors to maintain phosphoinositide levels despite high rates of hydrolysis by PLC, and suggest that phosphorylation of PtdIns4P to PtdIns(4,5)P2 is the rate-limiting step of the cycle. Highlighted Article: A range of genetically targeted lipid probes were used to track and quantify phosphoinositide turnover in the photoreceptors of completely intact living flies, with a precision unsurpassed in other systems.