In vivo tracking of phosphoinositides in Drosophila photoreceptors.

Roger C Hardie,Che-Hsiung Liu,Alexander S Randall,Sukanya Sengupta

doi:10.1242/jcs.180364

Roger C Hardie, Che-Hsiung Liu + Show 2 more

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https://doi.org/10.1242/jcs.180364

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Abstract

ABSTRACTIn 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.

Highlights

Phototransduction in Drosophila is mediated by a G-proteincoupled phospholipase C (PLC) cascade and is an influential model for phosphoinositide signalling (Hardie, 2012; Hardie and Juusola, 2015; Katz and Minke, 2009; Montell, 2012; Yau and Hardie, 2009)
These included GFP-tagged PLCδ1 pleckstrin homology (PH), which binds to both PtdIns(4,5)P2 and Ins(1,4,5)P3 (Hirose et al, Journal of Cell Science (2015) 128, 4328-4340 doi:10.1242/jcs
Since the introduction of PLCδ1-PH–GFP (Stauffer et al, 1998; Varnai and Balla, 1998), fluorescently tagged lipid-binding domains have been widely used to monitor phosphoinositide turnover, but in most cases their use has been restricted to cultured cells

Summary

Introduction

Phototransduction in Drosophila is mediated by a G-proteincoupled phospholipase C (PLC) cascade and is an influential model for phosphoinositide signalling (Hardie, 2012; Hardie and Juusola, 2015; Katz and Minke, 2009; Montell, 2012; Yau and Hardie, 2009). The electrical response to light is mediated by two Ca2+permeable cation channels: transient receptor potential (TRP) and TRP-like (TRPL) (Hardie and Minke, 1992; Montell and Rubin, 1989; Phillips et al, 1992). Like their mammalian TRP channel homologues, both are activated downstream of PLC. The channels are localised in a light-guiding ‘rhabdomere’ (a rod-like stack of ∼30,000 microvilli) along with other components of the phototransduction cascade, including rhodopsin, Gq protein and PLC (encoded by norpA) (Hardie, 2012; Huber et al, 1996). How hydrolysis of phosphatidylinositol (4,5)-bisphosphate [PtdIns(4,5) P2] by PLC activates TRP and TRPL channels remains debated.

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