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Endocytosis is required for internalization of micronutrients and turnover of membrane components. Endophilin has been assigned as a component of clathrin-mediated endocytosis. Here we show in mammalian cells that endophilin marks and controls a fast-acting tubulovesicular endocytic pathway that is independent of AP2 and clathrin, activated upon ligand binding to cargo receptors, inhibited by inhibitors of dynamin, Rac, phosphatidylinositol-3-OH kinase, PAK1 and actin polymerization, and activated upon Cdc42 inhibition. This pathway is prominent at the leading edges of cells where phosphatidylinositol-3,4-bisphosphate—produced by the dephosphorylation of phosphatidylinositol-3,4,5-triphosphate by SHIP1 and SHIP2—recruits lamellipodin, which in turn engages endophilin. This pathway mediates the ligand-triggered uptake of several G-protein-coupled receptors such as α 2a - and β 1 -adrenergic, dopaminergic D3 and D4 receptors and muscarinic acetylcholine receptor 4, the receptor tyrosine kinases EGFR, HGFR, VEGFR, PDGFR, NGFR and IGF1R, as well as interleukin-2 receptor. We call this new endocytic route fast endophilin-mediated endocytosis (FEME). This study describes a fast, clathrin-independent endocytic pathway mediated by endophilin, dynamin and actin; the pathway is activated by ligand binding to a variety of cargo receptors, and endophilin-mediated endocytosis occurs primarily at the leading edges of cells where lamellipodin and the lipid PtdIns(3,4)P 2 ensure endophilin targeting. Endocytosis and cell signalling Cells internalize nutrients and turnover membrane components through the process of endocytosis, which in most cases involves the protein clathrin. Endophilin has been thought to be a component of clathrin-mediated endocytosis, but two studies published in this issue of Nature show that this protein mediates a fast-acting, clathrin-independent form of endocytosis which involves formation of tubular vesicles. Emmanuel Boucrot et al . report that this pathway is triggered by binding of ligands to cargo receptors, and requires the proteins dynamin and actin. Endophilin-mediated endocytosis also seems to have distinct cellular homes, occurring at the leading edges of cells where the lipid PtdIns(3,4)P 2 ensures endophilin engagement. This form of endocytosis is shown to mediate the uptake of several physiological and disease-relevant receptors including G-protein-coupled receptors and receptor tyrosine kinases. In the second paper, Henri-François Renard et al . provide evidence that bacterial toxins take advantage of the same pathway to enter cells, and also find that endophilin-A2 acts together with dynamin and actin.

The phosphoinositide 3-kinase (PI3K)/AKT signalling pathway is hyperactivated in ~70% of breast cancers. Class I PI3K generates PtdIns(3,4,5)P3 at the plasma membrane in response to growth factor stimulation, leading to AKT activation to drive cell proliferation, survival and migration. PTEN negatively regulates PI3K/AKT signalling by dephosphorylating PtdIns(3,4,5)P3 to form PtdIns(4,5)P2. PtdIns(3,4,5)P3 can also be hydrolysed by the inositol polyphosphate 5-phosphatases (5-phosphatases) to produce PtdIns(3,4)P2. Interestingly, while PTEN is a bona fide tumour suppressor and is frequently mutated/lost in breast cancer, 5-phosphatases such as PIPP, SHIP2 and SYNJ2, have demonstrated more diverse roles in regulating mammary tumourigenesis. Reduced PIPP expression is associated with triple negative breast cancers and reduced relapse-free and overall survival. Although PIPP depletion enhances AKT phosphorylation and supports tumour growth, this also inhibits cell migration and metastasis in vivo, in a breast cancer oncogene-driven murine model. Paradoxically, SHIP2 and SYNJ2 are increased in primary breast tumours, which correlates with invasive disease and reduced survival. SHIP2 or SYNJ2 overexpression promotes breast tumourigenesis via AKT-dependent and independent mechanisms. This review will discuss how PTEN, PIPP, SHIP2 and SYNJ2 distinctly regulate multiple functional targets, and the mechanisms by which dysregulation of these distinct phosphoinositide phosphatases differentially affect breast cancer progression.

Inositol polyphosphate 5-phosphatase K (INPP5K) is a phosphatidylinositol (3,4,5)-trisphosphate phosphatase that increases glucose uptake and regulates myogenesis in the skeletal muscle. To understand the mechanism of its species-specific inhibition, we determined the 1.9-Å resolution crystal structure of human INPP5K in complex with a selective inhibitor, CPD-1 (IC 50  = 2.9 µM). The structure reveals that CPD-1 binds to a novel allosteric pocket, inducing a large conformational change in α-helix 3 that alters the active site and prevents substrate binding. This finding explains its unique, noncompetitive inhibitory mechanism. Crucially, while the inhibitor-binding residues are conserved, the key residue governing the allosteric transition is not conserved in mouse and rat INPP5K, which correlates with their insensitivity to CPD-1 (IC 50  > 100 µM). Based on these structural insights, we identified the hamster as a pharmacologically relevant preclinical model (IC 50  = 8.2 µM). These findings provide a structural basis for the rational design of next-generation INPP5K inhibitors and establish a suitable animal model for their evaluation.

Summary Kernel row number (KRN) is a major yield related trait for maize (Zea mays L.) and is also a major goal of breeders, as it can increase the number of kernels per plant. Thus, identifying new genetic factors involving in KRN formation may accelerate improving yield‐related traits genetically. We herein describe a new kernel number‐related gene (KRN5b) identified from KRN QTL qKRN5b and encoding an inositol polyphosphate 5‐phosphatase (5PTase). KRN5b has phosphatase activity towards PI(4,5)P2, PI(3,4,5)P3, and Ins(1,4,5)P3 in vitro. Knocking out KRN5b caused accumulation of PI(4,5)P2 and Ins(1,4,5)P3, resulting in disordered kernel rows and a decrease in the number of kernels and tassel branches. The introgression of the allele with higher expression abundance into different inbred lines could increase the ear weight of the inbred lines and the corresponding hybrids by 10.1%–12.2% via increasing KRN, with no adverse effects on other agronomic traits. Further analyses showed that KRN5b regulates inflorescence development through affecting the synthesis and distribution of hormones. Together, KRN5b contributes to spikelet pair meristem development through inositol phosphate and phosphatidylinositols, making it a selecting target for yield improvement.

Invadopodia are integrin-mediated adhesions with abundant PI(3,4) P₂. However, the functional role of PI(3,4)P₂ in adhesion signaling remains unclear. Here, we find that the PI(3,4)P₂ biogenesis regulates the integrin endocytosis at invadopodia. PI(3,4)P₂ is locally produced by PIK3CA and SHIP₂ and is concentrated at the trailing edge of the invadopodium arc. The PI(3,4)P₂-rich compartment locally forms small puncta (membrane buds) in a SNX9-dependent manner, recruits dynein activator Hook1 through AKTIP, and rear-ranges into micrometer-long tubular invaginations (membrane tubes). The uncurving membrane tube extends rapidly, follows the retrograde movement of dynein along microtubule tracks, and disconnects from the plasma membrane. Activated integrin-beta3 is locally internalized through the pathway of PI(3,4)P₂-mediated membrane invagination and is then actively recycled. Blockages of PI3K, SHIP₂, and SNX9 suppress integrin-beta3 endocytosis, delay adhesion turnover, and impede transwell invasion of MEF-Src and MDA-MB-231 cells. Thus, the production of PI(3,4)P₂ promotes invasive cell migration by stimulating the trafficking of integrin receptor at the invadopodium.

Endocytosis mediates the cellular uptake of micronutrients and the turnover of plasma membrane proteins. Clathrin-mediated endocytosis is the major uptake pathway in resting cells 1 , but several clathrin-independent endocytic routes exist in parallel 2 , 3 . One such pathway, fast endophilin-mediated endocytosis (FEME), is not constitutive but triggered upon activation of certain receptors, including the β 1 adrenergic receptor 4 . FEME activates promptly following stimulation as endophilin is pre-enriched by the phosphatidylinositol-3,4-bisphosphate-binding protein lamellipodin 4 , 5 . However, in the absence of stimulation, endophilin foci abort and disassemble after a few seconds. Looking for additional proteins involved in FEME, we found that 20 out of 65 BAR domain-containing proteins tested colocalized with endophilin spots. Among them, FBP17 and CIP4 prime the membrane of resting cells for FEME by recruiting the 5′-lipid phosphatase SHIP2 and lamellipodin to mediate the local production of phosphatidylinositol-3,4-bisphosphate and endophilin pre-enrichment. Membrane-bound GTP-loaded Cdc42 recruits FBP17 and CIP4, before being locally deactivated by RICH1 and SH3BP1 GTPase-activating proteins. This generates the transient assembly and disassembly of endophilin spots, which lasts 5–10 seconds. This mechanism periodically primes patches of the membrane for prompt responses upon FEME activation. Chan Wah Hak et al. show how plasma membrane patches are primed for fast endophilin-mediated endocytosis and disassembly, in the absence of receptor stimulation, through FBP17 and CIP4 binding to SHIP2 and lamellipodin.

The PI3K pathway is commonly activated in breast cancer, with PI3K-AKT pathway inhibitors used clinically. However, mechanisms that limit or enhance the therapeutic effects of PI3K-AKT inhibitors are poorly understood at a genome-wide level. Parallel CRISPR screens in 3 PTEN-null breast cancer cell lines identified genes mediating resistance to capivasertib (AKT inhibitor) and AZD8186 (PI3Kβ inhibitor). The dominant mechanism causing resistance is reactivated PI3K-AKT-mTOR signalling, but not other canonical signalling pathways. Deletion of TSC1/2 conferred resistance to PI3Kβi and AKTi through mTORC1. However, deletion of PIK3R2 and INPPL1 drove specific PI3Kβi resistance through AKT. Conversely deletion of PIK3CA , ERBB2 , ERBB 3 increased PI3Kβi sensitivity while modulation of RRAGC , LAMTOR1 , LAMTOR4 increased AKTi sensitivity. Significantly, we found that Mcl-1 loss enhanced response through rapid apoptosis induction with AKTi and PI3Kβi in both sensitive and drug resistant TSC1/2 null cells. The combination effect was BAK but not BAX dependent. The Mcl-1i + PI3Kβ/AKTi combination was effective across a panel of breast cancer cell lines with PIK3CA and PTEN mutations, and delivered increased anti-tumor benefit in vivo. This study demonstrates that different resistance drivers to PI3Kβi and AKTi converge to reactivate PI3K-AKT or mTOR signalling and combined inhibition of Mcl-1 and PI3K-AKT has potential as a treatment strategy for PI3Kβi/AKTi sensitive and resistant breast tumours.

Systemic sclerosis (SSc) is a heterogeneous multisystemic autoimmune disease, characterized by extensive skin fibrosis, vascular abnormalities, and autoantibodies against various cellular antigens. The integrated analysis of proteomic and metabolomic in SSc might shed light in the pathogenesis of the disease and reveal novel biomarkers. The aim of this study was to characterize the circulating proteomic and metabolomic profiles in SSc and their association with key clinical features. Serum NMR metabolomics (Nightingale; 250 metabolites covering glycolysis metabolites, amino acids and lipid measures), and proteomics, involving the analysis of 92 organ damage-related proteins [by proximity extension immunoassay (PEA, Olink)] were performed on serum from 72 SSc patients and 43 age-matched healthy donors (HD). Main disease complications in the SSC cohort, including lung fibrosis, skin fibrosis, renal, vascular, and esophageal involvement were assessed, and prevalence of circulating autoantibodies, along with standard demographic and inflammatory parameters were analyzed by multiple t-test. Unsupervised hierarchical clustering methodologies were applied using Metaboanalyst software to identify subgroups of patients based on their proteomic profiles. Gene ontology enrichment was used to interrogate the biological meaning of the distinctive molecular signatures identified. Sixteen circulating proteins related to organ damage (9 increased and 7 reduced) as well as 143 metabolites (37 increased and 101 reduced) were found altered in SSc patients in relation to HD. Unsupervised clustering analyses differentiated 3 patients clusters presenting different proteomic profiles. Clinically, patients belonging to cluster 1 (C1) were characterized by a significant prevalence of multiple organ involvement (84%) in relation to C2 (52%) and C3 (43%), mostly encompassing lung and skin fibrosis and esophageal dysmotility. Immunologically, C1 further displayed the highest percentage of positivity for anti-scl70 antibodies. Nineteen proteins were identified as significantly altered in cluster 1 (C1) in relation to C2 and C3, mostly involved in biological processes such as cell proliferation, apoptosis, cell adhesion, migration, and immune response. Among them, two were functionally linked with cutaneous diseases (CALR and BANK1), two with digestive disorders (FGR and STXBP3) and three with lung disfunction (FOSB, SMAD1 and FOXO1). Levels of some overexpressed proteins in C1 (BID, INPPL1, BANK1 and FOSB) were further related to the positivity for anti-scl70, the specific SSC-autoantibody mostly associated to a bad prognosis and multiple organ involvement in SSC. Interestingly, thirty-five metabolic markers were found deregulated when comparing those proteomic clusters of SSC, comprising lipoprotein subsets, sphingomyelins, cholesterol, lactate, and albumin. Moreover, significant correlations were identified among the levels of proteins and metabolites found deregulated, pointing at a concordance on their involvement in mechanisms underlying the disease process. 1)Serum proteomic and metabolomic signatures are significantly altered in SSC patients and associated to relevant clinical profiles.2)Clinical classification of SSc patients could be improved based on serum proteomic and metabolomic profile, adding new insights to the underlying pathophysiological mechanisms. Supported by ISCIII (PI21/0591, CD21/00187 and RICOR-RD21/0002/0033) co-financed by FEDER; Fundación Andaluza de Reumatología. None Declared.

Membrane-anchored and soluble inositol phospholipid species are critical mediators of intracellular cell signaling cascades. Alterations in their normal production or degradation are implicated in the pathology of a number of disorders including cancer and pro-inflammatory conditions. The SH2-containing 5′ inositol phosphatases, SHIP1 and SHIP2, play a fundamental role in these processes by depleting PI(3,4,5)P3, but also by producing PI(3,4)P2 at the inner leaflet of the plasma membrane. With the intent of targeting SHIP1 or SHIP2 selectively, or both paralogs simultaneously, small molecule inhibitors and agonists have been developed and tested in vitro and in vivo over the last decade in various disease models. These studies have shown promising results in various pre-clinical models of disease including cancer and tumor immunotherapy. In this review the potential use of SHIP inhibitors in cancer is discussed with particular attention to the molecular structure, binding site and efficacy of these SHIP inhibitors.

Background and objectives Maternal western-style diets that are high in glucose and fat have well-known cardiovascular effects on offspring, yet the combined influence of such diets during pregnancy is relatively less comprehended. This study investigates the impact of maternal high glucose and fat diet (HGF) on vascular constriction in offspring and the underlying mechanisms. Methods and results Pregnant Sprague–Dawley rats were provided with either HGF or control diets. The assessment of fetal and postnatal vascular function disclosed an enhanced sensitivity to angiotensin II-induced vascular constriction in the offspring exposed to HGF. This was ascribed to increased oxidative stress via upregulated NOX2 expression, which was due to downregulated SHIP2 expression that was influenced by upregulated miR-325-3p. The maternal HGF diet elevated miR-325-3p, suppressed SHIP2 and enhanced NOX2 expression in fetal vascular tissues, thereby resulting in vascular dysfunction. These alterations persist into adulthood, heightening the risk of vascular diseases. Conclusion The present study is the first to demonstrate that maternal HGF diet impairs vascular constriction function in offspring through the miR-325-3p/SHIP2/NOX2 pathway. These novel findings indicate that the detrimental effects of maternal HGF diet on fetal vascular function can persist into adulthood, advancing our knowledge on the impact of maternal diet on offspring vascular health and the early stages of fetal-origin vascular diseases.