Drosophila Photoreceptors Research Articles

The Roles of Transient Receptor Potential (TRP) Channels Underlying Aberrant Calcium Signaling in Blood-Retinal Barrier Dysfunction.

The inner blood-retinal barrier (iBRB) protects the retinal vasculature from the peripheral circulation. Endothelial cells (ECs) are the core component of the iBRB; their close apposition and linkage via tight junctions limit the passage of fluids, proteins, and cells from the bloodstream to the parenchyma. Dysfunction of the iBRB is a hallmark of many retinal disorders. Vascular endothelial growth factor (VEGF) has been identified as the primary driver leading to a dysfunctional iBRB, thereby becoming the main target for therapy. However, a complete understanding of the molecular mechanisms underlying iBRB dysfunction is elusive and alternative therapeutic targets remain unexplored. Calcium (Ca2+) is a universal intracellular messenger whose homeostasis and dynamics are dysregulated in many pathological disorders. Among the extensive components of the cellular Ca2+-signaling toolkit, cation-selective transient receptor potential (TRP) channels are broadly involved in cell physiology and disease and, therefore, are widely studied as possible targets for therapy. Albeit that TRP channels have been discovered in the photoreceptors of Drosophila and have been studied in the neuroretina, their presence and function in the iBRB have only recently emerged. Within this article, we discuss the structure and functions of the iBRB with a particular focus on Ca2+ signaling in retinal ECs and highlight the potential of TRP channels as new targets for retinal diseases.

Proximity labeling reveals interactions necessary to maintain the distinct apical domains of Drosophila photoreceptors.

Specialized membrane and cortical protein regions are common features of cells and are utilized to isolate differential cellular functions. In Drosophila photoreceptors, the apical membrane domain is defined by two distinct morphological membranes: the rhabdomere microvilli and the stalk membrane. To define the apical cortical protein complexes, we performed proximity labeling screens utilizing the rhabdomeric-specific protein PIP82 as bait. We found that the PIP82 interactome is enriched for actin-binding and cytoskeleton proteins, as well as proteins for cellular trafficking. Analysis of one target, Bifocal, with PIP82 revealed two independent pathways for localization to the rhabdomeric membrane and an additional mechanism of crosstalk between the protein complexes of the rhabdomeric and stalk membranes. The loss of Bifocal, and enhancement in the Bifocal/PIP82 double mutant, results in the additional distribution of Crumbs, an apical stalk membrane protein, to the lateral basal photoreceptor membrane. This phenotype is recapitulated by the knockdown of the catalytic subunit of Protein Phosphatase 1, a known interactor of Bifocal. Taken together, these results expand our understanding of the molecular mechanisms underlying the generation of the two distinct photoreceptor apical domains.

Histamine synthesis and transport are coupled in axon terminals via a dual quality control system

Monoamine neurotransmitters generated by de novo synthesis are rapidly transported and stored into synaptic vesicles at axon terminals. This transport is essential both for sustaining synaptic transmission and for limiting the toxic effects of monoamines. Here, synthesis of the monoamine histamine by histidine decarboxylase (HDC) and subsequent loading of histamine into synaptic vesicles are shown to be physically and functionally coupled within Drosophila photoreceptor terminals. This process requires HDC anchoring to synaptic vesicles via interactions with N-ethylmaleimide-sensitive fusion protein 1 (NSF1). Disassociating HDC from synaptic vesicles disrupts visual synaptic transmission and causes somatic accumulation of histamine, which leads to retinal degeneration. We further identified a proteasome degradation system mediated by the E3 ubiquitin ligase, purity of essence (POE), which clears mislocalized HDC from the soma, thus eliminating the cytotoxic effects of histamine. Taken together, our results reveal a dual mechanism for translocation and degradation of HDC that ensures restriction of histamine synthesis to axonal terminals and at the same time rapid loading into synaptic vesicles. This is crucial for sustaining neurotransmission and protecting against cytotoxic monoamines.

Golgi clustering by the deficiency of COPI-SNARE in Drosophila photoreceptors.

A comprehensive study of soluble N-ethylmaleimide-sensitive factor attachment protein receptors (SNAREs) in the fly genome by RNAi in Drosophila photoreceptors indicated that knockdown of any of the COPI-SNAREs, Syx18, Sec20, and Use1, resulted in the same characteristic phenotypes: Golgi stacks gathering on their trans-side, laterally expanded Golgi cisternae, and a reduced number of discrete Golgi stacks. These Golgi stacks are reminiscent of mammalian Golgi ribbons and Brefeldin A (BFA)-bodies in Drosophila S2 cells. As previously reported, BFA suppresses trans-Golgi network (TGN) fission and Golgi stack separation to form a BFA-body, which is a cluster of Golgi stacks cored by recycling endosomes. We found that the impairing each of COPI-SNAREs results in clustered Golgi stacks similar to BFA-bodies, indicating that COPI-SNAREs have a role to separate clustered Golgi stacks. These results further support the idea that the movement of Golgi stacks and the balance of fusion and fission of the TGN determine the level of clustering and ribbon formation of Golgi stacks within cells.

Mechanical force activates the light-dependent channels TRP and TRPL in excised patches from the rhabdomere of Drosophila photoreceptors

Drosophila phototransduction in light-sensitive microvilli involves a metabotropic signaling cascade. Photoisomerized rhodopsin couples to G-protein, activating phospholipase C, which cleaves phosphatidylinositol bisphosphate (PIP2) into inositol trisphosphate, diacylglycerol (DAG) and a proton. DAG is converted into phosphatidic acid by DAG-kinase and metabolized to L-linoleoyl glycerol (2-LG) by DAG-lipase. This complex enzyme cascade ultimately opens the light-dependent transient receptor potential channels, TRP and TRPL. PIP2, DAG, H+ and 2-LG are possible channel activators, either individually or combined, but their direct participation in channel-gating remains unresolved. Molecular interaction with the channels, modification of the channels’ lipid moiety and mechanical force on the channels by changes in the membrane structure derived from light-dependent changes in lipid composition are possible gating agents. In this regard, mechanical activation was suggested, based on a rapid light-dependent contraction of the photoreceptors mediated by the phototransduction cascade. Here, we further examined this possibility by applying force to inside-out patches from the microvilli membrane by changing the pressure in the pipette or pulling the membrane with a magnet through superparamagnetic nanospheres. The channels were opened by mechanical force, while mutant lacking both channels was insensitive to mechanical stimulation. Atomic Force Microscopy showed that the stiffness of an artificial phospholipid bilayer was increased by arachidonic acid and diacylglycerol whereas elaidic acid was ineffective, mirroring their relative effects in channel activity previously observed electrophysiologically. Together, the results are consistent with the notion that light-induced changes in lipid composition alter the membrane structure, generating mechanical force on the channels leading to channel opening.

A genetic screen to uncover mechanisms underlying lipid transfer protein function at membrane contact sites.

Lipid transfer proteins mediate the transfer of lipids between organelle membranes, and the loss of function of these proteins has been linked to neurodegeneration. However, the mechanism by which loss of lipid transfer activity leads to neurodegeneration is not understood. In Drosophila photoreceptors, depletion of retinal degeneration B (RDGB), a phosphatidylinositol transfer protein, leads to defective phototransduction and retinal degeneration, but the mechanism by which loss of this activity leads to retinal degeneration is not understood. RDGB is localized to membrane contact sites through the interaction of its FFAT motif with the ER integral protein VAP. To identify regulators of RDGB function in vivo, we depleted more than 300 VAP-interacting proteins and identified a set of 52 suppressors of rdgB The molecular identity of these suppressors indicates a role of novel lipids in regulating RDGB function and of transcriptional and ubiquitination processes in mediating retinal degeneration in rdgB9 The human homologs of several of these molecules have been implicated in neurodevelopmental diseases underscoring the importance of VAP-mediated processes in these disorders.

TsCRISPR based identification of Rab proteins required for the recycling of Drosophila TRPL ion channel.

In polarized cells, the precise regulation of protein transport to and from the plasma membrane is crucial to maintain cellular function. Dysregulation of intracellular protein transport in neurons can lead to neurodegenerative diseases such as Retinitis Pigmentosa, Alzheimer's and Parkinson's disease. Here we used the light-dependent transport of the TRPL (transient receptor potential-like) ion channel in Drosophila photoreceptor cells to study the role of Rab proteins in TRPL recycling. TRPL is located in the rhabdomeric membrane of dark-adapted flies, but it is transported out of the rhabdomere upon light exposure and localizes at the Endoplasmatic Reticulum within 12h. Upon subsequent dark adaptation, TRPL is recycled back to the rhabdomeric membrane within 90min. To screen for Rab proteins involved in TRPL recycling, we established a tissue specific (ts) CRISPR/Cas9-mediated knock-out of individual Rab genes in Drosophila photoreceptors and assessed TRPL localization using an eGFP tagged TRPL protein in the intact eyes of these mutants. We observed severe TRPL recycling defects in the knockouts of Rab3, Rab4, Rab7, Rab32, and RabX2. Using immunohistochemistry, we further showed that Rab3 and RabX2 each play a significant role in TRPL recycling and also influence TRPL transport. We localized Rab3 to the late endosome in Drosophila photoreceptors and observed disruption of TRPL transport to the ER in Rab3 knock-out mutants. TRPL transport from the ER to the rhabdomere ensues from the trans-Golgi where RabX2 is located. We observed accumulated TRPL at the trans-Golgi in RabX2 knock-out mutants. In summary, our study reveals the requirement of specific Rab proteins for different steps of TRPL transport in photoreceptor cells and provides evidence for a unique retrograde recycling pathway of TRPL from the ER via the trans-Golgi.

Combined inactivation of RB and Hippo converts differentiating Drosophila photoreceptors into eye progenitor cells through derepression of homothorax.

The retinoblastoma (RB) and Hippo pathways interact to regulate cell proliferation and differentiation. However, the mechanism of interaction is not fully understood. Drosophila photoreceptors with inactivated RB and Hippo pathways specify normally but fail to maintain their neuronal identity and dedifferentiate. We performed single-cell RNA sequencing to elucidate the cause of dedifferentiation and to determine the fate of these cells. We find that dedifferentiated cells adopt a progenitor-like fate due to inappropriate activation of the retinal differentiation suppressor homothorax (hth) by Yki/Sd. This results in the activation of a distinct Yki/Hth transcriptional program, driving photoreceptor dedifferentiation. We show that Rbf physically interacts with Yki and, together with the GAGA factor, inhibits the hth expression. Thus, RB and Hippo pathways cooperate to maintain photoreceptor differentiation by preventing inappropriate expression of hth in differentiating photoreceptors. Our work highlights the importance of both RB and Hippo pathway activities for maintaining the state of terminal differentiation.

Multifaceted luminance gain control beyond photoreceptors in Drosophila

Animals navigating in natural environments must handle vast changes in their sensory input. Visual systems, for example, handle changes in luminance at many timescales, from slow changes across the day to rapid changes during active behavior. To maintain luminance-invariant perception, visual systems must adapt their sensitivity to changing luminance at different timescales. We demonstrate that luminance gain control in photoreceptors alone is insufficient to explain luminance invariance at both fast and slow timescales and reveal the algorithms that adjust gain past photoreceptors in the fly eye. We combined imaging and behavioral experiments with computational modeling to show that downstream of photoreceptors, circuitry taking input from the single luminance-sensitive neuron type L3 implements gain control at fast and slow timescales. This computation is bidirectional in that it prevents the underestimation of contrasts in low luminance and overestimation in high luminance. An algorithmic model disentangles these multifaceted contributions and shows that the bidirectional gain control occurs at both timescales. The model implements a nonlinear interaction of luminance and contrast to achieve gain correction at fast timescales and a dark-sensitive channel to improve the detection of dim stimuli at slow timescales. Together, our work demonstrates how a single neuronal channel performs diverse computations to implement gain control at multiple timescales that are together important for navigation in natural environments.

Allnighter pseudokinase-mediated feedback links proteostasis and sleep in Drosophila

In nervous systems, retrograde signals are key for organizing circuit activity and maintaining neuronal homeostasis. We identify the conserved Allnighter (Aln) pseudokinase as a cell non-autonomous regulator of proteostasis responses necessary for normal sleep and structural plasticity of Drosophila photoreceptors. In aln mutants exposed to extended ambient light, proteostasis is dysregulated and photoreceptors develop striking, but reversible, dysmorphology. The aln gene is widely expressed in different neurons, but not photoreceptors. However, secreted Aln protein is retrogradely endocytosed by photoreceptors. Inhibition of photoreceptor synaptic release reduces Aln levels in lamina neurons, consistent with secreted Aln acting in a feedback loop. In addition, aln mutants exhibit reduced night time sleep, providing a molecular link between dysregulated proteostasis and sleep, two characteristics of ageing and neurodegenerative diseases.

Drosophila photoreceptor systems converge in arousal neurons and confer light responsive robustness.

Lateral ventral neurons (LNvs) in the fly circadian neural circuit mediate behaviors other than clock resetting, including light-activated acute arousal. Converging sensory inputs often confer functional redundancy. The LNvs have three distinct light input pathways: (1) cell autonomously expressed cryptochrome (CRY), (2) rhodopsin 7 (Rh7), and (3) synaptic inputs from the eyes and other external photoreceptors that express opsins and CRY. We explored the relative photoelectrical and behavioral input contributions of these three photoreceptor systems to determine their functional impact in flies. Patch-clamp electrophysiology measuring light evoked firing frequency (FF) was performed on large LNvs (l-LNvs) in response to UV (365 nm), violet (405 nm), blue (450 nm), or red (635 nm) LED light stimulation, testing controls versus mutants that lack photoreceptor inputs gl60j, cry-null, rh7-null, and double mutant gl60j-cry-null flies. For UV, violet, and blue short wavelength light inputs, all photoreceptor mutants show significantly attenuated action potential FF responses measured in the l-LNv. In contrast, red light FF responses are only significantly attenuated in double mutant gl60j-cry-null flies. We used a light-pulse arousal assay to compare behavioral responses to UV, violet, blue and red light of control and light input mutants, measuring the awakening arousal response of flies during subjective nighttime at two different intensities to capture potential threshold differences (10 and 400 μW/cm2). The light arousal behavioral results are similar to the electrophysiological results, showing significant attenuation of behavioral light responses for mutants compared to control. These results show that the different LNv convergent photoreceptor systems are integrated and together confer functional redundancy for light evoked behavioral arousal.

A conventional PKC critical for both the light-dependent and the light-independent regulation of the actin cytoskeleton in Drosophila photoreceptors

Pkc53E is the second conventional protein kinase C (PKC) gene expressed in Drosophila photoreceptors; it encodes at least six transcripts generating four distinct protein isoforms including Pkc53E-B whose mRNA is preferentially expressed in photoreceptors. By characterizing transgenic lines expressing Pkc53E-B-GFP, we show Pkc53E-B is localized in the cytosol and rhabdomeres of photoreceptors, and the rhabdomeric localization appears dependent on the diurnal rhythm. A loss of function of pkc53E-B leads to light-dependent retinal degeneration. Interestingly, the knockdown of pkc53E also impacted the actin cytoskeleton of rhabdomeres in a light-independent manner. Here the Actin-GFP reporter is mislocalized and accumulated at the base of the rhabdomere, suggesting that Pkc53E regulates depolymerization of the actin microfilament. We explored the light-dependent regulation of Pkc53E and demonstrated that activation of Pkc53E can be independent of the phospholipase C PLCβ4/NorpA as degeneration of norpAP24 photoreceptors was enhanced by a reduced Pkc53E activity. We further show that the activation of Pkc53E may involve the activation of Plc21C by Gqα. Taken together, Pkc53E-B appears to exert both constitutive and light-regulated activity to promote the maintenance of photoreceptors possibly by regulating the actin cytoskeleton.

Establishing the contribution of active histone methylation marks to the aging transcriptional landscape of Drosophila photoreceptors

Studies in multiple organisms have shown that aging is accompanied by several molecular phenotypes that include dysregulation of chromatin. Since chromatin regulates DNA-based processes such as transcription, alterations in chromatin modifications could impact the transcriptome and function of aging cells. In flies, as in mammals, the aging eye undergoes changes in gene expression that correlate with declining visual function and increased risk of retinal degeneration. However, the causes of these transcriptome changes are poorly understood. Here, we profiled chromatin marks associated with active transcription in the aging Drosophila eye to understand how chromatin modulates transcriptional outputs. We found that both H3K4me3 and H3K36me3 globally decrease across all actively expressed genes with age. However, we found no correlation with changes in differential gene expression. Downregulation of the H3K36me3 methyltransferase Set2 in young photoreceptors revealed significant changes in splicing events that overlapped significantly with those observed in aging photoreceptors. These overlapping splicing events impacted multiple genes involved in phototransduction and neuronal function. Since proper splicing is essential for visual behavior, and because aging Drosophila undergo a decrease in visual function, our data suggest that H3K36me3 could play a role in maintaining visual function in the aging eye through regulating alternative splicing.

Structural organization of RDGB (retinal degeneration B), a multi-domain lipid transfer protein: a molecular modelling and simulation based approach

Lipid transfer proteins (LTPs) that shuttle lipids at membrane contact sites (MCS) play an important role in maintaining cellular homeostasis. One such important LTP is the Retinal Degeneration B (RDGB) protein. RDGB is localized at the MCS formed between the endoplasmic reticulum (ER) and the apical plasma membrane (PM) in Drosophila photoreceptors where it transfers phosphatidylinositol (PI) during G-protein coupled phospholipase C signalling. Previously, the C-terminal domains of RDGB have been shown to be essential for its function and accurate localization. In this study, using in-silico integrative modelling we predict the structure of entire RDGB protein in complex with the ER membrane protein VAP. The structure of RDGB has then been used to decipher the structural features of the protein important for its orientation at the contact site. Using this structure, we identify two lysine residues in the C-terminal helix of the LNS2 domain important for interaction with the PM. Using molecular docking, we also identify an unstructured region USR1, immediately c-terminal to the PITP domain that is important for the interaction of RDGB with VAP. Overall the 10.06 nm length of the predicted RDGB-VAP complex spans the distance between the PM and ER and is consistent with the cytoplasmic gap between the ER and PM measured by transmission electron microscopy in photoreceptors. Overall our model explains the topology of the RDGB-VAP complex at this ER-PM contact site and paves the way for analysis of lipid transfer function in this setting. Communicated by Ramaswamy H. Sarma

Direct evaluation of neuroaxonal degeneration with the causative genes of neurodegenerative diseases in Drosophila using the automated axon quantification system, MeDUsA.

Drosophila is an excellent model organism for studying human neurodegenerative diseases (NDs). However, there is still almost no experimental system that could directly observe the degeneration of neurons and automatically quantify axonal degeneration. In this study, we created MeDUsA (a 'method for the quantification of degeneration using fly axons'), a standalone executable computer program based on Python that combines a pre-trained deep-learning masking tool with an axon terminal counting tool. This software automatically quantifies the number of retinal R7 axons in Drosophila from a confocal z-stack image series. Using this software, we were able to directly demonstrate that axons were degenerated by the representative causative genes of NDs for the first time in Drosophila. The fly retinal axon is an excellent experimental system that is capable of mimicking the pathology of axonal degeneration in human NDs. MeDUsA rapidly and accurately quantifies axons in Drosophila photoreceptor neurons. It enables large-scale research into axonal degeneration, including screening to identify genes or drugs that mediate axonal toxicity caused by ND proteins and diagnose the pathological significance of novel variants of human genes in axons.

Functions of neuronal Synaptobrevin in the post-Golgi transport of rhodopsin in Drosophila photoreceptors.

Polarized transport is essential for constructing multiple plasma membrane domains in the cell. Drosophila photoreceptors are an excellent model system to study the mechanisms of polarized transport. Rab11 is the key factor regulating the post-Golgi transport of rhodopsin 1 (Rh1), a photoreceptive protein, to the rhabdomere, a photoreceptive plasma membrane. Here, we found that neuronal Synaptobrevin (nSyb) colocalizes with Rab11 on the trans-side of Golgi stacks and post-Golgi vesicles at the rhabdomere-base, and nSyb-deficiency impairs rhabdomeric transport and induces accumulation of Rh1 and vesicles in the cytoplasm; this is similar to the effects of Rab11 loss. These results indicate that nSyb acts as a post-Golgi SNARE toward rhabdomeres. Surprisingly, in Rab11-, Rip11-, and nSyb-deficient photoreceptors, illumination enhances cytoplasmic accumulation of Rh1 colocalizing with Rab11, Rabenosyn5, nSyb, and Arrestin 1 (Arr1). Arr1 loss but not Rab5 dominant negative (Rab5DN) protein expression inhibits the light enhanced cytoplasmic Rh1 accumulation. Rab5DN inhibits the generation of Rh1 containing multi-vesicle bodies rather than Rh1 internalization. Overall, these results indicate that exocytic Rh1 mingle with endocytosed Rh1 and are then transported together to rhabdomeres.

Notch-dependent Abl signaling regulates cell motility during ommatidial rotation in Drosophila.

A collective cell motility event that occurs during Drosophila eye development, ommatidial rotation (OR), serves as a paradigm for signaling-pathway-regulated directed movement of cell clusters. OR is instructed by the EGFR and Notch pathways and Frizzled/planar cell polarity (Fz/PCP) signaling, all of which are associated with photoreceptor R3 and R4 specification. Here, we show that Abl kinase negatively regulates OR through its activity in the R3/R4 pair. Abl is localized to apical junctional regions in R4, but not in R3, during OR, and this apical localization requires Notch signaling. We demonstrate that Abl and Notch interact genetically during OR, and Abl co-immunoprecipitates in complexes with Notch in eye discs. Perturbations of Abl interfere with adherens junctional organization of ommatidial preclusters, which mediate the OR process. Together, our data suggest that Abl kinase acts directly downstream of Notch in R4 to fine-tune OR via its effect on adherens junctions.

Stratum is required for both apical and basolateral transport through stable expression of Rab10 and Rab35 in Drosophila photoreceptors.

Post-Golgi transport for specific membrane domains, also termed polarized transport, is essential for the construction and maintenance of polarized cells. Highly polarized Drosophila photoreceptors serve as a good model system for studying the mechanisms underlying polarized transport. The Mss4 Drosophila ortholog, Stratum (Strat), controls basal restriction of basement membrane proteins in follicle cells, and Rab8 acts downstream of Strat. We investigated the function of Strat in fly photoreceptors and found that polarized transport in both the basolateral and the rhabdomere membrane domains was inhibited in Strat-deficient photoreceptors. We also observed 79 and 55% reductions in Rab10 and Rab35 levels, respectively, but no reduction in Rab11 levels in whole-eye homozygous clones of Stratnull. Moreover, Rab35 was localized in the rhabdomere, and loss of Rab35 resulted in impaired Rh1 transport to the rhabdomere. These results indicate that Strat is essential for the stable expression of Rab10 and Rab35, which regulate basolateral and rhabdomere transport, respectively, in fly photoreceptors.

A Quantitative Model of Sporadic Axonal Degeneration in the Drosophila Visual System.

In human neurodegenerative diseases, neurons undergo axonal degeneration months to years before they die. Here, we developed a system modeling early degenerative events in Drosophila adult photoreceptor cells. Thanks to the stereotypy of their axonal projections, this system delivers quantitative data on sporadic and progressive axonal degeneration of photoreceptor cells. Using this method, we show that exposure of adult female flies to a constant light stimulation for several days overcomes the intrinsic resilience of R7 photoreceptors and leads to progressive axonal degeneration. This was not associated with apoptosis. We furthermore provide evidence that loss of synaptic integrity between R7 and a postsynaptic partner preceded axonal degeneration, thus recapitulating features of human neurodegenerative diseases. Finally, our experiments uncovered a role of postsynaptic partners of R7 to initiate degeneration, suggesting that postsynaptic cells signal back to the photoreceptor to maintain axonal structure. This model can be used to dissect cellular and circuit mechanisms involved in the early events of axonal degeneration, allowing for a better understanding of how neurons cope with stress and lose their resilience capacities.SIGNIFICANCE STATEMENT Neurons can be active and functional for several years. In the course of aging and in disease conditions leading to neurodegeneration, subsets of neurons lose their resilience and start dying. What initiates this turning point at the cellular level is not clear. Here, we developed a model allowing to systematically describe this phase. The loss of synapses and axons represents an early and functionally relevant event toward degeneration. Using the ordered distribution of Drosophila photoreceptor axon terminals, we assembled a system to study sporadic initiation of axon loss and delineated a role for non-cell-autonomous activity regulation in the initiation of axon degeneration. This work will help shed light on key steps in the etiology of nonfamilial cases of neurodegenerative diseases.

Novel mechanisms in phosphoinositide turnover

The hydrolysis of phosphatidylinositol 4,5‐ bisphosphate [PI(4,5)P2] at the plasma membrane by receptor activated phospholipase C (PLC) activity is a conserved mechanism of signal transduction. Given the low abundance of PI(4,5)P2 at the plasma membrane, its hydrolysis needs to be coupled to lipid resynthesis to ensure continued PLC activity during receptor activation. However, the mechanism by which PI(4,5)P2depletion during signalling is coupled to its resynthesis remains unknown. PI(4,5)P2synthesis is catalyzed by lipid kinase activity and the phosphorylation of phosphatidylinositol 4 phosphate (PI4P) by phosphatidylinositol 4 phosphate 5 kinase (PIP5K) is the final step in this process. In Drosophila photoreceptors, sensory transduction of photon absorption is transduced into PLC activity leading to an electrical response to light. During this process, PI(4,5)P2is resynthesized by a PIP5K activity but the mechanism by which the activity of this enzyme is coupled to PLC signalling is not known. In this study, we identify a unique protein isoform of dPIP5K,dPIP5KL that is both necessary and sufficient to mediate PI(4,5)P2synthesis during phototransduction. The activity of dPIP5KLin vitrois enhanced by depletion of PNUT, a non‐redundant subunit of the septin family of GTP binding proteins and in vivo, depletion of dPIP5KL rescues the effect of pnut depletion on the light response and PI(4,5)P2resynthesis during PLC signalling. Thus, our work defines a septin 7 mediated mechanism through which PIP5K activity is coupled to ongoing PLC mediated PI(4,5)P2 depletion.