@article {164007, title = {The multifaceted phosphatidylinositol 5 phosphate 4-kinase proteins: molecular properties and biological functions.}, journal = {Biochim Biophys Acta Mol Cell Biol Lipids}, volume = {1871}, year = {2026}, month = {2026 Jan}, pages = {159698}, abstract = {

Phosphoinositides are regulators of key cell biological processes such as plasma membrane function, vesicular transport, cytoskeletal and nuclear organization. In turn, their levels are tightly controlled by the function of lipid kinases and phosphatases that modify specific hydroxyl groups on the inositol ring. Phosphatidylinositol 5 phosphate 4-kinase (PIP4K) is one such lipid kinase. Although initially discovered as an enzyme that phosphorylates the 4th hydroxyl on the inositol ring of phosphatidylinositol 5 phosphate (PI5P) with exquisite specificity, it has recently emerged that PIP4K may also work on other substrates. Interestingly, recent studies have also proposed functions for this enzyme that do not require its catalytic activity. Although most elements of phosphoinositide signalling are conserved across all eukaryota, a limited number of phosphoinositide kinases and phosphatases including PIP4K seem to be a unique feature of the genomes of organisms that exist in a multicellular state but not unicellular eukaryotes. Genetic studies in model organisms implicate PIP4K function in key processes such as hormone regulated metabolic control as well as cell division and growth. Consequently, PIP4K function has important biomedical implications in the context of cancer, metabolic syndrome and autoimmune disorders. In this review, we analyze emerging findings on PIP4K function and reflect on the biochemical raison d{\textquoteright}{\^e}tre of how this protein regulates cell physiology in metazoans.

}, keywords = {Animals, Humans, Phosphatidylinositol Phosphates, Phosphotransferases (Alcohol Group Acceptor), Signal Transduction}, issn = {1879-2618}, doi = {10.1016/j.bbalip.2025.159698}, url = {https://pubmed.ncbi.nlm.nih.gov/41130585/}, author = {Krishnan, Harini and Venugopal, Aishwarya and Alex, Preethi and Raghu, Padinjat} } @article {155676, title = {Ca2+ binding to Esyt modulates membrane contact site density in Drosophila photoreceptors.}, journal = {J Cell Biol}, volume = {224}, year = {2025}, month = {2025 May 05}, abstract = {

Membrane contact sites (MCS) between the plasma membrane (PM) and endoplasmic reticulum (ER) regulate Ca2+ influx. However, the mechanisms by which cells modulate ER-PM MCS density are not understood, and the role of Ca2+, if any, in regulating these is unknown. We report that in Drosophila photoreceptors, MCS density is regulated by the Ca2+ channels, TRP and TRPL. Regulation of MCS density by Ca2+ is mediated by Drosophila extended synaptotagmin (dEsyt), a protein localized to ER-PM MCS and previously shown to regulate MCS density. We find that the Ca2+-binding activity of dEsyt is required for its function in vivo. dEsytCaBM, a Ca2+ non-binding mutant of dEsyt is unable to modulate MCS structure. Further, reconstitution of dEsyt null photoreceptors with dEsytCaBM is unable to rescue ER-PM MCS density and other key phenotypes. Thus, our data supports a role for Ca2+ binding to dEsyt in regulating ER-PM MCS density in photoreceptors thus tuning signal transduction during light-activated Ca2+ influx.

}, keywords = {Animals, Calcium, Calcium Signaling, Cell Membrane, Drosophila melanogaster, Drosophila Proteins, Endoplasmic Reticulum, Photoreceptor Cells, Invertebrate, Synaptotagmins, Transient Receptor Potential Channels}, doi = {10.1083/jcb.202407190}, author = {Nath, Vaisaly R and Krishnan, Harini and Mishra, Shirish and Raghu, Padinjat} } @article {161978, title = {Enhanced Notch dependent gliogenesis and delayed physiological maturation underlie neurodevelopmental defects in Lowe syndrome.}, journal = {EMBO Mol Med}, year = {2025}, month = {2025 Nov 11}, abstract = {

Coordination of cellular and physiological development by signaling is required for normal brain structure and function. Mutations in OCRL, a phosphatidylinositol 4,5 bisphosphate [PI(4,5)P], 5-phosphatase leads to Lowe Syndrome (LS). However, the mechanism by which mutations in OCRL leads to the neurodevelopmental phenotypes of LS is not understood. We find that on differentiation of LS patient iPSC, neural cultures show reduced excitability and enhanced GFAP levels. Multiomic single-nucleus RNA and ATACseq analysis of neural stem cells revealed enhanced numbers of cells with a gliogenic cell state. Analysis of snRNA seq revealed increased levels of DLK1, a Notch ligand in LS patient NSC associated increased levels of cleaved Notch and elevation of its transcriptional target HES5, indicating upregulated Notch signaling. Treatment of iPSC derived brain organoid with an inhibitor of PIP5K, the lipid kinase that synthesizes PI(4,5)P, was able to restore neuronal excitability and rescue Notch signaling defects in OCRL deficient organoids. Overall, our results demonstrate a role for PI(4,5)P dependent regulation of Notch signaling, cell fate specification and neuronal excitability regulated by OCRL.

}, issn = {1757-4684}, doi = {10.1038/s44321-025-00327-y}, author = {Sharma, Yojet and Bhatia, Priyanka and Rangappa, Gagana and Saha, Sankhanil and Raghu, Padinjat} } @article {162676, title = {Mitochondrial structure and function in OCRL depleted cells.}, journal = {Front Cell Dev Biol}, volume = {13}, year = {2025}, month = {2025}, pages = {1679675}, abstract = {

Lowe syndrome (LS) is an X-linked, recessive disease with a characteristic clinical triad of eye, brain, and kidney defects. LS results from mutations in the gene that encodes for inositol polyphosphate 5-phosphatase enzyme. The OCRL protein has been localized to multiple subcellular organelles including the plasma membrane and endo-lysosomal system, but the relevance of these to disease phenotypes is unclear. Previous studies have reported severe hypotonia at birth in LS patients along with structural changes in the mitochondria in muscle biopsies. These mitochondrial changes have been proposed to be secondary to renal tubular acidosis seen in LS patients. In this study, we find that neural stem cells and neurons differentiated from OCRL-depleted induced pluripotent stem cells (iPSCs) show mild defects in mitochondrial structure and function, whereas such defects are not seen in the iPSCs themselves. These mitochondrial phenotypes in neural stem cells and neurons were associated with modest changes in the mitochondrial transcriptome. Overall, our results indicate that loss of OCRL leads to mild cell autonomous defects in mitochondrial structure and function that is cell type-dependent.

}, issn = {2296-634X}, doi = {10.3389/fcell.2025.1679675}, author = {Philip, Ron George and Bhatia, Priyanka and Sharma, Yojet and Raghu, Padinjat} } @article {159722, title = {Phosphoinositide acyl chain diversity: comparative analysis across species and mouse tissues.}, journal = {Biochim Biophys Acta Mol Cell Biol Lipids}, volume = {1870}, year = {2025}, month = {2025 Aug}, pages = {159640}, abstract = {

Cells create acyl chain compositions for their phosphoinositide (PIPn) pools that are distinct from other phospholipid classes. While some lower eukaryotes present highly heterogeneous PIPn (e.g., yeast, fly), more complex organisms typically display PIPn enriched in fewer molecular species (e.g., the C38:4 species in fish, frog and mice). A comprehensive analysis of murine tissues (using both LC-MS/MS and MSI) confirms a general enrichment for C38:4-PIPn but also highlights the existence of several cell populations with strikingly divergent acyl chain compositions, characterised by the prevalence of shorter-chain, more saturated species (e.g., C32:0 in the testes and C34:1 in the prostate). The evolutionary pressures driving the creation of these specific acyl chain compositions are still unclear; current evidence suggests there is probably a balance to be achieved in different cell types between the biophysical constraints imposed by PIPn as membrane-captive {\textquoteright}messengers{\textquoteright} (e.g., flexibility in head group presentation in different membrane environments), the demand for substantial de novo lipid synthesis (e.g., in rapid membrane expansion), the need for acyl chain remodelling (e.g., in molecular segregation of functional pools) and fatty acid availability. Moreover, it would appear inevitable that this balance will be distorted under most cell culture conditions in vitro.

}, keywords = {Animals, Male, Mice, Phosphatidylinositols, Species Specificity, Tandem Mass Spectrometry}, issn = {1879-2618}, doi = {10.1016/j.bbalip.2025.159640}, author = {Barneda, David and Janardan, Vishnu and Swales, John and Ciaccia, Maria and Goodwin, Richard and Cosulich, Sabina and Raghu, Padinjat and Clark, Jonathan and Stephens, Len and Hawkins, Phillip} } @article {149383, title = {CDS2 expression regulates de novo phosphatidic acid synthesis.}, journal = {Biochem J}, volume = {481}, year = {2024}, month = {2024 Oct 16}, pages = {1449-1473}, abstract = {

CDS enzymes (CDS1 and 2 in mammals) convert phosphatidic acid (PA) to CDP-DG, an essential intermediate in the de novo synthesis of PI. Genetic deletion of CDS2 in primary mouse macrophages resulted in only modest changes in the steady-state levels of major phospholipid species, including PI, but substantial increases in several species of PA, CDP-DG, DG and TG. Stable isotope labelling experiments employing both 13C6- and 13C6D7-glucose revealed loss of CDS2 resulted in a minimal reduction in the rate of de novo PI synthesis but a substantial increase in the rate of de novo PA synthesis from G3P, derived from DHAP via glycolysis. This increased synthesis of PA provides a potential explanation for normal basal PI synthesis in the face of reduced CDS capacity (via increased provision of substrate to CDS1) and increased synthesis of DG and TG (via increased provision of substrate to LIPINs). However, under conditions of sustained GPCR-stimulation of PLC, CDS2-deficient macrophages were unable to maintain enhanced rates of PI synthesis via the {\textquoteright}PI cycle{\textquoteright}, leading to a substantial loss of PI. CDS2-deficient macrophages also exhibited significant defects in calcium homeostasis which were unrelated to the activation of PLC and thus probably an indirect effect of increased basal PA. These experiments reveal that an important homeostatic response in mammalian cells to a reduction in CDS capacity is increased de novo synthesis of PA, likely related to maintaining normal levels of PI, and provides a new interpretation of previous work describing pleiotropic effects of CDS2 deletion on lipid metabolism/signalling.

}, keywords = {Animals, Calcium, Diacylglycerol Cholinephosphotransferase, Macrophages, Mice, Mice, Inbred C57BL, Mice, Knockout, Phosphatidic Acids}, issn = {1470-8728}, doi = {10.1042/BCJ20240456}, author = {Collins, Daniel M and Janardan, Vishnu and Barneda, David and Anderson, Karen E and Niewczas, Izabella and Taylor, Diane and Qiu, Danye and Jessen, Henning J and Lopez-Clavijo, Andrea F and Walker, Simon and Raghu, Padinjat and Clark, Jonathan and Stephens, Len R and Hawkins, Phillip T} } @article {140649, title = {Challenges and opportunities for discovering the biology of rare genetic diseases of the brain.}, journal = {J Biosci}, volume = {49}, year = {2024}, month = {2024}, abstract = {

Diseases of the human nervous system are an important cause of morbidity and mortality worldwide. These disorders arise out of multiple aetiologies of which rare genetic mutations in genes vital to nervous system development and function are an important cause. The diagnosis of such rare disorders is challenging due to the close overlap of clinical presentations with other diseases that are not of genetic origin. Further, understanding the mechanisms by which mutations lead to altered brain structure and function is also challenging, given that the brain is not readily accessible for tissue biopsy. However, recent developments in modern technologies have opened up new opportunities for the analysis of rare genetic disorders of the brain. In this review, we discuss these developments and strategies by which they can be applied effectively for better understanding of rare diseases of the brain. This will lead to the development of new clinical strategies to manage brain disorders.

}, keywords = {Biology, Brain, Humans, Mutation, Rare Diseases}, issn = {0973-7138}, author = {Raghu, Padinjat and Sharma, Yojet and Suseela Devi, Aswathy Bhuvanendran Nair and Krishnan, Harini} } @article {142309, title = {A genetic screen to uncover mechanisms underlying lipid transfer protein function at membrane contact sites }, journal = {Life Sci Alliance}, volume = {7}, year = {2024}, month = {03/2024}, pages = {e202302525}, abstract = {

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.

}, doi = {10.26508/lsa.202302525}, author = {Mishra, S. and Manohar, V. and Chandel, S. and Manoj, T. and Bhattacharya, S. and Hegde, N. and Nath, V.R. and Krishnan, H. and Wendling, C. and Mattia, T.D. and Martinet, A. and Chimata, P. and Alpy, F. and Raghu, Padinjat} } @article {140659, title = {Immunogenicity of SARS-CoV-2 vaccines BBV152 (COVAXIN{\textregistered}) and ChAdOx1 nCoV-19 (COVISHIELD{\texttrademark}) in seronegative and seropositive individuals in India: a multicentre, nonrandomised observational study.}, journal = {Lancet Reg Health Southeast Asia}, volume = {22}, year = {2024}, month = {2024 Mar}, pages = {100361}, abstract = {

BACKGROUND: There are limited global data on head-to-head comparisons of vaccine platforms assessing both humoral and cellular immune responses, stratified by pre-vaccination serostatus. The COVID-19 vaccination drive for the Indian population in the age group 18-45 years began in April 2021 when seropositivity rates in the general population were rising due to the delta wave of COVID-19 pandemic during April-May\ 2021.

METHODS: Between June 30, 2021, and Jan 28, 2022, we enrolled 691 participants in the age group 18-45 years across four clinical sites in India. In this non-randomised and laboratory blinded study, participants received either two doses of Covaxin{\textregistered} (4 weeks apart) or two doses of Covishield{\texttrademark} (12 weeks apart) as per the national vaccination policy. The primary outcome was the seroconversion rate and the geometric mean titre (GMT) of antibodies against the SARS-CoV-2 spike and nucleocapsid proteins post two doses. The secondary outcome was the frequency of cellular immune responses pre- and post-vaccination.

FINDINGS: When compared to pre-vaccination baseline, both vaccines elicited statistically significant seroconversion and binding antibody levels in both seronegative and seropositive individuals. In the per-protocol cohort, Covishield{\texttrademark} elicited higher antibody responses than Covaxin{\textregistered} as measured by seroconversion rate (98.3\% vs 74.4\%, p\ \<\ 0.0001 in seronegative individuals; 91.7\% vs 66.9\%, p\ \<\ 0.0001 in seropositive individuals) as well as by anti-spike antibody levels against the ancestral strain (GMT 1272.1 vs 75.4 binding antibody units/ml [BAU/ml], p\ \<\ 0.0001 in seronegative individuals; 2089.07 vs 585.7 BAU/ml, p\ \<\ 0.0001 in seropositive individuals). As participants at all clinical sites were not recruited at the same time, site-specific immunogenicity was impacted by the timing of vaccination relative to the delta and omicron waves. Surrogate neutralising antibody responses against variants-of-concern including delta and omicron was higher in Covishield{\texttrademark} recipients than in Covaxin{\textregistered} recipients; and in seropositive than in seronegative individuals after both vaccination and asymptomatic infection (omicron variant). T cell responses are reported from only one of the four site cohorts where the vaccination schedule preceded the omicron wave. In seronegative individuals, Covishield{\texttrademark} elicited both CD4+ and CD8+ spike-specific cytokine-producing T cells whereas Covaxin{\textregistered} elicited mainly CD4+ spike-specific T cells. Neither vaccine showed significant post-vaccination expansion of spike-specific T cells in seropositive individuals.

INTERPRETATION: Covishield{\texttrademark} elicited immune responses of higher magnitude and breadth than Covaxin{\textregistered} in both seronegative individuals and seropositive individuals, across cohorts representing the pre-vaccination immune history of most of the vaccinated Indian population.

FUNDING: Corporate social responsibility (CSR) funding from Hindustan Unilever Limited (HUL) and Unilever India Pvt. Ltd. (UIPL).

}, issn = {2772-3682}, doi = {10.1016/j.lansea.2024.100361}, author = {Asokan, Mangaiarkarasi S and Joan, Roshni Florina and Babji, Sudhir and Dayma, Girish and Nadukkandy, Prajitha and Subrahmanyam, Vinutha and Pandey, Archana and Malagi, Girish and Arya, Pooja and Mahajan, Vibhuti and Bhavikatti, Jayateerth and Pawar, Ketakee and Thorat, Aishwarya and Shah, Priyanki and Goud, Ramakrishna B and Roy, Bishnudeo and Rajukutty, Shon and Immanuel, Sushil and Agarwal, Dhiraj and Saha, Sankhanil and Shivaraj, Akshatha and Panikulam, Patricia and Shome, Rajeshwari and Gulzar, Shah-E-Jahan and Sharma, Anusmrithi U and Naik, Ajinkya and Talashi, Shruti and Belekar, Madhuri and Yadav, Ritu and Khude, Poornima and V, Mamatha and Shivalingaiah, Sudarshan and Deshmukh, Urmila and Bhise, Chinmayee and Joshi, Manjiri and Inbaraj, Leeberk Raja and Chandrasingh, Sindhulina and Ghose, Aurnab and Jamora, Colin and Karumbati, Anandi S and Sundaramurthy, Varadharajan and Johnson, Avita and Ramesh, Naveen and Chetan, Nirutha and Parthiban, Chaitra and Ahmed, Asma and Rakshit, Srabanti and Adiga, Vasista and D{\textquoteright}Souza, George and Rale, Vinay and George, Carolin Elizabeth and John, Jacob and Kawade, Anand and Chaturvedi, Akanksha and Raghunathan, Anu and Dias, Mary and Bhosale, Anand and Raghu, Padinjat and Shashidhara, L S and Vyakarnam, Annapurna and Bal, Vineeta and Kang, Gagandeep and Mayor, Satyajit} } @article {137343, title = {IMPA1 dependent regulation of phosphatidylinositol 4,5-bisphosphate and calcium signalling by lithium.}, journal = {Life Sci Alliance}, volume = {7}, year = {2024}, month = {12/2023}, pages = {e202302425}, abstract = {

Lithium (Li) is widely used as a mood stabilizer to treat bipolar affective disorder. However, the molecular targets of Li that underpin its therapeutic effect remain unresolved. Inositol monophosphatase (IMPA1) is an enzyme involved in phosphatidylinositol 4,5-bisphosphate (PIP2) resynthesis after PLC signaling. In vitro, Li inhibits IMPA1, but the relevance of this inhibition within neural cells remains unknown. Here, we report that treatment with therapeutic concentrations of Li reduces receptor-activated calcium release from intracellular stores and delays PIP2 resynthesis. These effects of Li are abrogated in IMPA1 deleted cells. We also observed that in human forebrain cortical neurons, treatment with Li reduced neuronal excitability and calcium signals. After Li treatment of human cortical neurons, transcriptome analyses revealed down-regulation of signaling by glutamate, a key excitatory neurotransmitter in the human brain. Collectively, our findings suggest that inhibition of IMPA1 by Li reduces receptor-activated PLC signaling and neuronal excitability.

}, doi = {10.26508/lsa.202302425}, author = {Saha, S and Krishnan, H and Raghu, Padinjat} } @article {130539, title = {PI3P-dependent regulation of cell size and autophagy by phosphatidylinositol 5-phosphate 4-kinase.}, journal = {Life Sci Alliance}, volume = {6}, year = {2023}, month = {2023 Sep}, abstract = {

Phosphatidylinositol 3-phosphate (PI3P) and phosphatidylinositol 5-phosphate (PI5P) are low-abundance 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 role for PIP4K in regulating PI3P levels in Loss-of-function mutants of the only PIP4K gene show reduced cell size in salivary glands. PI3P levels are elevated in and reverting PI3P levels back towards WT, without changes in PI5P levels, can rescue the reduced cell size. mutants also show up-regulation in autophagy and the reduced cell size can be reverted by depleting Atg8a that is required for autophagy. Lastly, increasing PI3P levels in WT can phenocopy the reduction in cell size and associated autophagy up-regulation seen in Thus, our study reports a role for a PIP4K-regulated PI3P pool in the control of autophagy and cell size.

}, keywords = {Animals, Autophagy, Cell Size, Drosophila, Endosomes}, issn = {2575-1077}, doi = {10.26508/lsa.202301920}, author = {Ghosh, Avishek and Venugopal, Aishwarya and Shinde, Dhananjay and Sharma, Sanjeev and Krishnan, Meera and Mathre, Swarna and Krishnan, Harini and Saha, Sankhanil and Raghu, Padinjat} } @article {124445, title = {Structural organization of RDGB (retinal degeneration B), a multi-domain lipid transfer protein: a molecular modelling and simulation based approach.}, journal = {J Biomol Struct Dyn}, year = {2023}, month = {2023 Feb 20}, pages = {1-15}, abstract = {

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 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 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.

}, issn = {1538-0254}, doi = {10.1080/07391102.2023.2179545}, author = {Krishnan, Harini and Basak, Bishal and Nath, Vaisaly R and Mishra, Shirish and Raghu, Padinjat} } @article {128079, title = {Structural rationale to understand the effect of disease-associated mutations on Myotubularin.}, journal = {Curr Res Struct Biol}, volume = {5}, year = {2023}, month = {2023}, pages = {100100}, abstract = {

Myotubularin or MTM1 is a lipid phosphatase that regulates vesicular trafficking in the cell. The gene is mutated in a severe form of muscular disease, X-linked myotubular myopathy or XLMTM, affecting 1 in 50,000 newborn males worldwide. There have been several studies on the disease pathology of XLMTM, but the structural effects of missense mutations of MTM1 are underexplored due to the unavailability of a crystal structure. MTM1 consists of three domains-a lipid-binding N-terminal GRAM domain, the phosphatase domain and a coiled-coil domain which aids dimerisation of Myotubularin homologs. While most mutations reported to date map to the phosphatase domain of MTM1, the other two domains on the sequence are also frequently mutated in XLMTM. To understand the overall structural and functional effects of missense mutations on MTM1, we curated several missense mutations and performed and studies. Apart from significantly impaired binding to substrate, abrogation of phosphatase activity was observed for a few mutants. Possible long-range effects of mutations from non-catalytic domains on phosphatase activity were observed as well. Coiled-coil domain mutants have been characterised here for the first time in XLMTM literature.

}, issn = {2665-928X}, doi = {10.1016/j.crstbi.2023.100100}, author = {Bhattacharyya, Teerna and Ghosh, Avishek and Verma, Shailya and Raghu, Padinjat and Sowdhamini, Ramanathan} } @article {131285, title = {Validating saliva as a biological sample for cost-effective, rapid and routine screening for SARS-CoV-2}, journal = {INDIAN JOURNAL OF MEDICAL MICROBIOLOGY}, volume = {45}, year = {2023}, month = {09/2023}, abstract = {

Purpose: Compared to nasopharyngeal/oropharyngeal swabs (N/OPS-VTM), non-invasive saliva samples have enormous potential for scalability and routine population screening of SARS-CoV-2. In this study, we investigate the efficacy of saliva samples relative to N/OPS-VTM for use as a direct source for RT-PCR based SARS-CoV-2 detection. Methods: We collected paired nasopharyngeal/oropharyngeal swabs and saliva samples from suspected positive SARS-CoV-2 patients and tested using RT-PCR. We used generalized linear models to investigate factors that explain result agreement. Further, we used simulations to evaluate the effectiveness of saliva-based screening in restricting the spread of infection in a large campus such as an educational institution. Results: We observed a 75.4\% agreement between saliva and N/OPS-VTM, that increased drastically to 83\% in samples stored for less than three days. Such samples processed within two days of collection showed 74.5\% test sensitivity. Our simulations suggest that a test with 75\% sensitivity, but high daily capacity can be very effective in limiting the size of infection clusters in a workspace. Guided by these results, we successfully implemented a saliva-based screening in the Bangalore Life Sciences Cluster (BLiSC) campus. Conclusion: These results suggest that saliva may be a viable alternate source for SARS-CoV-2 surveillance if samples are processed immediately. Although saliva shows slightly lower sensitivity levels when compared to N/ OPS-VTM, saliva collection is logistically advantageous. We strongly recommend the implementation of salivabased screening strategies for large workplaces and in schools, as well as for population-level screening and routine surveillance as we learn to live with the SARS-CoV-2 virus.

}, doi = {10.1016/j.ijmmb.2023.100384}, author = {Ansil, B R and George, Carolin Elizabeth and Chandrasingh, Sindhulina and Viswanathan, Ashwin and Thattai, Mukund and Raghu, Padinjat and Devadiga, Santhosha and Harikumar, Arun Geetha and Harsha, Pulleri Kandi and Nair, Indu and Ramakrishnan, Uma and Mayor, Satyajit} } @article {108059, title = {Analysis of Lipid Signaling in Drosophila Photoreceptors using Mass Spectrometry.}, journal = {J Vis Exp}, year = {2022}, month = {2022 Mar 07}, abstract = {

The activation of phospholipase Cβ (PLCβ) is an essential step during sensory transduction in Drosophila photoreceptors. PLCβ activity results in the hydrolysis of the membrane lipid phosphatidylinositol 4,5 bisphosphate [PI(4,5)P2] leading ultimately to the activation of transient receptor potential (TRP) and TRP like (TRPL) channels. The activity of PLCβ also leads subsequently to the generation of many lipid species several of which have been proposed to play a role in TRP and TRPL activation. In addition, several classes of lipids have been proposed to play key roles in organizing the cell biology of photoreceptors to optimize signaling reactions for optimal sensory transduction. Historically, these discoveries have been driven by the ability to isolate Drosophila mutants for enzymes that control the levels of specific lipids and perform analysis of photoreceptor physiology in these mutants. More recently, powerful mass spectrometry methods for isolation and quantitative analysis of lipids with high sensitivity and specificity have been developed. These are particularly suited for use in Drosophila where lipid analysis is now possible from photoreceptors without the need for radionuclide labeling. In this article, the conceptual and practical considerations in the use of lipid mass spectrometry for the robust, sensitive, and accurate quantitative assessment of various signaling lipids in Drosophila photoreceptors are covered. Along with existing methods in molecular genetics and physiological analysis such lipid is likely to enhance the power of photoreceptors as a model system for discoveries in biology.

}, issn = {1940-087X}, doi = {10.3791/63516}, author = {Panda, Aniruddha and Thakur, Rajan and Kumari, Aastha and Raghu, Padinjat} } @article {105480, title = {A human stem cell resource to decipher the biochemical and cellular basis of neurodevelopmental defects in Lowe Syndrome.}, journal = {Biol Open}, year = {2022}, month = {2022 Jan 13}, abstract = {

Human brain development is a complex process where multiple cellular and developmental events are co-ordinated to generate normal structure and function. Alteration in any of these events can impact brain development, manifesting clinically as neurodevelopmental disorders. Human genetic disorders of lipid metabolism often present with features of altered brain function. Lowe syndrome (LS), is a X-linked recessive disease with features of altered brain function. LS results from mutations in OCRL1 that encodes a phosphoinositide 5-phosphatase enzyme. However, the cellular mechanisms by which loss of OCRL1 leads to brain defects remain unknown. Human brain development involves several cellular and developmental features not conserved in other species and understanding such mechanisms remains a challenge. Rodent models of LS have been generated, but failed to recapitulate features of the human disease. Here we describe the generation of human stem cell lines from LS patients. Further, we present biochemical characterization of lipid metabolism in patient cell lines and demonstrate their use as a "disease-in-a-dish" model for understanding the mechanism by which loss of OCRL1 leads to altered cellular and physiological brain development.

}, issn = {2046-6390}, doi = {10.1242/bio.059066}, author = {Akhtar, Bilal M and Bhatia, Priyanka and Acharya, Shubhra and Sharma, Sanjeev and Sharma, Yojet and Aswathy, B S and Ganapathy, Kavina and Vasudevan, Anil and Raghu, Padinjat} } @article {108052, title = {Septins tune lipid kinase activity and PI(4,5)P turnover during G-protein-coupled PLC signalling in vivo.}, journal = {Life Sci Alliance}, volume = {5}, year = {2022}, month = {2022 Jun}, abstract = {

Phosphatidylinositol 4,5-bisphosphate [PI(4,5)P] hydrolysis by phospholipase C (PLC) is a conserved mechanism of signalling. Given the low abundance of PI(4,5)P, its hydrolysis needs to be coupled to resynthesis to ensure continued PLC activity; however, the mechanism by which depletion is coupled to resynthesis remains unknown. PI(4,5)P synthesis is catalyzed by the phosphorylation of phosphatidylinositol 4 phosphate (PI4P) by phosphatidylinositol 4 phosphate 5 kinase (PIP5K). In photoreceptors, photon absorption is transduced into PLC activity and during this process, PI(4,5)P is resynthesized by a PIP5K. However, the mechanism by which PIP5K activity is coupled to PI(4,5)P hydrolysis is unknown. In this study, we identify a unique isoform dPIP5K, that is both necessary and sufficient to mediate PI(4,5)P synthesis during phototransduction. Depletion of PNUT, a non-redundant subunit of the septin family, enhances dPIP5K activity in vitro and PI(4,5)P resynthesis in vivo; co-depletion of dPIP5K reverses the enhanced rate of PI(4,5)P resynthesis in vivo. Thus, our work defines a septin-mediated mechanism through which PIP5K activity is coupled to PLC-mediated PI(4,5)P hydrolysis.

}, issn = {2575-1077}, doi = {10.26508/lsa.202101293}, author = {Kumari, Aastha and Ghosh, Avishek and Kolay, Sourav and Raghu, Padinjat} } @article {90472, title = {Emerging cell biological functions of phosphatidylinositol 5 phosphate 4 kinase.}, journal = {Curr Opin Cell Biol}, volume = {71}, year = {2021}, month = {2021 Mar 04}, pages = {15-20}, abstract = {

The generation of phosphoinositides (PIs) with spatial and temporal control is a key mechanism in cellular organization and signaling. The synthesis of PIs is mediated by PI kinases, proteins that are able to phosphorylate unique substrates at specific positions on the inositol headgroup to generate signaling molecules. Phosphatidylinositol 5 phosphate 4 kinase (PIP4K) is one such lipid kinase that is able to specifically phosphorylate phosphatidylinositol 5 phosphate, the most recently discovered PI to generate the well-known and abundant PI, phosphatidylinositol 4,5 bisphosphate [PI(4,5)P]. PIP4K appears to be encoded only in metazoan genomes, and several genetic studies indicate important physiological functions for these enzymes in metabolism, immune function, and growth control. PIP4K has recently been reported to localize to multiple cellular compartments, including the nucleus, plasma membrane, endosomal systems, and autophagosome. However, the biochemical activity of these enzymes that is relevant to these physiological functions remains elusive. We review recent developments in this area and highlight emerging roles for these enzymes in cellular organization.

}, issn = {1879-0410}, doi = {10.1016/j.ceb.2021.01.012}, author = {Raghu, Padinjat} } @article {94390, title = {Emerging perspectives on multidomain phosphatidylinositol transfer proteins.}, journal = {Biochim Biophys Acta Mol Cell Biol Lipids}, year = {2021}, month = {2021 Jun 04}, pages = {158984}, abstract = {

The phosphatidylinositol transfer protein domain (PITP) is an evolutionarily conserved protein that is able to transfer phosphatidylinositol between membranes in vitro and in vivo. However some animal genomes also include genes that encode proteins where the PITP is found in cis with a number of additional domains and recent large scale genome sequencing efforts indicate that this type of multidomain architecture is widespread in the animal kingdom. In Drosophila photoreceptors, the multidomain phosphatidylinositol transfer protein RDGB is required to regulate phosphoinositide turnover during G-protein activated phospholipase C signalling. Recent studies in flies and mammalian cell culture models have begun to elucidate functions for the non-PITP of RDGB and its vertebrate orthologs. We review emerging evidence on the genomics, functional and cell biological perspectives of these multi-domain PITP containing proteins.

}, issn = {1879-2618}, doi = {10.1016/j.bbalip.2021.158984}, author = {Raghu, Padinjat and Basak, Bishal and Krishnan, Harini} } @inbook {88400, title = {Label-Free Quantification of Phosphoinositides in Drosophila by Mass Spectrometry.}, booktitle = {Phosphoinositides}, volume = {2251}, number = {Methods Mol Biol}, year = {2021}, month = {2021}, pages = {19-37}, publisher = {Humana-Springer}, organization = {Humana-Springer}, abstract = {

Phosphoinositides (PIs), the seven phosphorylated derivatives of phosphatidylinositol, are recognized as key molecules in the control of multiple molecular events in eukaryotic cells. Within cells, PIs are low-abundance lipids making their detection and quantification challenging. While many methods that allow radiolabeling and quantification of PIs in the context of cultured cells are available, these are not useful in the context of in vivo animal models where cell and developmental processes are best studied. In this chapter, we describe radionuclide-free, mass spectrometry-based methods for the detection and quantification of PIs from Drosophila tissues in vivo. The use of these methods should facilitate the discovery of novel modes by which PIs regulate cellular and developmental processes in complex metazoans.

}, isbn = {978-1-0716-1141-8}, doi = {10.1007/978-1-0716-1142-5_2}, author = {Ghosh, Avishek and Raghu, Padinjat} } @article {79806, title = {Extended synaptotagmin regulates membrane contact site structure and lipid transfer function in~vivo.}, journal = {EMBO Rep}, year = {2020}, month = {2020 Jul 27}, pages = {e50264}, abstract = {

Inter-organelle communication between closely apposed membranes is proposed at membrane contact sites (MCS). However, the regulation of MCS structure and their functional relevance in\ vivo remain debated. The extended synaptotagmins (Esyt) are evolutionarily conserved proteins proposed to function at MCS. However, loss of all three Esyts in yeast or mammals shows minimal phenotypes questioning the functional importance of Esyt. We report that in Drosophila photoreceptors, MCS number is regulated by PLCβ activity. Photoreceptors of a null allele of Drosophila extended synaptotagmin (dEsyt) show loss of ER-PM MCS. Loss of dEsyt results in mislocalization of RDGB, an MCS localized lipid transfer protein, required for photoreceptor structure and function, ultimately leading to retinal degeneration. dEsyt depletion enhanced the retinal degeneration, reduced light responses and slower rates of plasma membrane PIP resynthesis seen in rdgB mutants. Thus, dEsyt function and PLCβ signaling regulate ER-PM MCS structure and lipid transfer in Drosophila photoreceptors.

}, issn = {1469-3178}, doi = {10.15252/embr.202050264}, author = {Nath, Vaisaly R and Mishra, Shirish and Basak, Bishal and Trivedi, Deepti and Raghu, Padinjat} } @article {78000, title = {Functional diversity in a lipidome.}, journal = {Proc Natl Acad Sci U S A}, volume = {117}, year = {2020}, month = {2020 May 26}, pages = {11191-11193}, issn = {1091-6490}, doi = {10.1073/pnas.2004764117}, author = {Raghu, Padinjat} } @article {67833, title = {A Genetic Screen in Drosophila To Identify Novel Regulation of Cell Growth by Phosphoinositide Signaling.}, journal = {G3 (Bethesda)}, volume = {10}, year = {2020}, month = {2019 Nov 08}, pages = {57-67}, abstract = {

Phosphoinositides are lipid signaling molecules that regulate several conserved sub-cellular processes in eukaryotes, including cell growth. Phosphoinositides are generated by the enzymatic activity of highly specific lipid kinases and phosphatases. For example, the lipid PIP, the Class I PI3 kinase that generates it and the phosphatase PTEN that metabolizes it are all established regulators of growth control in metazoans. To identify additional functions for phosphoinositides in growth control, we performed a genetic screen to identify proteins which when depleted result in altered tissue growth. By using RNA-interference mediated depletion coupled with mosaic analysis in developing eyes, we identified and classified additional candidates in the developing eye that regulate growth either cell autonomously or via cell-cell interactions. We report three genes: , and that are important for growth regulation and suggest that these are likely to act via cell-cell interactions in the developing eye. Our findings define new avenues for the understanding of growth regulation in metazoan tissue development by phosphoinositide metabolizing proteins.

}, issn = {2160-1836}, doi = {10.1534/g3.119.400851}, author = {Janardan, Vishnu and Sharma, Sanjeev and Basu, Urbashi and Raghu, Padinjat} } @article {86476, title = {A genome engineering resource to uncover principles of cellular organization and tissue architecture by lipid signalling.}, journal = {Elife}, volume = {9}, year = {2020}, month = {2020 Dec 15}, abstract = {

Phosphoinositides (PI) are key regulators of cellular organization in eukaryotes and genes that tune PI signalling are implicated in human disease mechanisms. Biochemical analyses and studies in cultured cells have identified a large number of proteins that can mediate PI signalling. However, the role of such proteins in regulating cellular processes and development in metazoans remains to be understood. Here we describe a set of CRISPR based genome engineering tools that allow the manipulation of each of these proteins with spatial and temporal control during metazoan development. We demonstrate the use of these reagents to deplete a set of 103 proteins individually in the eye and identify several new molecules that control eye development. Our work demonstrates the power of this resource in uncovering the molecular basis of tissue homeostasis during normal development and in human disease biology.

}, issn = {2050-084X}, doi = {10.7554/eLife.55793}, author = {Trivedi, Deepti and Cm, Vinitha and Bisht, Karishma and Janardan, Vishnu and Pandit, Awadhesh and Basak, Bishal and H, Shwetha and Ramesh, Navyashree and Raghu, Padinjat} } @article {78092, title = {In vitro human stem cell derived cultures to monitor calcium signaling in neuronal development and function}, journal = {Wellcome Open Res}, volume = {5}, year = {2020}, month = {2020}, pages = {16}, abstract = {

The development of the human brain involves multiple cellular processes including cell division, migration, and dendritic growth. These processes are triggered by developmental cues and lead to interactions of neurons and glial cells to derive the final complex organization of the brain. Developmental cues are transduced into cellular processes through the action of multiple intracellular second messengers including calcium. Calcium signals in cells are shaped by large number of proteins and mutations in several of these have been reported in human patients with brain disorders.\  However, the manner in which such mutations impact human brain development remains poorly understood. A key limitation in this regard is the need for a model system in which calcium signaling can be studied in neurons of patients with specific brain disorders. Here we describe a protocol to differentiate human neural stem cells into cortical neuronal networks that can be maintained as live cultures up to 120 days in a dish. Our protocol generates a 2D culture that exhibits molecular features of several layers of the human cerebral cortex. Using fluorescence imaging of intracellular calcium levels, we describe the development of neuronal activity as measured by intracellular calcium transients during development . These transients were dependent on the activity of voltage gated calcium channels and were abolished by blocking sodium channel activity. Using transcriptome analysis, we describe the full molecular composition of such cultures following differentiation thus offering an insight into the molecular basis of activity. Our approach will facilitate the understanding of calcium signaling defects during cortical neuron development in patients with specific brain disorders and a mechanistic analysis of these defects using genetic manipulations coupled with cell biological and physiological analysis.

}, issn = {2398-502X}, doi = {10.12688/wellcomeopenres.15626.1}, author = {Sharma, Yojet and Saha, Sankhanil and Joseph, Annu and Krishnan, Harini and Raghu, Padinjat} } @article {78021, title = {A PI4KIIIα protein complex is required for cell viability during Drosophila wing development.}, journal = {Dev Biol}, volume = {462}, year = {2020}, month = {2020 Jun 15}, pages = {208-222}, abstract = {

Phosphatidylinositol 4 phosphate (PI4P) and phosphatidylinositol 4,5 bisphosphate [PI(4,5)P] are enriched on the inner leaflet of the plasma membrane and proposed to be key determinants of its function. PI4P is also the biochemical precursor for the synthesis of PI(4,5)P but can itself also bind to and regulate protein function. However, the independent function of PI4P at the plasma membrane in supporting cell function in metazoans during development in vivo remains unclear. We find that conserved components of a multi-protein complex composed of phosphatidylinositol 4-kinase IIIα (PI4KIIIα), TTC7 and Efr3 is required for normal vein patterning and wing development. Depletion of each of these three components of the PI4KIIIα complex in developing wing cells results in altered wing morphology. These effects are associated with an increase in apoptosis and can be rescued by expression of an inhibitor of Drosophila caspase. We find that in contrast to previous reports, PI4KIIIα depletion does not alter key outputs of hedgehog signalling in developing wing discs. Depletion of PI4KIIIα results in reduced PI4P levels at the plasma membrane of developing wing disc cells while levels of PI(4,5)P, the downstream metabolite of PI4P, are not altered. Thus, PI4P itself generated by the activity of the PI4KIIIα complex plays an essential role in supporting cell viability in the developing Drosophila wing disc.

}, issn = {1095-564X}, doi = {10.1016/j.ydbio.2020.03.008}, author = {Basu, Urbashi and Balakrishnan, Sruthi S and Janardan, Vishnu and Raghu, Padinjat} } @article {51226, title = {Functional analysis of the biochemical activity of mammalian phosphatidylinositol 5 phosphate 4-kinase enzymes.}, journal = {Biosci Rep}, year = {2019}, month = {2019 Feb 04}, abstract = {

Phosphatidylinositol 5 phosphate 4-kinase (PIP4K) are enzymes that catalyse the phosphorylation of PI5P to generate PI(4,5)P Mammalian genomes contain three genes, PIP4K2A, 2B and 2C and murine knockouts for these suggest important physiological roles The proteins encoded by PIP4K2A, 2B and 2C show widely varying specific activity ; PIP4K2A is highly active and PIP4K2C 2000 times less active and the relationship between this biochemical activity and function is unknown. By contrast the genome encodes a single PIP4K (dPIP4K) that shows high specific activity and loss of this enzyme results in reduced salivary gland cell size We find that the kinase activity of dPIP4K is essential for normal salivary gland cell size Despite their highly divergent specific activity, we find that all three mammalian PIP4K isoforms are able to enhance salivary gland cell size in implying a lack of correlation between activity measurement and function. Further, the kinase activity of PIP4K2C, reported to be almost inactive is required for function. Our findings suggest the existence of unidentified factors that regulate PIP4K enzyme activity .

}, issn = {1573-4935}, doi = {10.1042/BSR20182210}, author = {Mathre, Swarna and Reddy, Balasankara and Ramya, Visvanathan and Krishnan, Harini and Ghosh, Avishek and Raghu, Padinjat} } @article {66852, title = {A novel mass assay to measure phosphatidylinositol-5-phosphate from cells and tissues.}, journal = {Biosci Rep}, volume = {39}, year = {2019}, month = {2019 Oct 30}, abstract = {

Phosphatidylinositol-5-phosphate (PI5P) is a low abundance lipid proposed to have functions in cell migration, DNA damage responses, receptor trafficking and insulin signalling in metazoans. However, studies of PI5P function are limited by the lack of scalable techniques to quantify its level from cells and tissues in multicellular organisms. Currently, PI5P measurement requires the use of radionuclide labelling approaches that are not easily applicable in tissues or in vivo samples. In the present study, we describe a simple and reliable, non-radioactive mass assay to measure total PI5P levels from cells and tissues of Drosophila, a genetically tractable multicellular model. We use heavy oxygen-labelled ATP (18O-ATP) to label PI5P from tissue extracts while converting it into PI(4,5)P2 using an in vitro kinase reaction. The product of this reaction can be selectively detected and quantified with high sensitivity using a liquid chromatography-tandem mass spectrometry (LC-MS/MS) platform. Further, using this method, we capture and quantify the unique acyl chain composition of PI5P from Drosophila cells and tissues. Finally, we demonstrate the use of this technique to quantify elevations in PI5P levels, from Drosophila larval tissues and cultured cells depleted of phosphatidylinositol 5 phosphate 4-kinase (PIP4K), that metabolizes PI5P into PI(4,5)P2 thus regulating its levels. Thus, we demonstrate the potential of our method to quantify PI5P levels with high sensitivity from cells and tissues of multicellular organisms thus accelerating understanding of PI5P functions in vivo.

}, issn = {1573-4935}, doi = {10.1042/BSR20192502}, author = {Ghosh, Avishek and Sharma, Sanjeev and Shinde, Dhananjay and Ramya, Visvanathan and Raghu, Padinjat} } @article {57783, title = {Phosphatidylinositol 5 Phosphate 4-Kinase Regulates Plasma-Membrane PIP Turnover and Insulin Signaling.}, journal = {Cell Rep}, volume = {27}, year = {2019}, month = {2019 May 14}, pages = {1979-1990.e7}, abstract = {

Phosphatidylinositol 3,4,5-trisphosphate (PIP) generation at the plasma membrane is a key event during activation of receptor tyrosine kinases such as the insulin receptor required for normal growth and metabolism. We report that in Drosophila, phosphatidylinositol 5 phosphate 4-kinase (PIP4K) is required to limit PIP levels during insulin receptor activation. Depletion of PIP4K increases the levels of PIP produced in response to insulin stimulation. We find that PIP4K function at the plasma membrane enhances class I phosphoinositide 3-kinase (PI3K) activity, although the catalytic ability of PIP4K to produce phosphatidylinositol 4,5-bisphosphate [PI(4,5)P] at the plasma membrane is dispensable for this regulation. Animals lacking PIP4K show enhanced insulin signaling-dependent phenotypes and are resistant to the metabolic consequences of a high-sugar diet, highlighting the importance of PIP4K in normal metabolism and development. Thus, PIP4Ks are key regulators of receptor tyrosine kinase signaling with implications for growth factor-dependent processes including tumor growth, T\ cell activation, and metabolism.

}, issn = {2211-1247}, doi = {10.1016/j.celrep.2019.04.084}, author = {Sharma, Sanjeev and Mathre, Swarna and Ramya, Visvanathan and Shinde, Dhananjay and Raghu, Padinjat} } @article {60328, title = {Regulation of Membrane Turnover by Phosphatidic Acid: Cellular Functions and Disease Implications.}, journal = {Front Cell Dev Biol}, volume = {7}, year = {2019}, month = {2019}, pages = {83}, abstract = {

Phosphatidic acid (PA) is a simple glycerophospholipid with a well-established role as an intermediate in phospholipid biosynthesis. In addition to its role in lipid biosynthesis, PA has been proposed to act as a signaling molecule that modulates several aspects of cell biology including membrane transport. PA can be generated in eukaryotic cells by several enzymes whose activity is regulated in the context of signal transduction and enzymes that can metabolize PA thus terminating its signaling activity have also been described. Further, several studies have identified PA binding proteins and changes in their activity are proposed to be mediators of the signaling activity of this lipid. Together these enzymes and proteins constitute a PA signaling toolkit that mediates the signaling functions of PA in cells. Recently, a number of novel genetic models for the analysis of PA function and analytical methods to quantify PA levels in cells have been developed and promise to enhance our understanding of PA functions. Studies of several elements of the PA signaling toolkit in a single cell type have been performed and are presented to provide a perspective on our understanding of the biochemical and functional organization of pools of PA in a eukaryotic cell. Finally, we also provide a perspective on the potential role of PA in human disease, synthesizing studies from model organisms, human disease genetics and analysis using recently developed PLD inhibitors.

}, issn = {2296-634X}, doi = {10.3389/fcell.2019.00083}, author = {Thakur, Rajan and Naik, Amruta and Panda, Aniruddha and Raghu, Padinjat} } @article {33717, title = {Discovery biology of neuropsychiatric syndromes (DBNS): a center for integrating clinical medicine and basic science.}, journal = {BMC Psychiatry}, volume = {18}, year = {2018}, month = {2018 Apr 18}, pages = {106}, abstract = {

BACKGROUND: There is emerging evidence that there are shared genetic, environmental and developmental risk factors in psychiatry, that cut across traditional diagnostic boundaries. With this background, the Discovery biology of neuropsychiatric syndromes (DBNS) proposes to recruit patients from five different syndromes (schizophrenia, bipolar disorder, obsessive compulsive disorder, Alzheimer{\textquoteright}s dementia and substance use disorders), identify those with multiple affected relatives, and invite these families to participate in this study. The families will be assessed: 1) To compare neuro-endophenotype measures between patients, first degree relatives (FDR) and healthy controls., 2) To identify cellular phenotypes which differentiate the groups., 3) To examine the longitudinal course of neuro-endophenotype measures., 4) To identify measures which correlate with outcome, and 5) To create a unified digital database and biorepository.

METHODS: The identification of the index participants will occur at well-established specialty clinics. The selected individuals will have a strong family history (with at least another affected FDR) of mental illness. We will also recruit healthy controls without family history of such illness. All recruited individuals (N = 4500) will undergo brief clinical assessments and a blood sample will be drawn for isolation of DNA and peripheral blood mononuclear cells (PBMCs). From among this set, a subset of 1500 individuals (300 families and 300 controls) will be assessed on several additional assessments [detailed clinical assessments, endophenotype measures (neuroimaging- structural and functional, neuropsychology, psychophysics-electroencephalography, functional near infrared spectroscopy, eye movement tracking)], with the intention of conducting repeated measurements every alternate year. PBMCs from this set will be used to generate lymphoblastoid cell lines, and a subset of these would be converted to induced pluripotent stem cell lines and also undergo whole exome sequencing.

DISCUSSION: We hope to identify unique and overlapping brain endophenotypes for major psychiatric syndromes. In a proportion of subjects, we expect these neuro-endophenotypes to progress over time and to predict treatment outcome. Similarly, cellular assays could differentiate cell lines derived from such groups. The repository of biomaterials as well as digital datasets of clinical parameters, will serve as a valuable resource for the broader scientific community who wish to address research questions in the area.

}, issn = {1471-244X}, doi = {10.1186/s12888-018-1674-2}, author = {Viswanath, Biju and Rao, Naren P and Narayanaswamy, Janardhanan C and Sivakumar, Palanimuthu T and Kandasamy, Arun and Kesavan, Muralidharan and Mehta, Urvakhsh Meherwan and Venkatasubramanian, Ganesan and John, John P and Mukherjee, Odity and Purushottam, Meera and Kannan, Ramakrishnan and Mehta, Bhupesh and Kandavel, Thennarasu and Binukumar, B and Saini, Jitender and Jayarajan, Deepak and Shyamsundar, A and Moirangthem, Sydney and Vijay Kumar, K G and Thirthalli, Jagadisha and Chandra, Prabha S and Gangadhar, Bangalore N and Murthy, Pratima and Panicker, Mitradas M and Bhalla, Upinder S and Chattarji, Sumantra and Benegal, Vivek and Varghese, Mathew and Reddy, Janardhan Y C and Raghu, Padinjat and Rao, Mahendra and Jain, Sanjeev} } @article {45418, title = {Functional analysis of mammalian phospholipase D enzymes.}, journal = {Biosci Rep}, year = {2018}, month = {2018 Oct 28}, abstract = {

Phosphatidylcholine-specific phospholipase D (PLD) hydrolyzes the phosphodiester bond of the phosphatidylcholine to generate phosphatidic acid (PA) and regulates several sub-cellular functions. Mammalian genomes contain two genes encoding distinct isoforms of PLD in contrast to invertebrate genomes that include a single PLD gene. However, the significance of two genes within a genome encoding the same biochemical activity remains unclear. Recently, loss of function in the only PLD gene in was reported to result in reduced PA levels and a PA dependent collapse of the photoreceptor plasma membrane due to defects in vesicular transport. Phylogenetic analysis reveals that human PLD1 (hPLD1)is evolutionarily closer to dPLD than humanPLD2 (hPLD2). In this study we expressed hPLD1 and hPLD2 in and found that while reconstitution of hPLD1 is able to completely rescue retinal degeneration in a loss of function dPLD mutant,hPLD2 was less effective in its ability to mediate a rescue. Using a newly developed analytical method, we determined the acyl chain composition of PA species produced by each enzyme. While dPLD was able to restore the levels of most PA species in cells, hPLD1 and hPLD2\  each were unable to restore the levels of a subset of unique species of PA. Finally, we found that in contrast to hPLD2, dPLD and hPLD1 are uniquely distributed to the sub-plasma membrane region in photoreceptors. In summary hPLD1 likely represents the ancestral PLD in mammalian genomes while hPLD2 represents neofunctionalization to generate PA at distinct sub-cellular membranes.

}, issn = {1573-4935}, doi = {10.1042/BSR20181690}, author = {Panda, Aniruddha and Thakur, Rajan and Krishnan, Harini and Naik, Amruta and Shinde, Dhananjay and Raghu, Padinjat} } @article {35863, title = {Phospholipid transport protein function at organelle contact sites.}, journal = {Curr Opin Cell Biol}, volume = {53}, year = {2018}, month = {2018 May 30}, pages = {52-60}, abstract = {

Phospholipids are synthesized at the endoplasmic reticulum (ER), the largest membrane bound organelle that forms membrane contact sites (MCS) with almost every other organelle. MCS are locations at which the membrane es of two organelles are closely positioned to provide a microenvironment where proteins in one membrane can interact with the opposite membrane. Thus, MCS provide an ideal location at which lipid transfer proteins (LTPs) can achieve the efficient transfer of individual classes of lipids from the ER to other organelles via non-vesicular transport. Here we provide an overview of emerging findings on the localization and biochemical activity of LTPs at MCS between the ER and other cellular membranes. The localization of LTPs at MCS offers an elegant cell biological solution to tune local lipid composition to ongoing cell physiology.

}, issn = {1879-0410}, doi = {10.1016/j.ceb.2018.04.011}, author = {Cockcroft, Shamshad and Raghu, Padinjat} } @article {29627, title = {Protein kinase D regulates metabolism and growth by controlling secretion of insulin like peptide.}, journal = {Dev Biol}, volume = {434}, year = {2018}, month = {2018 Feb 01}, pages = {175-185}, abstract = {

Mechanisms coupling growth and metabolism are conserved in Drosophila and mammals. In metazoans, such coupling is achieved across tissue scales through the regulated secretion of chemical messengers such as insulin that control the metabolism and growth of cells. Although the regulated secretion of Insulin like peptide (dILP) is key to normal growth and metabolism in Drosophila, the sub-cellular mechanisms that regulate dILP release remain poorly understood. We find that reduced function of the only protein kinase D in Drosophila (dPKDH) results in delayed larval growth and development associated with abnormal sugar and lipid metabolism, reduced insulin signalling and accumulation of dILP2 in the neurosecretory IPCs of the larval brain. These phenotypes are rescued by tissue-selective reconstitution of dPKD in the neurosecretory cells of dPKDH. Selective downregulation of dPKD activity in the neurosecretory IPCs phenocopies the growth defects, metabolic abnormalities and dILP2 accumulation seen in dPKDH. Thus, dPKD mediated secretion of dILP2 from neurosecretory cells during development is necessary for normal larval growth.

}, issn = {1095-564X}, doi = {10.1016/j.ydbio.2017.12.008}, author = {Ashe, Sudipta and Malhotra, Vivek and Raghu, Padinjat} } @article {28566, title = {RDGBα localization and function at membrane contact sites is regulated by FFAT-VAP interactions.}, journal = {J Cell Sci}, volume = {131}, year = {2018}, month = {2018 Jan 08}, abstract = {

Phosphatidylinositol transfer proteins (PITPs) are essential regulators of PLC signalling. The PI transfer domain (PITPd) of multi-domain PITPs is reported to be sufficient for in vivo function, questioning the relevance of other domains in the protein. In Drosophila photoreceptors, loss of RDGBα, a multi-domain PITP localized to membrane contact sites (MCSs), results in multiple defects during PLC signalling. Here, we report that the PITPd of RDGBα does not localize to MCSs and fails to support function during strong PLC stimulation. We show that the MCS localization of RDGBα depends on the interaction of its FFAT motif with dVAP-A. Disruption of the FFAT motif (RDGBFF/AA) or downregulation of dVAP-A, both result in mis-localization of RDGBα and are associated with loss of function. Importantly, the ability of the PITPd in full-length RDGBFF/AA to rescue mutant phenotypes was significantly worse than that of the PITPd alone, indicating that an intact FFAT motif is necessary for PITPd activity in vivo Thus, the interaction between the FFAT motif and dVAP-A confers not only localization but also intramolecular regulation on lipid transfer by the PITPd of RDGBα. This article has an associated First Person interview with the first author of the paper.

}, issn = {1477-9137}, doi = {10.1242/jcs.207985}, author = {Yadav, Shweta and Thakur, Rajan and Georgiev, Plamen and Deivasigamani, Senthilkumar and Krishnan, Harini and Ratnaparkhi, Girish and Raghu, Padinjat} } @article {38420, title = {Regulation of PI4P levels by PI4KIIIα during G-protein coupled PLC signaling in photoreceptors.}, journal = {J Cell Sci}, year = {2018}, month = {2018 Jul 06}, abstract = {

The activation of phospholipase C (PLC) is a conserved mechanism of receptor activated cell signaling at the plasma membrane. PLC hydrolyzes the minor membrane lipid phosphatidylinositol-4,5-bisphosphate [PI(4,5)P] and continued signaling requires the resynthesis and availability of PI(4,5)P at the plasma membrane. PI(4,5)P is synthesized by the phosphorylation of phosphatidylinositol-4-phosphate (PI4P). Thus, a continuous supply of PI4P is essential to support ongoing PLC signaling. While the enzyme PI4KA has been identified to perform this function in cultured mammalian cells, its function in the context of an physiological model has not been established. In this study, we show that in photoreceptors, PI4KIIIα activity is required to support signaling during G-protein coupled PLC activation. Depletion of PI4KIIIα results in impaired electrical responses to light and reduced plasma membrane levels of PI4P and PI(4,5)P Depletion of conserved proteins Efr3 and TTC7 that assemble PI4KIIIα at the plasma membrane also results in an impaired light response and reduced plasma membrane PI4P and PI(4,5)P levels. Thus PI4KIIIα activity at the plasma membrane generates PI4P and supports PI(4,5)P levels during receptor activated PLC signaling.

}, issn = {1477-9137}, doi = {10.1242/jcs.217257}, author = {Balakrishnan, Sruthi S and Basu, Urbashi and Shinde, Dhananjay and Thakur, Rajan and Jaiswal, Manish and Raghu, Padinjat} } @article {17518, title = {Phosphatidylinositol 5-phosphate 4-kinase regulates early endosomal dynamics during clathrin-mediated endocytosis.}, journal = {J Cell Sci}, year = {2017}, month = {2017 May 15}, abstract = {

Endocytic turnover is essential to regulate the protein composition and function of the plasma membrane thus regulating the plasma membrane levels of many receptors. In Drosophila photoreceptors, photon absorption by the GPCR rhodopsin 1 (Rh1) triggers its endocytosis by clathrin-mediated endocytosis (CME). We find that CME of Rh1 is regulated by phosphatidylinositol 5 phosphate 4-kinase (PIP4K). Flies lacking PIP4K show mislocalization of Rh1 on expanded endomembranes within the cell body. This mislocalization of Rh1 was dependent on the formation of an expanded Rab5 compartment. The Rh1 trafficking defect in dPIP4K-depleted cells could be suppressed by downregulating Rab5 function or by selectively reconstituting dPIP4K in the PI3P enriched early endosomal (EE) compartment of photoreceptors. We also found that loss of PIP4K was associated with increased CME and an enlarged Rab5 compartment in cultured Drosophila cells. Collectively our findings define PIP4K as a novel regulator of early endosomal homeostasis during CME.

}, issn = {1477-9137}, doi = {10.1242/jcs.202259}, author = {Kamalesh, Kumari and Trivedi, Deepti and Toscano, Sarah and Sharma, Sanjeev and Kolay, Sourav and Raghu, Padinjat} } @article {5300, title = {Control of diverse subcellular processes by a single multi-functional lipid phosphatidylinositol 4,5-bisphosphate [PI(4,5)P2].}, journal = {Biochem J}, volume = {473}, year = {2016}, month = {2016 Jun 15}, pages = {1681-92}, abstract = {

Phosphatidylinositol 4,5-bisphosphate [PI(4,5)P2] is a multi-functional lipid that regulates several essential subcellular processes in eukaryotic cells. In addition to its well-established function as a substrate for receptor-activated signalling at the plasma membrane (PM), it is now recognized that distinct PI(4,5)P2 pools are present at other organelle membranes. However, a long-standing question that remains unresolved is the mechanism by which a single lipid species, with an invariant functional head group, delivers numerous functions without loss of fidelity. In the present review, we summarize studies that have examined the molecular processes that shape the repertoire of PI(4,5)P2 pools in diverse eukaryotes. Collectively, these studies indicate a conserved role for lipid kinase isoforms in generating functionally distinct pools of PI(4,5)P2 in diverse metazoan species. The sophistication underlying the regulation of multiple functions by PI(4,5)P2 is also shaped by mechanisms that regulate its availability to enzymes involved in its metabolism as well as molecular processes that control its diffusion at nanoscales in the PM. Collectively, these mechanisms ensure the specificity of PI(4,5)P2 mediated signalling at eukaryotic membranes.

}, issn = {1470-8728}, doi = {10.1042/BCJ20160069}, author = {Kolay, Sourav and Basu, Urbashi and Raghu, Padinjat} } @article {3542, title = {The Drosophila photoreceptor as a model system for studying signalling at membrane contact sites.}, journal = {Biochem Soc Trans}, volume = {44}, year = {2016}, month = {2016 Apr 15}, pages = {447-51}, abstract = {

Several recent studies have demonstrated the existence of membrane contact sites (MCS) between intracellular organelles in eukaryotic cells. Recent exciting studies have also demonstrated the existence of biomolecular interactions at these contact sites in mediating changes in the membrane composition of the cellular compartments. However, the role of such contact sites in regulating organelle function and physiological processes remains less clear. In this review we discuss the existence of a contact site between the plasma membrane (PM) and the endoplasmic reticulum (ER) inDrosophilaphotoreceptors. Further, we discuss the role of specific proteins present at this location in regulating phospholipid turnover and its impact in regulating a physiological process, namely phototransduction.

}, issn = {1470-8752}, doi = {10.1042/BST20150256}, author = {Yadav, Shweta and Cockcroft, Shamshad and Raghu, Padinjat} } @article {11603, title = {Phospholipase D activity couples plasma membrane endocytosis with retromer dependent recycling.}, journal = {Elife}, volume = {5}, year = {2016}, month = {2016 Nov 16}, abstract = {

During illumination, the light-sensitive plasma membrane (rhabdomere) of Drosophila photoreceptors undergoes turnover with consequent changes in size and composition. However, the mechanism by which illumination is coupled to rhabdomere turnover remains unclear. We find that photoreceptors contain a light-dependent phospholipase D (PLD) activity. During illumination, loss of PLD resulted in an enhanced reduction in rhabdomere size, accumulation of Rab7 positive, rhodopsin1-containing vesicles (RLVs) in the cell body and reduced rhodopsin protein. These phenotypes were associated with reduced levels of phosphatidic acid, the product of PLD activity and were rescued by reconstitution with catalytically active PLD. In wild-type photoreceptors, during illumination, enhanced PLD activity was sufficient to clear RLVs from the cell body by a process dependent on Arf1-GTP levels and retromer complex function. Thus, during illumination, PLD activity couples endocytosis of RLVs with their recycling to the plasma membrane thus maintaining plasma membrane size and composition.

}, issn = {2050-084X}, doi = {10.7554/eLife.18515}, author = {Thakur, Rajan and Panda, Aniruddha and Coessens, Elise and Raj, Nikita and Yadav, Shweta and Balakrishnan, Sruthi and Zhang, Qifeng and Georgiev, Plamen and Basak, Bishal and Pasricha, Renu and Wakelam, Michael Jo and Ktistakis, Nicholas T and Raghu, Padinjat} } @article {11625, title = {sigma(2)-Adaptin Facilitates Basal Synaptic Transmission and Is Required for Regenerating Endo-Exo Cycling Pool Under High-Frequency Nerve Stimulation in Drosophila}, journal = {GENETICS}, volume = {1}, year = {2016}, month = {05/2016}, chapter = {369}, abstract = {

The functional requirement of adapter protein 2 (AP2) complex in synaptic membrane retrieval by clathrin-mediated endocytosis is not fully understood. Here we isolated and functionally characterized a mutation that dramatically altered synaptic development. Based on the aberrant neuromuscular junction (NMJ) synapse, we named this mutation angur (a Hindi word meaning "grapes"). Loss-of-function alleles of angur show more than twofold overgrowth in bouton numbers and a dramatic decrease in bouton size. We mapped the angur mutation to sigma(2)-adaptin, the smallest subunit of the AP2 complex. Reducing the neuronal level of any of the subunits of the AP2 complex or disrupting AP2 complex assembly in neurons phenocopied the sigma(2)-adaptin mutation. Genetic perturbation of sigma(2)-adaptin in neurons leads to a reversible temperature-sensitive paralysis at 38 degrees. Electrophysiological analysis of the mutants revealed reduced evoked junction potentials and quantal content. Interestingly, high-frequency nerve stimulation caused prolonged synaptic fatigue at the NMJs. The synaptic levels of subunits of the AP2 complex and clathrin, but not other endocytic proteins, were reduced in the mutants. Moreover, bone morphogenetic protein (BMP)/transforming growth factor beta (TGF beta) signaling was altered in these mutants and was restored by normalizing sigma(2)-adaptin in neurons. Thus, our data suggest that (1) while sigma(2)-adaptin facilitates synaptic vesicle (SV) recycling for basal synaptic transmission, its activity is also required for regenerating SVs during high-frequency nerve stimulation, and (2) sigma(2)-adaptin regulates NMJ morphology by attenuating TGF beta signaling.

}, doi = {10.1534/genetics.115.183863}, author = {Choudhury, Saumitra Dey and Mushtaq, Zeeshan and Reddy-Alla, Suneel and Balakrishnan, Sruthi S and Thakur, Rajan S and Krishnan, Kozhalmannom S. and Raghu, Padinjat and Ramaswami, Mani and Kumar, Vimlesh} } @article {11593, title = {Topological organisation of the phosphatidylinositol 4,5-bisphosphate-phospholipase C resynthesis cycle: PITPs bridge the ER-PM gap.}, journal = {Biochem J}, volume = {473}, year = {2016}, month = {2016 Dec 01}, pages = {4289-4310}, abstract = {

Phospholipase C (PLC) is a receptor-regulated enzyme that hydrolyses phosphatidylinositol 4,5-bisphosphate (PI(4,5)P2) at the plasma membrane (PM) triggering three biochemical consequences, the generation of soluble inositol 1,4,5-trisphosphate (IP3), membrane-associated diacylglycerol (DG) and the consumption of PM PI(4,5)P2 Each of these three signals triggers multiple molecular processes impacting key cellular properties. The activation of PLC also triggers a sequence of biochemical reactions, collectively referred to as the PI(4,5)P2 cycle that culminates in the resynthesis of this lipid. The biochemical intermediates of this cycle and the enzymes that mediate these reactions are topologically distributed across two membrane compartments, the PM and the endoplasmic reticulum (ER). At the PM, the DG formed during PLC activation is rapidly converted into phosphatidic acid (PA) that needs to be transported to the ER where the machinery for its conversion into PI is localised. Conversely, PI from the ER needs to be rapidly transferred to the PM where it can be phosphorylated by lipid kinases to regenerate PI(4,5)P2 Thus, two lipid transport steps between membrane compartments through the cytosol are required for the replenishment of PI(4,5)P2 at the PM. Here, we review the topological constraints in the PI(4,5)P2 cycle and current understanding how these constraints are overcome during PLC signalling. In particular, we discuss the role of lipid transfer proteins in this process. Recent findings on the biochemical properties of a membrane-associated lipid transfer protein of the PITP family, PITPNM proteins (alternative name RdgBα/Nir proteins) that localise to membrane contact sites are discussed. Studies in both Drosophila and mammalian cells converge to provide a resolution to the conundrum of reciprocal transfer of PA and PI during PLC signalling.

}, issn = {1470-8728}, doi = {10.1042/BCJ20160514C}, author = {Cockcroft, Shamshad and Raghu, Padinjat} } @article {5315, title = {σ2-Adaptin Facilitates Basal Synaptic Transmission and Is Required for Regenerating Endo-Exo Cycling Pool Under High-Frequency Nerve Stimulation in Drosophila.}, journal = {Genetics}, volume = {203}, year = {2016}, month = {2016 May}, pages = {369-85}, abstract = {

The functional requirement of adapter protein 2 (AP2) complex in synaptic membrane retrieval by clathrin-mediated endocytosis is not fully understood. Here we isolated and functionally characterized a mutation that dramatically altered synaptic development. Based on the aberrant neuromuscular junction (NMJ) synapse, we named this mutation angur (a Hindi word meaning "grapes"). Loss-of-function alleles of angur show more than twofold overgrowth in bouton numbers and a dramatic decrease in bouton size. We mapped the angur mutation to σ2-adaptin, the smallest subunit of the AP2 complex. Reducing the neuronal level of any of the subunits of the AP2 complex or disrupting AP2 complex assembly in neurons phenocopied the σ2-adaptin mutation. Genetic perturbation of σ2-adaptin in neurons leads to a reversible temperature-sensitive paralysis at 38{\textdegree}. Electrophysiological analysis of the mutants revealed reduced evoked junction potentials and quantal content. Interestingly, high-frequency nerve stimulation caused prolonged synaptic fatigue at the NMJs. The synaptic levels of subunits of the AP2 complex and clathrin, but not other endocytic proteins, were reduced in the mutants. Moreover, bone morphogenetic protein (BMP)/transforming growth factor β (TGFβ) signaling was altered in these mutants and was restored by normalizing σ2-adaptin in neurons. Thus, our data suggest that (1) while σ2-adaptin facilitates synaptic vesicle (SV) recycling for basal synaptic transmission, its activity is also required for regenerating SVs during high-frequency nerve stimulation, and (2) σ2-adaptin regulates NMJ morphology by attenuating TGFβ signaling.

}, issn = {1943-2631}, doi = {10.1534/genetics.115.183863}, author = {Choudhury, Saumitra Dey and Mushtaq, Zeeshan and Reddy-Alla, Suneel and Balakrishnan, Sruthi S and Thakur, Rajan S and Krishnan, Kozhalmannom S and Raghu, Padinjat and Ramaswami, Mani and Kumar, Vimlesh} } @article {877, title = {A dPIP5K dependent pool of phosphatidylinositol 4,5 bisphosphate (PIP2) is required for G-protein coupled signal transduction in Drosophila photoreceptors.}, journal = {PLoS Genet}, volume = {11}, year = {2015}, month = {2015 Jan}, pages = {e1004948}, abstract = {

Multiple PIP2 dependent molecular processes including receptor activated phospholipase C activity occur at the neuronal plasma membranes, yet levels of this lipid at the plasma membrane are remarkably stable. Although the existence of unique pools of PIP2 supporting these events has been proposed, the mechanism by which they are generated is unclear. In Drosophila photoreceptors, the hydrolysis of PIP2 by G-protein coupled phospholipase C activity is essential for sensory transduction of photons. We identify dPIP5K as an enzyme essential for PIP2 re-synthesis in photoreceptors. Loss of dPIP5K causes profound defects in the electrical response to light and light-induced PIP2 dynamics at the photoreceptor membrane. Overexpression of dPIP5K was able to accelerate the rate of PIP2 synthesis following light induced PIP2 depletion. Other PIP2 dependent processes such as endocytosis and cytoskeletal function were unaffected in photoreceptors lacking dPIP5K function. These results provide evidence for the existence of a unique dPIP5K dependent pool of PIP2 required for normal Drosophila phototransduction. Our results define the existence of multiple pools of PIP2 in photoreceptors generated by distinct lipid kinases and supporting specific molecular processes at neuronal membranes.

}, keywords = {Animals, Cell Membrane, Cytoskeleton, Drosophila, Drosophila melanogaster, Light Signal Transduction, Membrane Proteins, Ocular Physiological Phenomena, Phosphatidylinositol 4,5-Diphosphate, Phosphoinositide Phospholipase C, Phosphotransferases (Alcohol Group Acceptor), Photoreceptor Cells, Retina, Signal Transduction}, issn = {1553-7404}, doi = {10.1371/journal.pgen.1004948}, author = {Chakrabarti, Purbani and Kolay, Sourav and Yadav, Shweta and Kumari, Kamalesh and Nair, Amit and Trivedi, Deepti and Raghu, Padinjat} } @article {885, title = {Phosphoinositide signalling in Drosophila.}, journal = {Biochim Biophys Acta}, volume = {1851}, year = {2015}, month = {2015 Jun}, pages = {770-84}, abstract = {

Phosphoinositides (PtdInsPs) are lipids that mediate a range of conserved cellular processes in eukaryotes. These include the transduction of ligand binding to cell surface receptors, vesicular transport and cytoskeletal function. The nature and functions of PtdInsPs were initially elucidated through biochemical experiments in mammalian cells. However, over the years, genetic and cell biological analysis in a range of model organisms including S. cerevisiae, D. melanogaster and C. elegans have contributed to an understanding of the involvement of PtdInsPs in these cellular events. The fruit fly Drosophila is an excellent genetic model for the analysis of cell and developmental biology as well as physiological processes, particularly analysis of the complex relationship between the cell types of a metazoan in mediating animal physiology. PtdInsP signalling pathways are underpinned by enzymes that synthesise and degrade these molecules and also by proteins that bind to these lipids in cells. In this review we provide an overview of the current understanding of PtdInsP signalling in Drosophila. We provide a comparative genomic analysis of the PtdInsP signalling toolkit between Drosophila and mammalian systems. We also review some areas of cell and developmental biology where analysis in Drosophila might provide insights into the role of this lipid-signalling pathway in metazoan biology. This article is part of a Special Issue entitled Phosphoinositides.

}, keywords = {Animals, CDP-Diacylglycerol-Inositol 3-Phosphatidyltransferase, Drosophila melanogaster, Drosophila Proteins, Gene Expression Regulation, Isoenzymes, Phosphatidylinositol 4,5-Diphosphate, Phosphoinositide Phospholipase C, Phospholipid Transfer Proteins, Receptors, Cell Surface, Signal Transduction, Species Specificity, Substrate Specificity}, issn = {0006-3002}, doi = {10.1016/j.bbalip.2014.10.010}, author = {Balakrishnan, Sruthi S and Basu, Urbashi and Raghu, Padinjat} } @article {996, title = {RDGBα, a PtdIns-PtdOH transfer protein, regulates G-protein-coupled PtdIns(4,5)P2 signalling during Drosophila phototransduction.}, journal = {J Cell Sci}, volume = {128}, year = {2015}, month = {2015 Sep 1}, pages = {3330-44}, abstract = {

Many membrane receptors activate phospholipase C (PLC) during signalling, triggering changes in the levels of several plasma membrane lipids including phosphatidylinositol (PtdIns), phosphatidic acid (PtdOH) and phosphatidylinositol 4,5-bisphosphate [PtdIns(4,5)P2]. It is widely believed that exchange of lipids between the plasma membrane and endoplasmic reticulum (ER) is required to restore lipid homeostasis during PLC signalling, yet the mechanism remains unresolved. RDGBα (hereafter RDGB) is a multi-domain protein with a PtdIns transfer protein (PITP) domain (RDGB-PITPd). We find that, in vitro, the RDGB-PITPd binds and transfers both PtdOH and PtdIns. In Drosophila photoreceptors, which experience high rates of PLC activity, RDGB function is essential for phototransduction. We show that binding of PtdIns to RDGB-PITPd is essential for normal phototransduction; however, this property is insufficient to explain the in vivo function because another Drosophila PITP (encoded by vib) that also binds PtdIns cannot rescue the phenotypes of RDGB deletion. In RDGB mutants, PtdIns(4,5)P2 resynthesis at the plasma membrane following PLC activation is delayed and PtdOH levels elevate. Thus RDGB couples the turnover of both PtdIns and PtdOH, key lipid intermediates during G-protein-coupled PtdIns(4,5)P2 turnover.

}, issn = {1477-9137}, doi = {10.1242/jcs.173476}, author = {Yadav, Shweta and Garner, Kathryn and Georgiev, Plamen and Li, Michelle and Gomez-Espinosa, Evelyn and Panda, Aniruddha and Mathre, Swarna and Okkenhaug, Hanneke and Cockcroft, Shamshad and Raghu, Padinjat} }