Biology subjects

PADINJAT, R.

Publications and source records attributed to PADINJAT, R..

14 recordsLinked to original sources

bioRxiv2025

Phosphatidylinositol 5-phosphate 4-kinase (PIP4K) regulates sugar homeostasis in Drosophila

Metabolic adaptation such as sugar homeostasis is central to the response of living organisms to environmental changes and in insects, sugar homeostasis is an integral part of larval metabolism. Mutants of phosphatidylinositol 5-phosphate 4-kinase (dPIP4K) show altered growth and larval development. We find that dPIP4K mutants (dPIP4K29) show enhanced glucose uptake by the fat body, accompanied by increased circulating trehalose levels in feeding third instar larvae. Following acute starvation on a low sugar diet, dPIP4K29 larvae show reduced survival associated with high circulating trehalose levels, while under equivalent conditions, wild-type larvae undergo depletion of circulating trehalose. While Tret1-1PA transcripts were upregulated on starvation in muscle and brain tissue of controls, this enhancement was abrogated in dPIP4K29. Muscle specific depletion of Tret1-1PA in wild type larvae prevents reduction of circulating trehalose levels upon starvation, phenocopying that seen in dPIP4K29and selective reconstitution of dPIP4K in muscle of dPIP4K29 could rescue starvation responses to wild type levels. Together, these findings identify dPIP4K as a key regulator of sugar homeostasis in larval tissues and survival in larvae during starvation.

developmental biology↗

bioRxiv2025

OCRL regulates lysosomal function and endolysosomal homeostasis in Drosophila nephrocytes

The OCRL gene encodes a lipid phosphatase that dephosphorylates phosphatidylinositol 4,5 bisphosphate [(PI(4,5)P2]. Mutations in OCRL lead to a rare human genetic disorder, Lowe syndrome (LS) that affects the eye, kidney and brain. OCRL is widely expressed in cells and is localized to multiple organelles, including the plasma membrane, endosomes, Golgi and lysosomes. Although multiple defects in the endo-lysosomal system have been reported in OCRL depleted cells, the primary site of action of OCRL is unclear. Here we present a Drosophila nephrocyte model of LS; depletion of Drosophila OCRL (dOCRL) manifests with defects in endocytic uptake, altered endosomal compartments as well as expanded but dysfunctional lysosomes. Reconstitution of dOCRL depleted nephrocytes with a lysosome targeted version of the enzyme rescues not only the lysosomal defects but surprisingly also defects in endosomal structure and function. These findings suggest that the primary defect in LS cells is likely to be altered lysosome structure and function. Therefore, regulation of PI(4,5)P2 homeostasis at the lysosome membrane by OCRL is critical to homeostasis of the endosomal system.

cell biology↗

bioRxiv2025

Neurodevelopmental origin of seizures in Lowe syndrome

Lowe syndrome (LS) is a rare X-linked monogenic disorder resulting from mutations in the OCRL1 gene that encodes a phosphatidylinositol 4,5-bisphosphate 5 phosphatase enzyme. Patients with LS exhibit a range of neurological symptoms, including neurodevelopmental delays, hypotonia, febrile seizures, and behavioural abnormalities; however, the cellular and developmental origins of LS remain poorly understood. The Drosophila genome encodes a single homolog of OCRL (docrl). Here, we report that a germline null allele of docrl (docrlKO) shows heat induced seizures reminiscent of the febrile seizures in LS patients. Cell type specific deletion of docrl in neurons was sufficient to recapitulate the heat induced seizures seen in docrlKO indicating a cell autonomous requirement of docrl in neurons to prevent seizures. Temporally controlled deletion of docrl showed that heat induced seizure in adults were predetermined by a requirement of docrl in neural stem cells during embryonic neurogenesis. Collectively, our findings demonstrate the developmental origin of the neurological manifestations of LS highlighting the need to target potential therapeutic interventions during this developmental time window.

neuroscience↗

bioRxiv2025

Phosphatidylinositol 5 phosphate 4 kinase regulates phosphatidylinositol 3,4 bisphosphate levels in vivo.

In Drosophila, loss of phosphatidylinositol 5 phosphate 4 kinase (PIP4K) results in a reduction in larval salivary gland cell size. Previous studies have shown that this reduction in cell size is not correlated with the levels of phosphatidylinositol 5 phosphate (PI5P), the canonical substrate of PIP4K but to the levels of phosphatidylinositol 3 phosphate (PI3P) a substrate that is used less effectively by the PIP4K enzyme in vitro. The phosphorylation of PI3P by PIP4K generates phosphatidylinositol 3,4 bisphosphate [PI(3,4)P2]. Using a biosensor for PI(3,4)P2, surprisingly, we find that depletion of PIP4K leads to an elevation of intracellular PI(3,4)P2 punctae in salivary gland cells. This elevation in PI(3,4)P2 punctae was not dependent on the catalytic activity of dPIP4K. Rather, we found that the elevation of PI(3,4)P2 was dependent on the catalytic activity of Class II phosphatidylinositol 3 kinase (Class II PI3K). Thus, the PIP4K protein regulates an intracellular pool of PI(3,4)P2 via Class II PI3K activity in Drosophila cells.

cell biology↗

bioRxiv2025

Ceramide transfer protein regulates G-protein coupled phospholipase signalling in Drosophila photoreceptors.

The non-vesicular transfer of lipids between organelles at membrane contact sites (MCS) has been proposed as a key principle in the regulation of cell physiology. While several proteins with lipid transfer activity have been identified and localized to MCS, their functional significance for supporting physiology is poorly understood. Ceramide transfer protein (CERT) is one such molecule that can transfer ceramide between membranes in vitro. However, evidence for the mechanism and in vivo significance of CERT function is limited. In this study, we have analyzed the function of the only gene (dcert) encoding CERT in Drosophila. We find that loss of function alleles of dcert (dcert1), show elevated levels of short chain ceramide species along with a reduction in the levels of its metabolite phosphatidyl ethanolamine ceramide. Physiological analysis of dcert1 mutant alleles showed reduced electrical responses in the eye to light stimulation although photoreceptors did not undergo retinal degeneration, and this phenotype could be rescued by reconstitution of dcert1 with the wild type gene. The altered light response in dcert1 was associated with a reduction in the rate of phosphatidylinositol 4,5 bisphosphate (PIP2) resynthesis following light induced phospholipase C (PLC) stimulation. The reduced electrical response of dcert1 could be suppressed by reducing ceramide synthesis at the ER. Taken together, our findings suggest that ceramide synthesized at the ER and transferred to the Golgi by CERT regulates G-protein coupled phospholipase C signaling in vivo.

cell biology↗

bioRxiv2024

The accessory domains of Phospholipase D regulate its localization and activity in Drosophila photoreceptors

Phospholipase D (PLD) is activated during the response of animal cells to a large number of extracellular signals including growth factors, hormones and other extracellular stimuli. The product of PLD activity, phosphatidic acid (PA) is thought to act as a signalling molecule in cells. Since both the substrate, phosphatidylcholine and product phosphatidic acid (PA) are membrane anchored lipids, the precise localization and activation of PLD at specific locations in cells is critical. Here we report that that the precise localization of Drosophila PLD (dPLD) in photoreceptors is critical to maintain the structure of these cells during illumination. This localization of dPLD is dependent on its N-terminal PH domain; deletion of the PH domain (dPLD{Delta}PH) results in mis localization of the protein, loss of functional activity and dPLD{Delta}PHis unable to rescue the phenotypes of dPLD loss of function. By contrast, deletion of the PX domain (dPLD{Delta}PX) does not impact the localization of dPLD but enhances the functional activity of the protein. Thus the PH and PX domains of dPLD regulate its localization and activity respectively.

cell biology↗

bioRxiv2024

The conserved biochemical activity and function of an early metazoan phosphatidylinositol 5 phosphate 4-kinase regulates growth and development.

The ability to co-ordinate function between multiple cells is a critical requirement for multi-cellularity. This co-ordination is mediated by hormones or growth factors, molecules secreted by one cell type that can convey information to the other cells and influence their behaviour. Hormone-dependent signalling is mediated by second messenger systems;phosphoinositides (PIs) generated by lipid kinase activity are one such key second messenger system. Phosphatidylinositol 5 phosphate 4-kinase (PIP4K) is a lipid kinase that phosphorylates phosphatidylinositol 5-phosphate (PI5P) to generate phosphatidylinositol 4,5 bisphosphate [PI(4,5)P2]. Following a comprehensive bioinformatics analysis of ca. 23296 proteomes covering the tree of life, we find that PIP4K is a metazoan-specific enzyme, although its homologs are also found in choanoflagellate genomes. To understand their function in early metazoans, we experimentally analysed the biochemical activity and physiological function of PIP4K from several early metazoans. We find that the PIP4K enzyme from an early branching metazoan sponge Amphimedon queenslandica (AqPIP4K), regarded as the earliest evolved metazoan, shows a biochemical activity highly conserved with human PIP4K; AqPIP4K is able to selectively phosphorylate PI5P to generate PI(4,5)P2 just as effectively as the human enzyme. Further, AqPIP4K was able to rescue the reduced cell size, growth and development phenotype in larvae of a null mutant in Drosophila PIP4K. These phenotypes are regulated through activity of the hormone insulin, acting via the cell surface insulin receptor, a member of the receptor tyrosine kinase family, that is unique to metazoans. Together, our findings indicate that in early metazoans, AqPIP4K is likely to function in a signal transduction pathway that is required for receptor tyrosine kinase signalling. Overall, our work defines PIP4K as a signal transduction motif required to regulate receptor tyrosine kinase signalling for intercellular communication in the earliest forms of metazoa.

biochemistry↗

bioRxiv2024

Ca2+ binding to Esyt is required to modulate membrane contact site density in Drosophila photoreceptors

Membrane Contact Sites (MCS) between the plasma membrane (PM) and endoplasmic reticulum (ER) have been shown to regulate Ca2+ influx into animal cells. However, the mechanisms by which cells modulate ER-PM MCS density is not understood and the role of Ca2+, if any, in regulating this process is not known. We report that in Drosophila photoreceptors, MCS density is dependent on the activity of the Ca2+ permeable channels-TRP and TRPL. This regulation of MCS density by Ca2+ is mediated by extended synaptotagmin (dEsyt), a protein localised to ER-PM MCS in photoreceptors and previously shown to regulate MCS density. We find that the Ca2+ binding activity of dEsyt is required for its functional activity in vivo. dEsytCaBM, a Ca2+ non-binding mutant of dEsyt is unable to modulate MCS structure in a manner equivalent to its wild type counterpart. Further, when reconstituted in dEsyt null photoreceptors, in contrast to wild type dEsyt, dEsytCaBM is unable to rescue ER-PM MCS density and other key phenotypes. Finally, when expressed in wild type photoreceptors, dEsytCaBM phenocopies loss of dEsyt function. Taken together, our data supports a role for the Ca2+ binding activity of dEsyt in regulating the ER-PM MCS density in photoreceptors thus tuning signal transduction in response to Ca2+ influx triggered by ambient illumination. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=196 SRC="FIGDIR/small/588361v1_ufig1.gif" ALT="Figure 1"> View larger version (38K): org.highwire.dtl.DTLVardef@f8faacorg.highwire.dtl.DTLVardef@114009org.highwire.dtl.DTLVardef@8337f0org.highwire.dtl.DTLVardef@cd7c2f_HPS_FORMAT_FIGEXP M_FIG C_FIG

cell biology↗

bioRxiv2024

A genetic and physiological model of renal dysfunction in Lowe syndrome

Lowe syndrome (LS) is an X-linked recessive genetic disorder characterized by renal dysfunction, neurodevelopmental defects, and cataract. The affected gene, OCRL encodes for a polyphosphoinositide 5-phosphatase. OCRL is localized to multiple sub-cellular locations in the endolysosomal system and defects in these organelles have been described in human cells depleted of OCRL. However, the relationship of the endolysosomal defects in OCRL depleted cells to the altered physiology of kidney cells of LS patients has not been completely determined. Here we model the kidney phenotypes of LS using a Drosophila nephrocyte model. Using this model system, we demonstrate that OCRL plays a cell-autonomous role in nephrocyte function. Deletion of the only OCRL ortholog in Drosophila (dOCRL) leads to cell-autonomous defects in larval nephrocyte structure and function. Null mutants of dOCRL (dOCRLKO) show defects in the endolysosomal system of larval nephrocytes that are associated with physiological defects in nephrocyte function. These defects could be rescued by reconstitution with a human OCRL transgene but not with a phosphatase dead version or a human LS patient derived mutation. Overall, this work provides a model system to understand the mechanisms by which the sub-cellular changes from loss of OCRL leads to defects in kidney function in human patients.

cell biology↗

bioRxiv2023

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

Lipid transfer proteins mediate the transfer of lipids between organelle membranes in eukaryotes and loss of function in these has been linked to neurodegenerative disorders. However, the mechanism by which loss of lipid transfer protein function leads to neurodegeneration is not understood. In Drosophila photoreceptors, depletion of Retinal Degeneration B (RDGB), a phosphatidylinositol transfer protein localized to endoplasmic reticulum-plasma membrane contact sites leads to defective phototransduction and retinal degeneration but the mechanism by which RDGB function is regulated and the process by which loss of this activity leads to retinal degeneration is not understood. RDGB is localized to membrane contact sites (MCS) and this depends in 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 for novel lipids in regulating RDGB function and for transcriptional and ubiquitination processes in mediating retinal degeneration in rdgB. The human homologs of several of these molecules have been implicated in neurodevelopmental diseases underscoring the importance of VAP mediated processes in these disorders.

neuroscience↗

bioRxiv2022

PI3P dependent regulation of cell size by phosphatidylinositol 5-phosphate 4-kinase

Phosphatidylinositol 3-phosphate (PI3P) and Phosphatidylinositol 5-phosphate (PI5P) are low abundant phosphoinositides crucial for key cellular events such as endosomal trafficking and autophagy. Phosphatidylinositol 5-phosphate 4-kinase (PIP4K) is an enzyme that regulates PI5P in vivo but can act on both PI5P and PI3P, in vitro. In this study, we report a novel role for PIP4K in regulating PI3P levels in Drosophila tissues. Loss-of-function mutants of the only PIP4K gene in Drosophila (dPIP4K29) show reduced cell size in larval salivary glands. We find that PI3P levels are elevated in dPIP4K29 tissues and that reverting PI3P levels back towards wild type, without changes in PI5P levels, can also rescue the reduced cell size phenotype. dPIP4K29 mutants also show an upregulation in autophagy and the reduced cell size can be reverted by decreasing Atg8a, that is required for autophagosome maturation. Lastly, increasing PI3P levels in wild type salivary glands can phenocopy the reduction in cell size and associated upregulation of autophagy seen in dPIP4K29. Thus, our study reports for the first time, a role for a PIP4K-regulated PI3P pool in the control of autophagy and cell size regulation that may explain the reported role of PIP4K in regulating neurodegeneration and tumour growth.

cell biology↗

bioRxiv2021

Genomic sequencing of Lowe syndrome trios reveal a mechanism for the heterogeneity of neurodevelopmental phenotypes

Lowe syndrome is an X-linked recessive monogenic disorder resulting from mutations in the OCRL gene that encodes a phosphatidylinositol 4,5 bisphosphate 5-phosphatase. The disease affects three organs-the kidney, brain and eye and clinically manifests as proximal renal tubule dysfunction, neurodevelopmental delay and congenital cataract. Although Lowe syndrome is a monogenic disorder, there is considerable heterogeneity in clinical presentation; some individuals show primarily renal symptoms with minimal neurodevelopmental impact whereas others show neurodevelopmental defect with minimal renal symptoms. However, the molecular and cellular mechanisms underlying this clinical heterogeneity remain unknown. Here we analyze a Lowe syndrome family in whom affected members show clinical heterogeneity with respect to the neurodevelopmental phenotype despite carrying an identical mutation in the OCRL gene. Genome sequencing and variant analysis in this family identified a large number of damaging variants in each patient. Using novel analytical pipelines and segregation analysis we prioritize variants uniquely present in the patient with the severe neurodevelopmental phenotype compared to those with milder clinical features. The identity of genes carrying such variants underscore the role of additional gene products enriched in the brain or highly expressed during brain development that may be determinants of the neurodevelopmental phenotype in Lowe syndrome. We also identify a heterozygous variant in CEP290, previously implicated in ciliopathies that underscores the potential role of OCRL in regulating ciliary function that may impact brain development. More generally, our findings demonstrate analytic approaches to identify high-confidence genetic variants that could underpin the phenotypic heterogeneity observed in monogenic disorders.

genomics↗

bioRxiv2021

Septin function tunes lipid kinase activity and phosphatidylinositol 4,5 bisphosphate turnover during G-protein coupled PLC signaling in vivo

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)P2 depletion during signalling is coupled to its resynthesis remains unknown. PI(4,5)P2 synthesis 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)P2 is 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)P2 synthesis during phototransduction. The activity of dPIP5KL in vitro is enhanced by depletion of PNUT, a non-redundant subunit of the septin family of GTP binding proteins and in vivo, depletion of pnut rescues the effect of dPIP5KL depletion on the light response and PI(4,5)P2 resynthesis during PLC signalling. Lastly we find that depletion of Septin Interacting Protein 1 (Sip1), previously shown to bind PNUT, phenocopies the effect of dPIP5KL depletion in vivo. Thus, our work defines a septin 7 and Sip1 mediated mechanism through which PIP5K activity is coupled to ongoing PLC mediated PI(4,5)P2 depletion.

cell biology↗

bioRxiv2020

An evolutionarily conserved metallophosphodiesterase is a determinant of lifespan in Drosophila

Evolutionarily conserved genes usually have a critical role to play during organismal aging and longevity. Here, we show that a previously uncharacterized Class III metallophosphoesterase in Drosophila, an ortholog of the MPPED1 and MPPED2 proteins in mammals, is necessary for optimal lifespan. dMPPED is the product of the gene CG16717 and hydrolyzed a variety of phosphodiester substrates in a metal-dependent manner. dMPPED was expressed widely during development and in the adult fly. Deletion of the gene in flies dramatically reduced lifespan, without affecting development or fecundity. Longevity was restored on ubiquitous expression of the protein, and neuronal expression of both wild type and the catalytically inactive form of dMPPED was also able to restore normal lifespan. Overexpression of the protein, both ubiquitously and neuronally in wild type flies extended lifespan by ~ 20%. RNA-seq analysis of dMPPEDKO flies revealed mis-regulation of innate immune pathways, a number of transcription factors and genes earlier reported to affect aging and lifespan. Importantly, neuronal expression of mammalian MPPED2 was able to rescue lifespan in dMPPEDKO flies, but not extend lifespan in wild type flies. This reports the first description of the biological role of an evolutionarily conserved metallophosphoesterase that may serve as a scaffolding protein in diverse signaling pathways to modulate longevity in the fly.

developmental biology↗