Mutant/mutation
The mutant expresses a fusion protein consisting of the PH domain of human phospholipase Cδ1 (hPLCδ1) fused to yellow fluorescent protein (YFP) and cyan fluorescent protein (CFP). The gene encoding this fusion protein is expressed under the control of the constitutive eef1α promoter and introduced into the parasites as episomal plasmids.

Protein (function)
The study analyses Ca²⁺ mobilization in (activated) gametocytes by phosphoinositide specific phospholipase C (PI-PLC; PBANKA_121190,  PF10_0132), which hydrolyses the minor membrane lipid phosphatidylinositol-(4,5)-bisphosphate (PIP₂), producing the secondary messengers inositol-(1,4,5)-trisphosphate (IP3) and diacylglycerol (DAG); IP₃ then triggers Ca²⁺ release into the cytosol by binding to IP₃-gated Ca²⁺ channels localized predominately in the ER membrane.
In the paper evidence is presented that activation of gametocytes (to produce gametes) is triggered by the hydrolysis of PIP₂ and the production of the secondary messenger IP₃ in activated gametocytes. Both processes were selectively blocked by a PI-PLC inhibitor, which also reduced the early Ca²⁺ signal.

Dynamic changes in cellular PIP2 have been monitored successfully in cultured mammalian cells by single cell imaging of a fluorescent reporter protein fused to the PH domain of human phospholipase Cδ1 (hPLCδ1). PH domains can bind both, PIP₂ and IP₃. Resting cells contain low IP₃ levels and a PH domain-containing reporter protein is targeted mostly to the plasma membrane where PIP₂ resides. PI-PLC activation and IP₃ production then leads to translocation of the probe to the cytoplasm

Phenotype
In most resting gametocytes of either sex the CFP–PH–YFP protein was clearly detectable in the periphery of the cells, consistent with a localization at the plasma membrane. By time lapse microscopy it was observed that within two minutes of activation of gametocytes, the CFP–PH–YFP protein began to redistribute to the cytosol, a process that was typically complete 5 min after gametocyte activation. A quantitative analysis in randomly selected macrogametocytes found that CFP–PH–YFP redistributed to the cytosol in about half of the cells. A few cells showed a high proportion of peripherally located CFP–PH–YFP that did not change upon activation; these cells may have been immature gametocytes still unable to be activated. Other gametocytes had a relatively high level of cytosolic fluorescence that remained unchanged.The latter response was typical of the cytosolic localization in the control cell line expressing GFP without a PH domain. CFP–PH–YFP expressing cells with  cytosolic localization of the marker may have responded already during the minute that typically elapsed between gametocyte activation and recording of the first image. Male and female gametocytes both showed redistribution of the CFP–PH–YFP reporter constructs, but due to the choice of promoter the reporter protein was more strongly expressed and easier to detect in female gametocytes.

Additional information

Other mutants