Although the regulation of branching morphogenesis by spatially distributed cues is well established, the underlying intracellular signaling mechanisms are not well understood. The development of the lacrimal gland is driven by fibroblast growth factor (FGF) signaling, which activates phospholipase C gamma (PLCγ). Here, we showed that mutating the PLCγ1 binding site on Fgfr2 leads to ectopic branching and hyperplasia in the lacrimal gland, which was phenocopied by either deleting PLCγ1 or disabling any of its SH2 domains. PLCγ1 inactivation did not change the level of Fgfr2 or affect mitogen-activated protein kinase (MAPK) signaling but instead led to sustained AKT phosphorylation due to increased phosphatidylinositol 3,4,5-trisphosphate (PIP3) production. Consistent with this, the PLCγ1 mutant phenotype can be reproduced by the elevation of phosphatidylinositol 3-kinase (PI3K) signaling in Pten knockout and attenuated by blocking AKT signaling. Our findings demonstrate that FGF-activated PLCγ modulates PI3K signaling by shifting phosphoinositide metabolism, revealing the crucial role of PLCγ in branching morphogenesis and organ size control.

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 'messengers' (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.

Insulin and other growth factors are key regulators of liver gene expression, including in metabolic diseases. Most of the phosphoinositide 3-kinase (PI3K) activity induced by insulin is considered to be dependent on PI3Kα. We used mice lacking p110α, the catalytic subunit of PI3Kα, to investigate its role in the regulation of liver gene expression in health and in metabolic dysfunction-associated steatotic liver disease (MASLD). The absence of hepatocyte PI3Kα reduced maximal insulin-induced PI3K activity and signaling, promoted glucose intolerance in lean mice and significantly regulated liver gene expression, including insulin-sensitive genes, in ad libitum feeding. Some of the defective regulation of gene expression in response to hepatocyte-restricted insulin receptor deletion was related to PI3Kα signaling. In addition, though PI3Kα deletion in hepatocytes promoted insulin resistance, it was protective against steatotic liver disease in diet-induced obesity. In the absence of hepatocyte PI3Kα, the effect of diet-induced obesity on liver gene expression was significantly altered, with changes in rhythmic gene expression in liver. Altogether, this study highlights the specific role of p110α in the control of liver gene expression in physiology and in the metabolic rewiring that occurs during MASLD.

Metastatic uveal melanoma is an aggressive disease with limited effective therapeutic options. To comprehensively map monogenic and digenic dependencies, we performed CRISPR-Cas9 screening in ten extensively profiled human uveal melanoma cell line models. Analysis involved genome-wide single-gene and combinatorial paired-gene CRISPR libraries. Among our 76 uveal melanoma-specific essential genes and 105 synthetic lethal gene pairs, we identified and validated the CDP-diacylglycerol synthase 2 gene (CDS2) as a genetic dependency in the context of low CDP-diacylglycerol synthase 1 gene (CDS1) expression. We further demonstrate that CDS1/CDS2 forms a synthetic lethal interaction in vivo and reveal that CDS2 knockout results in the disruption of phosphoinositide synthesis and increased cellular apoptosis and that re-expression of CDS1 rescues this cell fitness defect. We extend our analysis using pan-cancer data, confirming increased CDS2 essentiality in diverse tumor types with low CDS1 expression. Thus, the CDS1/CDS2 axis is a therapeutic target across a range of cancers.

Receptor tyrosine kinases such as EGF receptor (EGFR) stimulate phosphoinositide 3 kinases to convert phosphatidylinositol-4,5-bisphosophate [PtdIns(4,5)P2] into phosphatidylinositol-3,4,5-trisphosphate [PtdIns(3,4,5)P3]. PtdIns(3,4,5)P3 then remodels actin and gene expression, and boosts cell survival and proliferation. PtdIns(3,4,5)P3 partly achieves these functions by triggering activation of the kinase Akt, which phosphorylates targets like Tsc2 and GSK3β. Consequently, unchecked upregulation of PtdIns(3,4,5)P3-Akt signaling promotes tumor progression. Interestingly, 50–70% of PtdIns and PtdInsPs have stearate and arachidonate at sn-1 and sn-2 positions of glycerol, respectively, forming a species known as 38:4-PtdIns/PtdInsPs. LCLAT1 and MBOAT7 acyltransferases partly enrich PtdIns in this acyl format. We previously showed that disruption of LCLAT1 lowered PtdIns(4,5)P2 levels and perturbed endocytosis and endocytic trafficking. However, the role of LCLAT1 in receptor tyrosine kinase and PtdIns(3,4,5)P3 signaling was not explored. Here, we show that LCLAT1 silencing in MDA-MB-231 and ARPE-19 cells abated the levels of PtdIns(3,4,5)P3 in response to EGF signaling. Importantly, LCLAT1-silenced cells were also impaired for EGF-driven and insulin-driven Akt activation and downstream signaling. Thus, our work provides first evidence that the LCLAT1 acyltransferase is required for receptor tyrosine kinase signaling.

Although the regulation of branching morphogenesis by spatially distributed cues is well established, the role of intracellular signaling in determining the branching pattern remains poorly understood. In this study, we investigated the regulation and function of phospholipase C gamma (PLCγ) in Fibroblast Growth Factor (FGF) signaling in lacrimal gland development. We showed that deletion of PLCγ1 in the lacrimal gland epithelium leads to ectopic branching and acinar hyperplasia, which was phenocopied by either mutating the PLCγ1 binding site on Fgfr2 or disabling any of its SH2 domains. PLCγ1 inactivation did not change the level of Fgfr2 or affect MAPK signaling, but instead led to sustained AKT phosphorylation due to increased PIP3 production. Consistent with this, PLCγ1 mutant phenotype can be reproduced by elevation of PI3K signaling in Pten knockout and attenuated by blocking AKT signaling. This study demonstrated that PLCγ modulates PI3K signaling by shifting phosphoinositide metabolism, revealing an important role of signaling dynamics in conjunction with spatial cues in shaping branching morphogenesis.

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 ‘PI cycle’, 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.

Insulin and other growth factors are key regulators of liver gene expression, including in metabolic diseases. Most of the phosphoinositide 3-kinase (PI3K) activity induced by insulin is dependent on PI3Kα. We used mice lacking p110α, the catalytic subunit of PI3Kα, to investigate its role in the regulation of liver gene expression. The absence of hepatocyte PI3Kα signaling promoted glucose intolerance in lean mice and significantly regulated liver gene expression, including insulin-sensitive genes, in ad libitum feeding. Some of the defective regulation of gene expression in response to hepatocyte-restricted insulin receptor deletion was related to PI3Kα signaling. In addition, though PI3Kα deletion in hepatocytes promoted insulin resistance, it was protective against steatotic liver disease in diet-induced obesity. In the absence of hepatocyte PI3Kα, the effect of diet-induced obesity on liver gene expression was significantly altered, with changes in rhythmic gene expression in liver. Therefore, this study highlights the specific role of membrane signaling dependent on hepatocyte PI3Kα in the control of liver gene expression in physiology and in Metabolic dysfunction-Associated Steatotic Liver Disease (MASLD).Highlights Hepatocyte p110α is required for liver growth, glucose homeostasis, and regulation of liver gene expression.Hepatocyte p110α is dispensable for the regulation of carbohydrate sensing by ChREBP and fatty acid sensing by PPARα.Hepatocyte p110α-mediated regulation of gene expression is related to both insulin receptor-dependent and -independent pathways.Hepatocyte p110α is required for lipid homeostasis and the rewiring of gene expression that occurs during diet-induced obesity.### Competing Interest StatementThe authors have declared no competing interest.

The PIP3/PI3K network is a central regulator of metabolism and is frequently activated in cancer, commonly by loss of the PIP3/PI(3,4)P2 phosphatase, PTEN. Despite huge research investment, the drivers of the PI3K network in normal tissues and how they adapt to overactivation are unclear. We find that in healthy mouse prostate PI3K activity is driven by RTK/IRS signaling and constrained by pathway feedback. In the absence of PTEN, the network is dramatically remodeled. A poorly understood YXXM- and PIP3/PI(3,4)P2-binding PH domain-containing adaptor, PLEKHS1, became the dominant activator and was required to sustain PIP3, AKT phosphorylation, and growth in PTEN-null prostate. This was because PLEKHS1 evaded pathway-feedback and experienced enhanced PI3K- and Src-family kinase-dependent phosphorylation of Y258XXM, eliciting PI3K activation. hPLEKHS1 mRNA and activating Y419 phosphorylation of hSrc correlated with PI3K pathway activity in human prostate cancers. We propose that in PTEN-null cells receptor-independent, Src-dependent tyrosine phosphorylation of PLEKHS1 creates positive feedback that escapes homeostasis, drives PIP3 signaling, and supports tumor progression.

Myeloproliferative neoplasms (MPNs) are characterized by the activated JAK2/STAT pathway. Pleckstrin-2 (Plek2) is a downstream target of the JAK2/STAT5 pathway and is overexpressed in patients with MPNs. We previously revealed that Plek2 plays critical roles in the pathogenesis of JAK2-mutated MPNs. The nonessential roles of Plek2 under physiologic conditions make it an ideal target for MPN therapy. Here, we identified first-in-class Plek2 inhibitors through an in silico high-throughput screening approach and cell-based assays, followed by the synthesis of analogs. Plek2-specific small-molecule inhibitors showed potent inhibitory effects on cell proliferation. Mechanistically, Plek2 interacts with and enhances the activity of Akt through the recruitment of downstream effector proteins. The Plek2-signaling complex also includes Hsp72, which protects Akt from degradation. These functions were blocked by Plek2 inhibitors via their direct binding to the Plek2 dishevelled, Egl-10 and pleckstrin (DEP) domain. The role of Plek2 in activating Akt signaling was further confirmed in vivo using a hematopoietic-specific Pten-knockout mouse model. We next tested Plek2 inhibitors alone or in combination with an Akt inhibitor in various MPN mouse models, which showed significant therapeutic efficacies similar to that seen with the genetic depletion of Plek2. The Plek2 inhibitor was also effective in reducing proliferation of CD34-positive cells from MPN patients. Our studies reveal a Plek2/Akt complex that drives cell proliferation and can be targeted by a class of antiproliferative compounds for MPN therapy.

e21585 Background: Despite low rates of local recurrence in primary uveal melanomas following radical treatment, approximately half of all patients will develop metastatic disease. There are limited effective systemic therapeutic options and survival outcomes remain poor. Genome-wide CRISPR-Cas9 knockout screening enables appropriate molecular targets and pathways that underlie cancer development to be identified. Methods: Genome-wide single gRNA CRISPR knockout screens were performed using Human Improved Genome-wide Knockout CRISPR Library v1.1. Eight human uveal melanoma cell lines were screened for 14 days to identify gene dependencies. Prior to screening, all cell lines underwent RNA-Seq to quantify gene expression. CRISPR screen hits were validated using single gRNA competitive growth assays. In vitro functional assessment of CDS2 knockout was performed using inducible CDS2 knockout isogenic cell lines in colony forming assays and Annexin V apoptosis assays. To determine the effects of CDS2 loss on phosphoinositide synthesis, quantification of phosphatidic acid and phosphoinositides was performed using HPLC-MS. To test the association between CDS2 essentiality and CDS1 expression across all cancer types, we analysed gene essentiality data from Project Score and Project Achilles with gene expression data obtained from Cell Model Passports and the DepMap download portal (22Q4). Cell lines with CDS1 expression log2(TPM+1) < 1 were annotated as low-expressors, and the rest were annotated as high-expressors. Single group data were assessed using Student's t test. Results: We identified CDS2, which encodes enzymes that maintain phosphoinositide levels during PLC signalling, to be an essential gene in 7 out of 8 screened cell lines. All cell lines were found to have low expression of its paralogous gene, CDS1. Doxycycline-inducible CDS2 knockout clones were generated from cell lines MP41 and OMM2.5. Reduced colony formation, with a significant decrease in surviving fraction was observed after doxycycline compared with treatment with a DMSO control in both cell lines (P < 0.0001). Apoptotic and dead cells increased significantly when doxycycline was added in MP41(13.6 ± 0.9% to 47.4 ± 3.3%; P= 0.0006) and OMM2.5 (3.7 ± 0.4% to 60.4± 0.7%, P< 0.0001). CDS2 knockout resulted in an increase in the precursor phosphatidic acid, and a significant reduction of the phosphoinositides PI (MP41, P= 0.018; OMM2.5, P= 0.0005) and PIP (MP41, P= 0.033; OMM2.5, P= 0.006). CDS2 was more essential in CDS1 low-expressors in both Project Score ( P< 0.0001) and Project Achilles ( P< 0.0001) datasets, compared with CDS1 high-expressors. Conclusions: Disruption of CDS2 results in reduced cellular fitness in uveal melanoma. Given the synthetic lethal relationship between CDS1 and CDS2, in the context of low CDS1 gene expression as observed in uveal melanomas and in other tumour types, CDS2 is a potential novel therapeutic target.

Harnessing the potential beneficial effects of kinase signalling through the generation of direct kinase activators remains an underexplored area of drug development1–5. This also applies to the PI3K signalling pathway, which has been extensively targeted by inhibitors for conditions with PI3K overactivation, such as cancer and immune dysregulation. Here we report the discovery of UCL-TRO-1938 (referred to as 1938 hereon), a small-molecule activator of the PI3Kα isoform, a crucial effector of growth factor signalling. 1938 allosterically activates PI3Kα through a distinct mechanism by enhancing multiple steps of the PI3Kα catalytic cycle and causes both local and global conformational changes in the PI3Kα structure. This compound is selective for PI3Kα over other PI3K isoforms and multiple protein and lipid kinases. It transiently activates PI3K signalling in all rodent and human cells tested, resulting in cellular responses such as proliferation and neurite outgrowth. In rodent models, acute treatment with 1938 provides cardioprotection from ischaemia–reperfusion injury and, after local administration, enhances nerve regeneration following nerve crush. This study identifies a chemical tool to directly probe the PI3Kα signalling pathway and a new approach to modulate PI3K activity, widening the therapeutic potential of targeting these enzymes through short-term activation for tissue protection and regeneration. Our findings illustrate the potential of activating kinases for therapeutic benefit, a currently largely untapped area of drug development. A new specific, small-molecule activator of the PI3Kα isoform (UCL-TRO-1938) identified through high-throughput screening can transiently activate PI3K signalling and biological responses in cells and tissues, with potential therapeutic applications in tissue protection and regeneration.

Despite their low abundance, phosphoinositides play a central role in membrane traffic and signalling. PtdIns(3,4,5)P-3 and PtdIns(3,4)P-2 are uniquely important, as they promote cell growth, survival and migration. Pathogenic organisms have developed means to subvert phosphoinositide metabolism to promote successful infection and their survival in host organisms. We demonstrate that PtdIns(3,4)P-2 is a major product generated in host cells by the effectors of the enteropathogenic bacteria Salmonella and Shigella. Pharmacological, gene silencing and heterologous expression experiments revealed that, remarkably, the biosynthesis of PtdIns(3,4)P-2 occurs independently of phosphoinositide 3-kinases. Instead, we found that the Salmonella effector SopB, heretofore believed to be a phosphatase, generates PtdIns(3,4)P-2 de novo via a phosphotransferase/phosphoisomerase mechanism. Recombinant SopB is capable of generating PtdIns(3,4,5)P-3 and PtdIns(3,4)P-2 from PtdIns(4,5)P-2 in a cell-free system. Through a remarkable instance of convergent evolution, bacterial effectors acquired the ability to synthesize 3-phosphorylated phosphoinositides by an ATP- and kinase-independent mechanism, thereby subverting host signalling to gain entry and even provoke oncogenic transformation. Walpole et al. show that the Salmonella effector SopB generates phosphatidylinositol 3,4-bisphosphate de novo via a phosphotransferase mechanism, independently of phosphoinositide 3-kinases and ATP.

Li et al present the results of a proximity‐interaction screen in mammalian cells for the effector proteins of 25 members of the Arf family of small GTPases. This study has generated an important resource for those working in several areas of cell biology and provided an initial characterisation of two new cellular roles for some of the least well studied members of this family, the regulation of PLD1 by ARL11/14 in phagocytosis, and the regulation of PI4KB by ARL5A/5B in the Golgi.

Upon antigen binding, the B cell receptor (BCR) undergoes clustering to form a signalosome that propagates downstream signaling required for normal B cell development and physiology. BCR clustering is dependent on remodeling of the cortical actin network, but the mechanisms that regulate actin remodeling in this context remain poorly defined. In this study, we identify the inositol 5-phosphatase INPP5B as a key regulator of actin remodeling, BCR clustering, and downstream signaling in antigen-stimulated B cells. INPP5B acts via dephosphorylation of the inositol lipid PI(4,5)P-2 that in turn is necessary for actin disassembly, BCR mobilization, and cell spreading on immobilized surface antigen. These effects can be explained by increased actin severing by cofilin and loss of actin linking to the plasma membrane by ezrin, both of which are sensitive to INPP5B-dependent PI(4,5)P-2 hydrolysis. INPP5B is therefore a new player in BCR signaling and may represent an attractive target for treatment of B cell malignancies caused by aberrant BCR signaling.

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 ‘re‐cycling pathway’, 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 ‘metabolic channelling’ from PIP2 to PI via pools of intermediates (DG, PA and CDP‐DG) common to other lipid metabolic pathways.

Antibody-mediated cross-presentation is known to elicit strong CD8+ T cell responses, particularly relevant in cancer therapy. Anti-tumor antibodies are known to rapidly kill cancer cells by antibody-dependent cellular cytotoxicity (ADCC), but the long-term immune control of tumors is based on induction of CD8+ T cell response, which relays on anti-tumor antibody-mediated cross-presentation. The molecular mechanisms by which this pathway of cross-presentation allows to poor cross-presenting cells, such as type 2 dendritic cells (DCs) and monocyte-derived DCs (moDCs), to efficiently cross present are not well understood. We demonstrated that enzymatic activity of p84/p110g complex of PI3Kg regulates the assembly of NADPH oxidase NOX2 and ROS production in type 2 DCs from mouse spleen and murine bone marrow derived DCs, enhancing thus the cross-presentation of immune complexes by these cells. Our results suggest that the association between anti-tumor monoclonal antibodies and PI3Kg inhibitors might block the antibody-mediated cross-presentation of tumor antigens and should be avoided in clinical therapy of cancers.

Phosphoinositide 3-kinases (PI3Ks) play a central role in adaptive immunity by transducing signals from the T cell antigen receptor (TCR) via production of PIP3. PI3Kδ is a heterodimer composed of a p110δ catalytic subunit associated with a p85α or p85β regulatory subunit and is preferentially engaged by the TCR upon T cell activation. The molecular mechanisms leading to PI3Kδ recruitment and activation at the TCR signalosome remain unclear. In this study, we have used quantitative mass spectrometry, biochemical approaches and CRISPR-Cas9 gene editing to uncover the p110δ interactome in primary CD4+ T cells. Moreover, we have determined how the PI3Kδ interactome changes upon the differentiation of small naïve T cells into T cell blasts expanded in the presence of IL-2. Our interactomic analyses identified multiple constitutive and inducible PI3Kδ-interacting proteins, some of which were common to naïve and previously-activated T cells. Our data reveals that PI3Kδ rapidly interacts with as many as seven adaptor proteins upon TCR engagement, including the Gab-family proteins, GAB2 and GAB3, a CD5-CBL signalosome and the transmembrane proteins ICOS and TRIM. Our results also suggest that PI3Kδ pre-forms complexes with the adaptors SH3KBP1 and CRKL in resting cells that could facilitate the localization and activation of p110δ at the plasma membrane by forming ternary complexes during early TCR signalling. Furthermore, we identify interactions that were not previously known to occur in CD4+ T cells, involving BCAP, GAB3, IQGAP3 and JAML. We used CRISPR-Cas9-mediated gene knockout in primary T cells to confirm that BCAP is a positive regulator of PI3K-AKT signalling in CD4+ T cell blasts. Overall, our results provide evidence for a large protein network that regulates the recruitment and activation of PI3Kδ in T cells. Finally, this work shows how the PI3Kδ interactome is remodeled as CD4+ T cells differentiate from naïve T cells to activated T cell blasts. These activated T cells upregulate additional PI3Kδ adaptor proteins, including BCAP, GAB2, IQGAP3 and ICOS. This rewiring of TCR-PI3K signalling that occurs upon T cell differentiation may serve to reduce the threshold of activation and diversify the inputs for the PI3K pathway in effector T cells.

Our friend and colleague, Michael Wakelam, the Director of the Babraham Institute, passed away from respiratory complications arising from suspected Covid-19 infection on 31 March 2020. Michael’s career spanned more than 40 years and 200 papers, during which he made many friends and was heavily involved in scientific leadership. He was also a great supporter of the Biochemical Society and its journals.Michael obtained his BSc in Medical Biochemistry (1977) and PhD in Biochemistry (1980) from the University of Birmingham, having worked on the expression of hepatic glucokinase. At Birmingham Michael also met his life-long partner, Jane Fensome, who later became his wife. Together they moved to the University of Konstanz where Michael undertook a post-doc with Dirk Pette, studying inositol lipid turnover during myoblast fusion; this shift towards signal transduction was prompted by Bob Michell’s hypotheses around inositol lipid turnover, and Bob and Michael remained firm friends. In 1983, Michael moved to Imperial College London, as a Beit Memorial Fellow, studying the RAS oncoproteins with the late Chris Marshall and Allan Hall at the Institute of Cancer Research.In 1985, Michael became a lecturer in biochemistry at the University of Glasgow. Here, Michael’s lab discovered that RAS could regulate a phospholipase C, though the full significance of this was not recognized until 15 years later, when PLC-ε was identified as a RAS effector. Michael’s lab, together with those of Miles Houslay and Graeme Milligan, was vibrant and exciting, with a strong work ethic; experiments ran 7 days a week and into the evening. Results were discussed as they came off the scintillation counter, and digested over beers on Byres Road. Michael’s excitement for science was infectious and several significant scientific careers were forged, including Shireen Davies, Susan Pyne and Robin Plevin. Michael also took his first steps in lipid analysis by mass spectrometry (MS) demonstrating that PLD and PLC generate DAG species with distinct fatty acyl chains.Michael’s interest in lipid-MS really took off in 1993, when he returned to Birmingham as Professor of Molecular Pharmacology in the Institute for Cancer Studies. With Trevor Pettitt, Michael developed the methods that made lipid-MS routine, providing unparalleled new insights into lipid signalling; the days of scraping radiolabelled lipids off silica thin layer chromatography (TLC) plates were finally gone! With Matt Hodgkin, Dale Powner, Khalid Saqib, Mark McDermott and Neil Shimwell, Michael also discovered key elements in the regulation and functions of PLD1 and PLD2. Michael became increasingly involved in science leadership, sitting on the MRC Council as Chair of the Molecular and Cellular Medicine Board and serving on Site Visit Assessment panels in the UK and abroad. It was also during this time that Jane and Michael welcomed their sons Alex and Patrick to their world.Michael joined the Babraham Institute as Director in 2007, attracted by the opportunity to lead a world-class research institute that pioneered work on lipid signalling. Michael’s lab continued to work on PLD, autotaxin/LPA and lipid metabolism. Michael also provided the vision and drive to create a fantastic lipid-MS facility, run by Qifeng Zhang and, latterly, by Andrea Lopez. This increased capability and Michael’s encyclopaedic knowledge supported many significant collaborations, including: Eyal Gottlieb and Almut Schulze (cancer), Linda Partridge and David Gems (ageing) and Rob Semple (overgrowth syndromes). His pre-eminence in lipid signalling and lipidomics was recognized by the award of the Biochemical Society’s Morton Lecture in 2018.The further development of the Babraham Research Campus is the lasting legacy of Michael’s leadership at Brabaham. Michael worked with Derek Jones and the Biotechnology and Biological Sciences Research Council (BBSRC) to bend the ear of successive science ministers, including David Willetts, securing significant public investment so that Babraham is now home to both a world-renowned institute and 60 biotech/drug discovery companies, and is the model for all BBSRC UK Research and Innovation Campuses.Michael was a tireless supporter of Brabaham science, ever present at seminars and student posters, always asking questions and always approachable. He is fondly remembered for turning up slightly late, often out of breath, but immediately ‘hooking in’ to the scientific discussion. Although the Director, he was still Michael, talking science over a pint at the Social Club or running on the treadmill in the gym. His humanity always shone through, supporting students, post-docs, staff and collaborators through difficult scientific or personal situations.For all his achievements, Michael’s greatest joy and comfort was as a husband and father. He regaled us with his sons’ latest exploits, and we all sensed his growing pride as they progressed to university and chose their own paths in life. Their first child, Ellen, tragically died at birth and was always remembered as a member of the family. There was a very strong bond in the Wakelam family and we, his colleagues, offer our deepest condolences and best wishes to Jane, Patrick and Alex.