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Fig. 1. SigD expression depletes plasma membrane PtdIns(4,5)P₂. (A) Simplified PtdIns pathway. PtdIns (PI) can be phosphorylated by PtdIns 4-kinases (PI4K), such as Drosophila Fwd, to yield PtdIns(4)P [PI4P]. PtdIns(4)P is phosphorylated by PtdIns(4)P 5-kinases (PIP5K), such as Drosophila Sktl, to produce PtdIns(4,5)P₂ (PIP2). PtdIns(4,5)P₂ is hydrolyzed by phospholipase C (PLC), generating the second messengers inositol trisphosphate (InsP₃, IP3) and diacylglycerol (DAG). InsP₃ can lead to release of Ca²⁺ from intracellular stores (via the InsP₃ receptor) and to synthesis of other inositol polyphosphates, such as inositol hexakisphosphate (InsP₆, IP6). Alternatively, PtdIns(4,5)P₂ can be sequentially dephosphorylated by 5-phosphatases (5-P'ase), including Salmonella SigD, to regenerate PtdIns(4)P, which in turn can be dephosphorylated by 4-phosphatases (4-P'ase), such as Drosophila Sac1, to produce PtdIns. (B-E') Phase-contrast (B-E) and corresponding fluorescence (B'-E') images of male germ cells expressing PLC ${delta}$ -PH-GFP (B,C,E) or PLC ${delta}$ -PH-RFP (D), which bind PtdIns(4,5)P₂. (B') PtdIns(4,5)P₂ is found in the plasma membrane (arrow) of wild-type male germ cells. (C') SigD expression diminishes PtdIns(4,5)P₂ at the plasma membrane and causes accumulation of PLC ${delta}$ -PH-GFP puncta in the cytoplasm (arrow). (D') Co-expression of Sktl partially restores plasma membrane PtdIns(4,5)P₂ (arrow), although some cells exhibit non-specific PLC ${delta}$ -PH-RFP localization in the cytoplasm. (E') Expression of SigD^dead has no effect on PtdIns(4,5)P₂ accumulation. (F-G') Phase-contrast (F,G) and corresponding fluorescence (F',G') images of male germ cells expressing RFP-PH-FAPP to detect PtdIns(4)P. (F') PtdIns(4)P localizes to the Golgi in spermatocytes (arrowhead) and to the acroblast in spermatids (arrow). (G') SigD expression increases PtdIns(4)P levels in both spermatocytes (arrowhead) and spermatids (arrow). Note that, although the exposure times for F' and G' were identical, the contrast in F' was increased by 50% to make the PtdIns(4)P localization more obvious. Scale bars: 20 µm.

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