@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 {113527, title = {Acyl chain selection couples the consumption and synthesis of phosphoinositides.}, journal = {EMBO J}, year = {2022}, month = {2022 Jun 30}, pages = {e110038}, abstract = {

Phosphoinositides (PIPn) in mammalian tissues are enriched in the stearoyl/arachidonoyl acyl chain species ("C38:4"), but its functional significance is unclear. We have used metabolic tracers (isotopologues of inositol, glucose and water) to study PIPn synthesis in cell lines in which this enrichment is preserved to differing relative extents. We show that PIs synthesised from glucose are initially enriched in shorter/more saturated acyl chains, but then rapidly remodelled towards the C38:4 species. PIs are also synthesised by a distinct {\textquoteright}re-cycling pathway{\textquoteright}, which utilises existing precursors and exhibits substantial selectivity for the synthesis of C38:4-PA and -PI. This re-cycling pathway is rapidly stimulated during receptor activation of phospholipase-C, both allowing the retention of the C38:4 backbone and the close coupling of PIPn consumption to its resynthesis, thus maintaining pool sizes. These results suggest that one property of the specific acyl chain composition of PIPn is that of a molecular code, to facilitate {\textquoteright}metabolic channelling{\textquoteright} from PIP2 to PI via pools of intermediates (DG, PA and CDP-DG) common to other lipid metabolic pathways.

}, issn = {1460-2075}, doi = {10.15252/embj.2021110038}, author = {Barneda, David and Janardan, Vishnu and Niewczas, Izabella and Collins, Daniel M and Cosulich, Sabina and Clark, Jonathan and Stephens, Len R and Hawkins, Phillip T} } @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 {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} }