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Phosphoinositides (PIPs) are phospholipids and components of the cellular membrane. In mammals, seven phosphorylated derivatives of PIPs have been identified. Among them, phosphatidylinositol 3,4-bisphosphate [PI(3,4)P2] is produced by lipid phosphatases (e.g., SHIP2) or by lipid kinases PI3KC2α and PI3KC2β. Although PI(3,4)P2 is undetectable in normal mouse or human tissues and common cell lines, it appears in a mouse prostate cancer model and in cells exposed to oxidative stress, indicating that PI(3,4)P2 is involved in the pathogenesis of some diseases. Here, I summarize recent findings on the cellular roles and pathophysiological significance of PI(3,4)P2., (© The Author(s) 2024. Published by Oxford University Press on behalf of the Japanese Biochemical Society. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Aggressive solid malignancies, including pancreatic ductal adenocarcinoma (PDAC), can exploit lysosomal exocytosis to modify the tumor microenvironment, enhance motility, and promote invasiveness. However, the molecular pathways through which lysosomal functions are co-opted in malignant cells remain poorly understood. In this study, we demonstrate that inositol polyphosphate 4-phosphatase, Type II (INPP4B) overexpression in PDAC is associated with PDAC progression. We show that INPP4B overexpression promotes peripheral dispersion and exocytosis of lysosomes resulting in increased migratory and invasive potential of PDAC cells. Mechanistically, INPP4B overexpression drives the generation of PtdIns(3,5)P2 on lysosomes in a PIKfyve-dependent manner, which directs TRPML-1 to trigger the release of calcium ions (Ca2+). Our findings offer a molecular understanding of the prognostic significance of INPP4B overexpression in PDAC through the discovery of a novel oncogenic signaling axis that orchestrates migratory and invasive properties of PDAC via the regulation of lysosomal phosphoinositide homeostasis., (© 2024 Saffi et al.)

Exomer is a protein complex that facilitates trafficking between the Golgi and the plasma membrane (PM). Schizosaccharomyces pombe exomer is composed of Cfr1 and Bch1, and we have found that full activation of the cell integrity pathway (CIP) in response to osmotic stress requires exomer. In the wild-type, the CIP activators Rgf1 (Rho1 GEF) and Pck2 (PKC homologue) and the MEK kinase Mkh1 localize in the PM, internalize after osmotic shock and re-localize after adaptation. This re-localization is inefficient in exomer mutants. Overexpression of the PM-associated 1-phosphatidylinositol 4-kinase stt4+ , and deletion of the nem1+ phosphatase suppress the defects in Pck2 dynamics in exomer mutants, but not their defect in CIP activation, demonstrating that exomer regulates CIP in additional ways. Exomer mutants accumulate PI4P in the TGN, and increasing the expression of the Golgi-associated 1-phosphatidylinositol 4-kinase pik1+ suppresses their defect in Pck2 dynamics. These findings suggest that efficient PI4P transport from the Golgi to the PM requires exomer. Mutants lacking clathrin adaptors are defective in CIP activation, but not in Pck2 dynamics or in PI4P accumulation in the Golgi. Hence, traffic from the Golgi regulates CIP activation, and exomer participates in this regulation through an exclusive mechanism.

The erlin1/erlin2 (E1/E2) complex is an endoplasmic reticulum membrane-located assemblage of the proteins erlin1 and erlin2. Here, we demonstrate direct and selective binding of phosphatidylinositol 3-phosphate (PI(3)P) to recombinant erlins and that disruption or deletion of the E1/E2 complex reduces HeLa cell PI(3)P levels by ∼50 %. This reduction correlated with a decrease in autophagic flux, with no effect on the endocytic pathway, and was not due to reduced VPS34 kinase activity, which is critical for maintaining steady-state PI(3)P levels. Pharmacological inhibition of VPS34 and suppression of PI(3)P levels caused a similar reduction in autophagic flux. Overall, these data indicate that by binding to PI(3)P, the E1/E2 complex plays an important role in maintaining the steady-state levels of PI(3)P and, thus, sustains some key PI(3)P-dependent processes, e.g., autophagy., Competing Interests: Declaration of competing interest None. The authors have no competing interest., (Copyright © 2024 Elsevier Inc. All rights reserved.)

Enterovirus 71 (EV71) belongs to the family of Picornaviridae ; it could cause a variety of illnesses and pose a great threat to public health worldwide. Currently, there is no specific drug treatment for this virus, and a better understanding of virus-host interaction is crucial for novel antiviral development. Here, we find that the class III phosphatidylinositol 3-kinase, VPS34, is an essential host factor for EV71 infection. VPS34 inhibition with either shRNA or specific chemical inhibitor significantly reduces EV71 infection. Meanwhile, EV71 infection upregulates phosphatidylinositol 3-phosphate (PI3P) production in viral replication organelles (ROs), while the depletion of PI3P by phosphatase overexpression inhibits EV71 infection. In addition, the PI3P-binding protein, double FYVE-containing protein 1 (DFCP1), is also required for an efficient replication of EV71. DFCP1 could interact with viral 2C protein and facilitate viral association with lipid droplets (LDs), which are important lipid sources for viral RO biogenesis. Taken together, these results indicate that EV71 virus exploits the VPS34-PI3P-DFCP1-LDs pathway to promote viral RO formation and viral infection, and they also illuminate novel targets for antiviral development.IMPORTANCEEnterovirus 71 (EV71) is a major pathogen that causes hand-foot-and-mouth disease (HFMD) and other serious complications, which are big threats to children under 5 years old. Unravelling the interactions between virus and the host cells will open new avenues in antiviral research. Here, we found the class III phosphatidylinositol 3-kinase, VPS34, and its effector, double FYVE-containing protein 1 (DFCP1), were essential for EV71 infection, both of which could support EV71 viral replication by enhancing the biogenesis of viral replication organelles (ROs). As DFCP1 localizes to lipid droplets, hijacking of these host factors will enable viral utilization of lipids from LDs for the generation of membrane structures during RO biogenesis. In addition, the VPS34 kinase inhibitor was found to be potent against EV71 infection; therefore, this study also brings up a novel target for future anti-EV71 drug development., Competing Interests: The authors declare no conflict of interest.

Target of Myb1 (TOM1) facilitates the transport of endosomal ubiquitinated proteins destined for lysosomal degradation; however, the mechanisms regulating TOM1 during this process remain unknown. Here, we identified an adjacent DXXLL motif-containing region to the TOM1 VHS domain, which enhances its affinity for ubiquitin and can be modulated by phosphorylation. TOM1 is an endosomal phosphatidylinositol 5-phosphate (PtdIns5P) effector under Shigella flexneri infection. We pinpointed a consensus PtdIns5P-binding motif in the VHS domain. We show that PtdIns5P binding by TOM1 is pH-dependent, similarly observed in its binding partner TOLLIP. Under acidic conditions, TOM1 retained its complex formation with TOLLIP, but was unable to bind ubiquitin. S. flexneri infection inhibits pH-dependent endosomal maturation, leading to reduced protein degradation. We propose a model wherein pumping of H + to the cytosolic side of endosomes contributes to the accumulation of TOM1, and possibly TOLLIP, at these sites, thereby promoting PtdIns5P- and pH-dependent signaling, facilitating bacterial survival., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2024 Elsevier Inc. All rights reserved.)

The FAM83 ( Fam ily with sequence similarity 83 ) family is highly conserved in vertebrates, but little is known of the functions of these proteins beyond their association with oncogenesis. Of the family, FAM83F is of particular interest because it is the only membrane-targeted FAM83 protein. When overexpressed, FAM83F activates the canonical Wnt signalling pathway and binds to and stabilizes p53; it therefore interacts with two pathways often dysregulated in disease. Insights into gene function can often be gained by studying the roles they play during development, and here we report the generation of fam83f knock-out (KO) zebrafish, which we have used to study the role of Fam83f in vivo . We show that endogenous fam83f is most strongly expressed in the hatching gland of developing zebrafish embryos, and that fam83f KO embryos hatch earlier than their wild-type (WT) counterparts, despite developing at a comparable rate. We also demonstrate that fam83f KO embryos are more sensitive to ionizing radiation than WT embryos-an unexpected finding, bearing in mind the previously reported ability of FAM83F to stabilize p53. Transcriptomic analysis shows that loss of fam83f leads to downregulation of phosphatidylinositol-3-phosphate (PI(3)P) binding proteins and impairment of cellular degradation pathways, particularly autophagy, a crucial component of the DNA damage response. Finally, we show that Fam83f protein is itself targeted to the lysosome when overexpressed in HEK293T cells, and that this localization is dependent upon a C' terminal signal sequence. The zebrafish lines we have generated suggest that Fam83f plays an important role in autophagic/lysosomal processes, resulting in dysregulated hatching and increased sensitivity to genotoxic stress in vivo .

Phosphatidylinositol phosphates are powerful signaling molecules that orchestrate signaling and direct membrane trafficking in the cytosol. Interestingly, phosphatidylinositol phosphates also localize within the membrane-less compartments of the cell nucleus, where they participate in the regulation of gene expression. Nevertheless, current models of gene expression, which include condensates of proteins and nucleic acids, do not include nuclear phosphatidylinositol phosphates. This gap is partly a result of the missing detailed analysis of the subnuclear distribution of phosphatidylinositol phosphates and their relationships with gene expression. Here, we used quantitative dual-color direct stochastic optical reconstruction microscopy to analyze the nanoscale co-patterning between RNA polymerase II transcription initiation and elongation markers with respect to phosphatidylinositol 4,5- or 3,4-bisphosphate in the nucleoplasm and nuclear speckles and compared it with randomized data and cells with inhibited transcription. We found specific co-patterning of the transcription initiation marker P-S5 with phosphatidylinositol 4,5-bisphosphate in the nucleoplasm and with phosphatidylinositol 3,4-bisphosphate at the periphery of nuclear speckles. We showed the specific accumulation of the transcription elongation marker PS-2 and of nascent RNA in the proximity of phosphatidylinositol 3,4-bisphosphate associated with nuclear speckles. Taken together, this shows that the distinct spatial associations between the consecutive stages of RNA polymerase II transcription and nuclear phosphatidylinositol phosphates exhibit specificity within the gene expression compartments. Thus, in analogy to the cellular membranes, where phospholipid composition orchestrates signaling pathways and directs membrane trafficking, we propose a model in which the phospholipid identity of gene expression compartments orchestrates RNA polymerase II transcription., (© 2024 The Authors. The FEBS Journal published by John Wiley & Sons Ltd on behalf of Federation of European Biochemical Societies.)

In macroautophagy, lysosomes fuse with closed autophagosomes but not with unclosed ones. This is achieved, at least in part, by the temporally regulated recruitment of the autophagosomal SNARE STX17 (syntaxin 17) to only mature autophagosomes. However, the molecular mechanism by which STX17 recognizes autophagosomal maturation remains unknown. Our recent study revealed that STX17 recruitment is regulated by the electrostatic interaction between the positively charged C-terminal region of STX17 and the autophagosomal membrane, which becomes negatively charged during maturation due to the accumulation of phosphatidylinositol-4-phosphate (PtdIns4P). Here, we propose an electrostatic maturation model of the autophagosome.

As key mediators in a wide array of signaling events, phosphoinositides (PIPs) orchestrate the recruitment of proteins to specific cellular locations at precise moments. This intricate spatiotemporal regulation of protein activity often necessitates the localized enrichment of the corresponding PIP. We investigate the extent and thermal stabilities of phosphatidylinositol-4-phosphate (PI(4)P), phosphatidylinositol-4,5-bisphosphate (PI(4,5)P 2 and phosphatidylinositol-3,4,5-trisphosphate (PI(3,4,5)P 3 ) clusters with calcium and magnesium ions. We observe negligible or minimal clustering of all examined PIPs in the presence of Mg 2+ ions. While PI(4)P shows in the presence of Ca 2+ no clustering, PI(4,5)P 2 forms with Ca 2+ strong clusters that exhibit stablity up to at least 80°C. The extent of cluster formation for the interaction of PI(3,4,5)P 3 with Ca 2+ is less than what was observed for PI(4,5)P 2 , yet we still observe some clustering up to 80°C. Given that cholesterol has been demonstrated to enhance PIP clustering, we examined whether bivalent cations and cholesterol synergistically promote PIP clustering. We found that the interaction of Mg 2+ or Ca 2+ with PI(4)P remains extraordinarily weak, even in the presence of cholesterol. In contrast, we observe synergistic interaction of cholesterol and Ca 2+ with PI(4,5)P 2 . Also, in the presence of cholesterol, the interaction of Mg 2+ with PI(4,5)P 2 remains weak. PI(3,4,5)P 3 does not show strong clustering with cholesterol for the experimental conditions of our study and the interaction with Ca 2+ and Mg 2+ was not influenced by the presence of cholesterol., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Phospholipase A 2 's (PLA 2 's) constitute a superfamily of enzymes that hydrolyze the sn-2 fatty acyl chain on glycerophospholipids. We have previously reported that each PLA 2 Type shows a unique substrate specificity for the molecular species it hydrolyzes, especially the acyl chain that is cleaved from the sn-2 position and to some extent the polar group. However, phosphatidylinositol (PI) and PI phosphates (PIPs) have not been as well studied as substrates as other phospholipids because the PIPs require adaptation of the standard analysis methods, but they are important in vivo. We determined the in vitro activity of the three major types of human PLA 2 's, namely the cytosolic (c), calcium-independent (i), and secreted (s) PLA 2 ' s toward PI, PI-4-phosphate (PI(4)P), and PI-4,5-bisphosphate (PI(4,5)P 2 ). The in vitro assay revealed that Group IVA cPLA 2 (GIVA cPLA 2 ) showed relatively high activity toward PI and PI(4)P among the tested PLA 2 's; nevertheless, the highly hydrophilic headgroup disrupted the interaction between the lipid surface and the enzyme. GIVA cPLA 2 and GVIA iPLA 2 showed detectable activity toward PI(4,5)P 2 , but it appeared to be a poorer substrate for all of the PLA 2 's tested. Furthermore, molecular dynamics (MD) simulations demonstrated that Thr416 and Glu418 of GIVA cPLA 2 contribute significantly to accommodating the hydrophilic head groups of PI and PI(4)P, which could explain some selectivity for PI and PI(4)P. These results indicated that GIVA cPLA 2 can accommodate PI and PI(4)P in its active site and hydrolyze them, suggesting that the GIVA cPLA 2 may best account for the PI and PIP hydrolysis in living cells., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Authors. Published by Elsevier B.V. All rights reserved.)

Membrane contact sites (MCS) facilitate communication between organelles. Casler et al. (https://doi.org/10.1083/jcb.202308144) show that tripartite MCS between mitochondria, the endoplasmic reticulum (ER), and the plasma membrane (PM) regulate mitochondrial division and the distribution of phosphatidylinositol 4-phosphate [PI(4)P] on the PM., (© 2024 Posor and Haucke.)

The mitochondria-ER-cortex anchor (MECA) forms a tripartite membrane contact site between mitochondria, the endoplasmic reticulum (ER), and the plasma membrane (PM). The core component of MECA, Num1, interacts with the PM and mitochondria via two distinct lipid-binding domains; however, the molecular mechanism by which Num1 interacts with the ER is unclear. Here, we demonstrate that Num1 contains a FFAT motif in its C-terminus that interacts with the integral ER membrane protein Scs2. While dispensable for Num1's functions in mitochondrial tethering and dynein anchoring, the FFAT motif is required for Num1's role in promoting mitochondrial division. Unexpectedly, we also reveal a novel function of MECA in regulating the distribution of phosphatidylinositol-4-phosphate (PI(4)P). Breaking Num1 association with any of the three membranes it tethers results in an accumulation of PI(4)P on the PM, likely via disrupting Sac1-mediated PI(4)P turnover. This work establishes MECA as an important regulatory hub that spatially organizes mitochondria, ER, and PM to coordinate crucial cellular functions., (© 2024 Casler et al.)

Receptor tyrosine kinases such as EGF receptor (EGFR) stimulate phosphoinositide 3 kinases to convert phosphatidylinositol-4,5-bisphosophate [PtdIns(4,5)P 2 ] into phosphatidylinositol-3,4,5-trisphosphate [PtdIns(3,4,5)P 3 ]. PtdIns(3,4,5)P 3 then remodels actin and gene expression, and boosts cell survival and proliferation. PtdIns(3,4,5)P 3 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)P 3 -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)P 2 levels and perturbed endocytosis and endocytic trafficking. However, the role of LCLAT1 in receptor tyrosine kinase and PtdIns(3,4,5)P 3 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)P 3 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.

Rho GTPases are a family of highly conserved G proteins that regulate numerous cellular processes, including cytoskeleton organisation, migration, and proliferation. The 20 canonical Rho GTPases are regulated by ∼85 guanine nucleotide exchange factors (GEFs), with the largest family being the 71 Diffuse B-cell Lymphoma (Dbl) GEFs. Dbl GEFs promote GTPase activity through the highly conserved Dbl homology domain. The specificity of GEF activity, and consequently GTPase activity, lies in the regulation and structures of the GEFs themselves. Dbl GEFs contain various accessory domains that regulate GEF activity by controlling subcellular localisation, protein interactions, and often autoinhibition. This review focuses on the two phosphatidylinositol (3,4,5)-trisphosphate (PI(3,4,5)P3)-dependent Rac exchangers (P-Rex), particularly the structural basis of P-Rex1 autoinhibition and synergistic activation. First, we discuss structures that highlight the conservation of P-Rex catalytic and phosphoinositide binding activities. We then explore recent breakthroughs in uncovering the structural basis for P-Rex1 autoinhibition and detail the proposed minimal two-step model of how PI(3,4,5)P3 and Gβγ synergistically activate P-Rex1 at the membrane. Additionally, we discuss the further layers of P-Rex regulation provided by phosphorylation and P-Rex2-PTEN coinhibitory complex formation, although these mechanisms remain incompletely understood. Finally, we leverage the available data to infer how cancer-associated mutations in P-Rex2 destabilise autoinhibition and evade PTEN coinhibitory complex formation, leading to increased P-Rex2 GEF activity and driving cancer progression and metastasis., (© 2024 The Author(s).)

Activity-regulated cytoskeleton-associated protein (Arc/Arg3.1) is an immediate early gene that plays a vital role in learning and memory. Arc protein has structural and functional properties similar to viral Group-specific antigen (Gag) protein and mediates the intercellular RNA transfer through virus-like capsids. However, the regulators and secretion pathway through which Arc capsids maneuver cargos are unclear. Here, we identified that phosphatidylinositol-3-phosphate (PI3P) mediates Arc capsid assembly and secretion through the endosomal-multivesicular body (MVB) pathway. Indeed, reconstituted Arc protein preferably binds to PI3P. In HEK293T cells, Arc forms puncta that colocalize with FYVE, an endosomal PI3P marker, as well as Rab5 and CD63, early endosomal and MVB markers, respectively. Superresolution imaging resolves Arc accumulates within the intraluminal vesicles of MVB. CRISPR double knockout of RalA and RalB, crucial GTPases for MVB biogenesis and exocytosis, severely reduces the Arc-mediated RNA transfer efficiency. RalA/B double knockdown in cultured rat cortical neurons increases the percentage of mature dendritic spines. Intake of extracellular vesicles purified from Arc-expressing wild-type, but not RalA/B double knockdown, cells in mouse cortical neurons reduces their surface GlutA1 levels. These results suggest that unlike the HIV Gag, whose membrane targeting requires interaction with plasma-membrane-specific phosphatidyl inositol (4,5) bisphosphate (PI(4,5)P2), the assembly of Arc capsids is mediated by PI3P at endocytic membranes. Understanding Arc's secretion pathway helps gain insights into its role in intercellular cargo transfer and highlights the commonality and distinction of trafficking mechanisms between structurally resembled capsid proteins., Competing Interests: Competing interests statement:The authors declare no competing interest.

Autophagy is a highly conserved process from yeast to mammals in which intracellular materials are engulfed by a double-membrane organelle called autophagosome and degrading materials by fusing with the lysosome. The process of autophagy is regulated by sequential recruitment and function of autophagy-related (Atg) proteins. Genetic hierarchical analyses show that the ULK1 complex comprised of ULK1-FIP200-ATG13-ATG101 translocating from the cytosol to autophagosome formation sites as a most upstream ATG factor; this translocation is critical in autophagy initiation. However, how this translocation occurs remains unclear. Here, we show that ULK1 is palmitoylated by palmitoyltransferase ZDHHC13 and translocated to the autophagosome formation site upon autophagy induction. We find that the ULK1 palmitoylation is required for autophagy initiation. Moreover, the ULK1 palmitoylated enhances the phosphorylation of ATG14L, which is required for activating PI3-Kinase and producing phosphatidylinositol 3-phosphate, one of the autophagosome membrane's lipids. Our results reveal how the most upstream ULK1 complex translocates to the autophagosome formation sites during autophagy., (© 2024. The Author(s).)

Despite recent studies implicating liquid-like biomolecular condensates in diverse cellular processes, many biomolecular condensates exist in a solid-like state, and their function and regulation are less understood. We show that the tumor suppressor Merlin, an upstream regulator of the Hippo pathway, localizes to both cell junctions and medial apical cortex in Drosophila epithelia, with the latter forming solid-like condensates that activate Hippo signaling. Merlin condensation required phosphatidylinositol-4-phosphate (PI4P)-mediated plasma membrane targeting and was antagonistically controlled by Pez and cytoskeletal tension through plasma membrane PI4P regulation. The solid-like material properties of Merlin condensates are essential for physiological function and protect the condensates against external perturbations. Collectively, these findings uncover an essential role for solid-like condensates in normal physiology and reveal regulatory mechanisms for their formation and disassembly.

Numerous lipids are heterogeneously distributed among organelles. Most lipid trafficking between organelles is achieved by a group of lipid transfer proteins (LTPs) that carry lipids using their hydrophobic cavities. The human genome encodes many intracellular LTPs responsible for lipid trafficking and the function of many LTPs in defining cellular lipid levels and distributions is unclear. Here, we created a gene knockout library targeting 90 intracellular LTPs and performed whole-cell lipidomics analysis. This analysis confirmed known lipid disturbances and identified new ones caused by the loss of LTPs. Among these, we found major sphingolipid imbalances in ORP9 and ORP11 knockout cells, two proteins of previously unknown function in sphingolipid metabolism. ORP9 and ORP11 form a heterodimer to localize at the ER- trans -Golgi membrane contact sites, where the dimer exchanges phosphatidylserine (PS) for phosphatidylinositol-4-phosphate (PI(4)P) between the two organelles. Consequently, loss of either protein causes phospholipid imbalances in the Golgi apparatus that result in lowered sphingomyelin synthesis at this organelle. Overall, our LTP knockout library toolbox identifies various proteins in control of cellular lipid levels, including the ORP9-ORP11 heterodimer, which exchanges PS and PI(4)P at the ER-Golgi membrane contact site as a critical step in sphingomyelin synthesis in the Golgi apparatus., Competing Interests: BC, SB, JA, NB, AM, RK, MG, JN No competing interests declared, (© 2023, Cabukusta et al.)

Two-pore channels are pathophysiologically important Na + - and Ca 2+ -permeable channels expressed in lysosomes and other acidic organelles. Unlike most other ion channels, their permeability is malleable and ligand-tuned such that when gated by the signaling lipid PI(3,5)P 2 , they are more Na + -selective than when gated by the Ca 2+ mobilizing messenger nicotinic acid adenine dinucleotide phosphate. However, the structural basis that underlies such plasticity and single-channel behavior more generally remains poorly understood. A recent Cryo-electron microscopy (cryo-EM) structure of TPC2 bound to PI(3,5)P 2 in a proposed open-channel conformation provided an opportunity to address this via molecular dynamics (MD) simulation. To our surprise, simulations designed to compute conductance through this structure revealed almost no Na + permeation events even at very high transmembrane voltages. However further MD simulations identified a spontaneous transition to a dramatically different conformation of the selectivity filter that involved expansion and a flip in the orientation of two core asparagine residues. This alternative filter conformation was remarkably stable and allowed Na + to flow through the channel leading to a conductance estimate that was in very good agreement with direct single-channel measurements. Furthermore, this conformation was more permeable for Na + over Ca 2+ . Our results have important ramifications not just for understanding the control of ion selectivity in TPC2 channels but also more broadly in terms of how ion channels discriminate ions., Competing Interests: Competing interests statement:The authors declare no competing interest.

Little is known about eukaryotic chemorepulsion. The enzymes phosphatase and tensin homolog (PTEN) and CnrN dephosphorylate phosphatidylinositol 3,4,5-trisphosphate [PI(3,4,5)P3] to phosphatidylinositol 4,5-bisphosphate [PI(4,5)P2]. Dictyostelium discoideum cells require both PTEN and CnrN to induce chemorepulsion of cells away from the secreted chemorepellent protein AprA. How D. discoideum cells utilize two proteins with redundant phosphatase activities in response to AprA is unclear. Here, we show that D. discoideum cells require both PTEN and CnrN to locally inhibit Ras activation, decrease basal levels of PI(3,4,5)P3 and increase basal numbers of macropinosomes, and AprA prevents this increase. AprA requires both PTEN and CnrN to increase PI(4,5)P2 levels, decrease PI(3,4,5)P3 levels, inhibit proliferation, decrease myosin II phosphorylation and increase filopod sizes. PTEN, but not CnrN, decreases basal levels of PI(4,5)P2, and AprA requires PTEN, but not CnrN, to induce cell roundness. Together, our results suggest that CnrN and PTEN play unique roles in AprA-induced chemorepulsion., Competing Interests: Competing interests The authors declare no competing or financial interests., (© 2024. Published by The Company of Biologists Ltd.)

PIP 3 -dependent Rac exchanger 1 (P-Rex1) is abundantly expressed in neutrophils and plays central roles in chemotaxis and cancer metastasis by serving as a guanine-nucleotide exchange factor (GEF) for Rac. The enzyme is synergistically activated by PIP 3 and heterotrimeric Gβγ subunits, but mechanistic details remain poorly understood. While investigating the regulation of P-Rex1 by PIP 3 , we discovered that Ins(1,3,4,5)P 4 (IP 4 ) inhibits P-Rex1 activity and induces large decreases in backbone dynamics in diverse regions of the protein. Cryo-electron microscopy analysis of the P-Rex1·IP 4 complex revealed a conformation wherein the pleckstrin homology (PH) domain occludes the active site of the Dbl homology (DH) domain. This configuration is stabilized by interactions between the first DEP domain (DEP1) and the DH domain and between the PH domain and a 4-helix bundle (4HB) subdomain that extends from the C-terminal domain of P-Rex1. Disruption of the DH-DEP1 interface in a DH/PH-DEP1 fragment enhanced activity and led to a more extended conformation in solution, whereas mutations that constrain the occluded conformation led to decreased GEF activity. Variants of full-length P-Rex1 in which the DH-DEP1 and PH-4HB interfaces were disturbed exhibited enhanced activity during chemokine-induced cell migration, confirming that the observed structure represents the autoinhibited state in living cells. Interactions with PIP 3 -containing liposomes led to disruption of these interfaces and increased dynamics protein-wide. Our results further suggest that inositol phosphates such as IP 4 help to inhibit basal P-Rex1 activity in neutrophils, similar to their inhibitory effects on phosphatidylinositol-3-kinase., Competing Interests: SR, SA, SL, CC, MC, JS, JC, JT No competing interests declared, (© 2023, Ravala et al.)

Depending on their phosphorylation status, derivatives of phosphatidylinositol play important roles in vesicle identity, recognition and intracellular trafficking processes. In eukaryotic cells, phosphatidylinositol-4 phosphate pools generated by specific kinases are key determinants of the conventional secretion pathways. Earlier work in yeast has classified phosphatidylinositol-4 kinases in two types, Stt4p and Pik1p belonging to type III and Lsb6p to type II, with distinct cellular localizations and functions. Eurotiomycetes appear to lack Pik1p homologues. In Aspergillus nidulans, unlike homologues in other fungi, AnLsb6 is associated to late Golgi membranes and when heterologously overexpressed, it compensates for the thermosensitive phenotype in a Saccharomyces cerevisiae pik1 mutant, whereas its depletion leads to disorganization of Golgi-associated PH OSBP -labelled membranes, that tend to aggregate dependent on functional Rab5 GTPases. Evidence provided herein, indicates that the single type II phosphatidylinositol-4 kinase AnLsb6 is the main contributor for decorating secretory vesicles with relevant phosphatidylinositol-phosphate species, which navigate essential cargoes following the route of apical polarization via endocytic recycling., (© 2024. The Author(s).)

The autophagosomal SNARE STX17 (syntaxin 17) promotes lysosomal fusion and degradation, but its autophagosomal recruitment is incompletely understood. Notably, PtdIns4P is generated on autophagosomes and promotes fusion through an unknown mechanism. Here we show that soluble recombinant STX17 is spontaneously recruited to negatively charged liposomes and adding PtdIns4P to liposomes containing neutral lipids is sufficient for its recruitment. Consistently, STX17 colocalizes with PtdIns4P-positive autophagosomes in cells, and specific inhibition of PtdIns4P synthesis on autophagosomes prevents its loading. Molecular dynamics simulations indicate that C-terminal positively charged amino acids establish contact with membrane bilayers containing negatively charged PtdIns4P. Accordingly, Ala substitution of Lys and Arg residues in the C terminus of STX17 abolishes membrane binding and impairs its autophagosomal recruitment. Finally, only wild type but not Ala substituted STX17 expression rescues the autophagosome-lysosome fusion defect of STX17 loss-of-function cells. We thus identify a key step of autophagosome maturation that promotes lysosomal fusion. Abbreviations: Cardiolipin: 1',3'-bis[1-palmitoyl-2-oleoyl-sn-glycero-3-phospho]-glycerol; DMSO: dimethyl sulfoxide; GST: glutathione S-transferase; GUV: giant unilamellar vesicles; LAMP1: lysosomal associated membrane protein 1; MAP1LC3/LC3: microtubule associated protein 1 light chain 3; PA: 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphate; PC/POPC: 1-palmitoyl-2-oleoyl-glycero-3-phosphocholine; PG: 1-palmitoyl-2-linoleoyl-sn-glycero-3-phospho-(1'-rac-glycerol); PI: L-α-phosphatidylinositol; PI4K2A: phosphatidylinositol 4-kinase type 2 alpha; PIK3C3/VPS34: phosphatidylinositol 3-kinase catalytic subunit type 3; POPE/PE: 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine; PS: 1-stearoyl-2-linoleoyl-sn-glycero-3-phospho-L-serine; PtdIns(3,5)P 2 : 1,2-dioleoyl-sn-glycero-3-phospho-(1"-myo-inositol-3',5'-bisphosphate); PtdIns3P: 1,2- dioleoyl-sn-glycero-3-phospho-(1'-myo-inositol-3'-phosphate); PtdIns4P: 1,2-dioleoyl-sn-glycero-3-phospho-(1"-myo-inositol-4'-phosphate); SDS-PAGE: sodium dodecyl sulfate-polyacrylamide gel electrophoresis; STX17: syntaxin 17.

Together with its β-subunit OSTM1, ClC-7 performs 2Cl - /H + exchange across lysosomal membranes. Pathogenic variants in either gene cause lysosome-related pathologies, including osteopetrosis and lysosomal storage. CLCN7 variants can cause recessive or dominant disease. Different variants entail different sets of symptoms. Loss of ClC-7 causes osteopetrosis and mostly neuronal lysosomal storage. A recently reported de novo CLCN7 mutation (p.Tyr715Cys) causes widespread severe lysosome pathology (hypopigmentation, organomegaly, and delayed myelination and development, "HOD syndrome"), but no osteopetrosis. We now describe two additional HOD individuals with the previously described p.Tyr715Cys and a novel p.Lys285Thr mutation, respectively. Both mutations decreased ClC-7 inhibition by PI(3,5)P 2 and affected residues lining its binding pocket, and shifted voltage-dependent gating to less positive potentials, an effect partially conferred to WT subunits in WT/mutant heteromers. This shift predicts augmented pH gradient-driven Cl - uptake into vesicles. Overexpressing either mutant induced large lysosome-related vacuoles. This effect depended on Cl - /H + -exchange, as shown using mutants carrying uncoupling mutations. Fibroblasts from the p.Y715C patient also displayed giant vacuoles. This was not observed with p.K285T fibroblasts probably due to residual PI(3,5)P 2 sensitivity. The gain of function caused by the shifted voltage-dependence of either mutant likely is the main pathogenic factor. Loss of PI(3,5)P 2 inhibition will further increase current amplitudes, but may not be a general feature of HOD. Overactivity of ClC-7 induces pathologically enlarged vacuoles in many tissues, which is distinct from lysosomal storage observed with the loss of ClC-7 function. Osteopetrosis results from a loss of ClC-7, but osteoclasts remain resilient to increased ClC-7 activity., Competing Interests: Conflict of interest The authors declare that they have no conflicts of interest with the contents of this article., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

Autophagy is relevant for diverse processes in eukaryotic cells, making its regulation of fundamental importance. The formation and maturation of autophagosomes require a complex choreography of numerous factors. The endosomal sorting complex required for transport (ESCRT) is implicated in the final step of autophagosomal maturation by sealing of the phagophore membrane. ESCRT-III components were shown to mediate membrane scission by forming filaments that interact with cellular membranes. However, the molecular mechanisms underlying the recruitment of ESCRTs to non-endosomal membranes remain largely unknown. Here we focus on the ESCRT-associated protein ALG2-interacting protein X (ALIX) and identify Ca 2+ -dependent lipid binding protein 1 (CaLB1) as its interactor. Our findings demonstrate that CaLB1 interacts with AUTOPHAGY8 (ATG8) and PI(3)P, a phospholipid found in autophagosomal membranes. Moreover, CaLB1 and ALIX localize with ATG8 on autophagosomes upon salt treatment and assemble together into condensates. The depletion of CaLB1 impacts the maturation of salt-induced autophagosomes and leads to reduced delivery of autophagosomes to the vacuole. Here, we propose a crucial role of CaLB1 in augmenting phase separation of ALIX, facilitating the recruitment of ESCRT-III to the site of phagophore closure thereby ensuring efficient maturation of autophagosomes., (© 2024. The Author(s).)

Cells depend on their endolysosomal system for nutrient uptake and downregulation of plasma membrane proteins. These processes rely on endosomal maturation, which requires multiple membrane fusion steps. Early endosome fusion is promoted by the Rab5 GTPase and its effector, the hexameric CORVET tethering complex, which is homologous to the lysosomal HOPS. How these related complexes recognize their specific target membranes remains entirely elusive. Here, we solve the structure of CORVET by cryo-electron microscopy and revealed its minimal requirements for membrane tethering. As expected, the core of CORVET and HOPS resembles each other. However, the function-defining subunits show marked structural differences. Notably, we discover that unlike HOPS, CORVET depends not only on Rab5 but also on phosphatidylinositol-3-phosphate (PI3P) and membrane lipid packing defects for tethering, implying that an organelle-specific membrane code enables fusion. Our data suggest that both shape and membrane interactions of CORVET and HOPS are conserved in metazoans, thus providing a paradigm how tethering complexes function., (© 2024. The Author(s).)

Epithelial remodeling of the Drosophila retina depends on the pulsatile contraction and expansion of apical contacts between the cells that form its hexagonal lattice. Phosphoinositide PI(3,4,5)P 3 (PIP 3 ) accumulates around tricellular adherens junctions (tAJs) during contact expansion and dissipates during contraction, but with unknown function. Here, we found that manipulations of Pten or PI3-kinase (PI3K) that either decreased or increased PIP 3 resulted in shortened contacts and a disordered lattice, indicating a requirement for PIP 3 dynamics and turnover. These phenotypes are caused by a loss of branched actin, resulting from impaired activity of the Rac1 Rho GTPase and the WAVE regulatory complex (WRC). We additionally found that during contact expansion, PI3K moves into tAJs to promote the cyclical increase of PIP 3 in a spatially and temporally precise manner. Thus, dynamic control of PIP 3 by Pten and PI3K governs the protrusive phase of junctional remodeling, which is essential for planar epithelial morphogenesis., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2024 Elsevier Inc. All rights reserved.)

During macroautophagy, cytoplasmic constituents are engulfed by autophagosomes. Lysosomes fuse with closed autophagosomes but not with unclosed intermediate structures. This is achieved in part by the late recruitment of the autophagosomal SNARE syntaxin 17 (STX17) to mature autophagosomes. However, how STX17 recognizes autophagosome maturation is not known. Here, we show that this temporally regulated recruitment of STX17 depends on the positively charged C-terminal region of STX17. Consistent with this finding, mature autophagosomes are more negatively charged compared with unclosed intermediate structures. This electrostatic maturation of autophagosomes is likely driven by the accumulation of phosphatidylinositol 4-phosphate (PI4P) in the autophagosomal membrane. Accordingly, dephosphorylation of autophagosomal PI4P prevents the association of STX17 to autophagosomes. Furthermore, molecular dynamics simulations support PI4P-dependent membrane insertion of the transmembrane helices of STX17. Based on these findings, we propose a model in which STX17 recruitment to mature autophagosomes is temporally regulated by a PI4P-driven change in the surface charge of autophagosomes., Competing Interests: SS, YS, TM, MU, IK, JS, HY No competing interests declared, NM Reviewing editor, eLife, (© 2023, Shinoda et al.)

Myogenesis is a multistep process that requires a spatiotemporal regulation of cell events resulting finally in myoblast fusion into multinucleated myotubes. Most major insights into the mechanisms underlying fusion seem to be conserved from insects to mammals and include the formation of podosome-like protrusions (PLPs) that exert a driving force toward the founder cell. However, the machinery that governs this process remains poorly understood. In this study, we demonstrate that MTM1 is the main enzyme responsible for the production of phosphatidylinositol 5-phosphate, which in turn fuels PI5P 4-kinase α to produce a minor and functional pool of phosphatidylinositol 4,5-bisphosphate that concentrates in PLPs containing the scaffolding protein Tks5, Dynamin-2, and the fusogenic protein Myomaker. Collectively, our data reveal a functional crosstalk between a PI-phosphatase and a PI-kinase in the regulation of PLP formation., Competing Interests: Competing interests statement:The authors declare no competing interest.

Phosphoinositides are a small family of phospholipids that act as signaling hubs and key regulators of cellular function. Detecting their subcellular distribution is crucial to gain insights into membrane organization and is commonly done by the overexpression of biosensors. However, this leads to cellular perturbations and is challenging in systems that cannot be transfected. Here, we present a toolkit for the reliable, fast, multiplex, and super-resolution detection of phosphoinositides in fixed cells and tissue, based on recombinant biosensors with self-labeling SNAP tags. These are highly specific and reliably visualize the subcellular distributions of phosphoinositides across scales, from 2D or 3D cell culture to Drosophila tissue. Further, these probes enable super-resolution approaches, and using STED microscopy, we reveal the nanoscale organization of PI(3)P on endosomes and PI(4)P on the Golgi. Finally, multiplex staining reveals an unexpected presence of PI(3,5)P2-positive membranes in swollen lysosomes following PIKfyve inhibition. This approach enables the versatile, high-resolution visualization of multiple phosphoinositide species in an unprecedented manner., (© 2024 Maib et al.)

Autophagy, an essential cellular process for maintaining cellular homeostasis and eliminating harmful cytoplasmic objects, involves the de novo formation of double-membraned autophagosomes that engulf and degrade cellular debris, protein aggregates, damaged organelles, and pathogens. Central to this process is the phagophore, which forms from donor membranes rich in lipids synthesized at various cellular sites, including the endoplasmic reticulum (ER), which has emerged as a primary source. The ER-associated omegasomes, characterized by their distinctive omega-shaped structure and accumulation of phosphatidylinositol 3-phosphate (PI3P), play a pivotal role in autophagosome formation. Omegasomes are thought to serve as platforms for phagophore assembly by recruiting essential proteins such as DFCP1/ZFYVE1 and facilitating lipid transfer to expand the phagophore. Despite the critical importance of phagophore biogenesis, many aspects remain poorly understood, particularly the complete range of proteins involved in omegasome dynamics, and the detailed mechanisms of lipid transfer and membrane contact site formation., (© 2024 The Authors. BioEssays published by Wiley Periodicals LLC.)

Phosphoinositide 3-kinases regulate many cellular functions, including migration, growth, proliferation, and cell survival. Early studies equated the inhibition of Class I PI3Ks with loss of; phosphatidylinositol 3,4,5-trisphosphate (PIP3), but over time, it was realised that these; treatments also depleted phosphatidylinositol 3,4-bisphosphate (PI(3,4)P2). In recent years, the; use of better tools and an improved understanding of its metabolism have allowed for the; identification of specific roles of PI(3,4)P2. This includes the production of PI(3,4)P2 and the; activation of its effector Akt2 in response to growth factor signalling. In contrast, a lysosomal pool of PI(3,4)P2 is a negative regulator of mTORC1 during growth factor deprivation. A growing body of literature also demonstrates that PI(3,4)P2 controls many dynamic plasmalemmal processes. The significance of PI(3,4)P 2 in cell biology is increasingly evident., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Author(s). Published by Elsevier Ltd.. All rights reserved.)

The Hippo pathway blocks epithelial-mesenchymal transition and metastasis in cancer mediated by the transcriptional coactivator YAP. In this issue of Science Signaling , Palamiuc et al. demonstrate that phosphatidylinositol 5-phosphate (PI5P) enhances Hippo pathway activation and that simultaneously the Hippo pathway initiates a positive feedback loop by inhibiting the conversion of PI5P into PIP 2 .

Phosphatidylinositol 3-kinase α (PI3Kα) is a heterodimer of p110α catalytic and p85 adaptor subunits that is activated by agonist-stimulated receptor tyrosine kinases. Although p85α recruits p110α to activated receptors on membranes, p85α loss, which occurs commonly in cancer, paradoxically promotes agonist-stimulated PI3K/Akt signaling. p110α localizes to microtubules via microtubule-associated protein 4 (MAP4), facilitating its interaction with activated receptor kinases on endosomes to initiate PI3K/Akt signaling. Here, we demonstrate that in response to agonist stimulation and p85α knockdown, the residual p110α, coupled predominantly to p85β, exhibits enhanced recruitment with receptor tyrosine kinases to endosomes. Moreover, the p110α C2 domain binds PI3-phosphate, and this interaction is also required to recruit p110α to endosomes and for PI3K/Akt signaling. Stable knockdown of p85α, which mimics the reduced p85α levels observed in cancer, enhances cell growth and tumorsphere formation, and these effects are abrogated by MAP4 or p85β knockdown, underscoring their role in the tumor-promoting activity of p85α loss., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

As a key component for NADPH oxidase 2 (NOX2) activation, the peripheral membrane protein p47 phox translocates a cytosolic activating complex to the membrane through its PX domain. This study elucidates a potential regulatory mechanism of p47 phox recruitment and NOX2 activation by inositol hexaphosphate (IP6). Through NMR, fluorescence polarization, and FRET experimental results, IP6 is shown to be capable of breaking the lipid binding and membrane anchoring events of p47 phox -PX with low micromolar potency. Other phosphorylated inositol species such as IP5(1,3,4,5,6), IP4(1,3,4,5), and IP3(1,3,4) show weaker binding and no ability to inhibit lipid interactions in physiological concentration ranges. The low micromolar potency of IP6 inhibition of the p47 phox membrane anchoring suggests that physiologically relevant concentrations of IP6 serve as regulators, as seen in other membrane anchoring domains. The PX domain of p47 phox is known to be promiscuous to a variety of phosphatidylinositol phosphate (PIP) lipids, and this regulation may help target the domain only to the membranes most highly enriched with the highest affinity PIPs, such as the phagosomal membrane, while preventing aberrant binding to other membranes with high and heterogeneous PIP content, such as the plasma membrane. This study provides insight into a potential novel regulatory mechanism behind NOX2 activation and reveals a role for small-molecule regulation in this important NOX2 activator.

Phosphatidylinositol (3,4,5)-trisphosphate (PIP3) is a signaling lipid on the plasma membrane that plays a fundamental role in cell signaling with a strong impact on cell physiology and diseases. It is responsible for the protruding edge formation, cell polarization, macropinocytosis, and other membrane remodeling dynamics in cells. It has been shown that the membrane confinement and curvature affects the wave formation of PIP3 and F-actin. But, even in the absence of F-actin, a complex self-organization of the spatiotemporal PIP3 waves is observed. In recent findings, we have shown that these waves can be guided and pinned on strongly bended Dictyostelium membranes caused by molecular crowding and curvature-limited diffusion. Based on these experimental findings, we investigate the spatiotemporal PIP3 wave dynamics on realistic three-dimensional cell-like membranes to explore the effect of curvature-limited diffusion, as observed experimentally. We use an established stochastic reaction-diffusion model with enzymatic Michaelis-Menten-type reactions that mimics the dynamics of Dictyostelium cells. As these cells mimic the three-dimensional shape and size observed experimentally, we found that the PIP3 wave directionality can be explained by a Hopf-like and a reverse periodic-doubling bifurcation for uniform diffusion and curvature-limited diffusion properties. Finally, we compare the results with recent experimental findings and discuss the discrepancy between the biological and numerical results., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2024 Biophysical Society. Published by Elsevier Inc. All rights reserved.)

The Hippo pathway effectors Yes-associated protein 1 (YAP) and its homolog TAZ are transcriptional coactivators that control gene expression by binding to TEA domain (TEAD) family transcription factors. The YAP/TAZ-TEAD complex is a key regulator of cancer-specific transcriptional programs, which promote tumor progression in diverse types of cancer, including breast cancer. Despite intensive efforts, the YAP/TAZ-TEAD complex in cancer has remained largely undruggable due to an incomplete mechanistic understanding. Here, we report that nuclear phosphoinositides function as cofactors that mediate the binding of YAP/TAZ to TEADs. The enzymatic products of phosphoinositide kinases PIPKIα and IPMK, including phosphatidylinositol 4,5-bisphosphate (PI(4,5)P 2 ) and phosphatidylinositol 3,4,5-trisphosphate (P(I3,4,5)P 3 ), bridge the binding of YAP/TAZ to TEAD. Inhibiting these kinases or the association of YAP/TAZ with PI(4,5)P 2 and PI(3,4,5)P 3 attenuates YAP/TAZ interaction with the TEADs, the expression of YAP/TAZ target genes, and breast cancer cell motility. Although we could not conclusively exclude the possibility that other enzymatic products of IPMK such as inositol phosphates play a role in the mechanism, our results point to a previously unrecognized role of nuclear phosphoinositide signaling in control of YAP/TAZ activity and implicate this pathway as a potential therapeutic target in YAP/TAZ-driven breast cancer., (© 2024. The Author(s).)

Insulin is packaged into secretory granules that depart the Golgi and undergo a maturation process that involves changes in the protein and lipid composition of the granules. Here, we show that insulin secretory granules form physical contacts with the endoplasmic reticulum and that the lipid exchange protein oxysterol-binding protein (OSBP) is recruited to these sites in a Ca 2+ -dependent manner. OSBP binding to insulin granules is positively regulated by phosphatidylinositol-4 (PI4)-kinases and negatively regulated by the PI4 phosphate (PI(4)P) phosphatase Sac2. Loss of Sac2 results in excess accumulation of cholesterol on insulin granules that is normalized when OSBP expression is reduced, and both acute inhibition and small interfering RNA (siRNA)-mediated knockdown of OSBP suppress glucose-stimulated insulin secretion without affecting insulin production or intracellular Ca 2+ signaling. In conclusion, we show that lipid exchange at endoplasmic reticulum (ER)-granule contact sites is involved in the exocytic process and propose that these contacts act as reaction centers with multimodal functions during insulin granule maturation., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2024 The Author(s). Published by Elsevier Inc. All rights reserved.)

Autophagy eliminates cytoplasmic material by engulfment in membranous vesicles targeted for lysosome degradation. Nonselective autophagy coordinates sequestration of bulk cargo with the growth of the isolation membrane (IM) in a yet-unknown manner. Here, we show that in the budding yeast Saccharomyces cerevisiae, IMs expand while maintaining a rim sufficiently wide for sequestration of large cargo but tight enough to mature in due time. An obligate complex of Atg24/Snx4 with Atg20 or Snx41 assembles locally at the rim in a spatially extended manner that specifically depends on autophagic PI(3)P. This assembly stabilizes the open rim to promote autophagic sequestration of large cargo in correlation with vesicle expansion. Moreover, constriction of the rim by the PI(3)P-dependent Atg2-Atg18 complex and clearance of PI(3)P by Ymr1 antagonize rim opening to promote autophagic maturation and consumption of small cargo. Tight regulation of membrane rim aperture by PI(3)P thus couples the mechanism and physiology of nonselective autophagy., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2024 Elsevier Inc. All rights reserved.)

Background: Primary cilia are static microtubule-based structures protruding from the cell surface and present on most vertebrate cells. The appropriate localization of phospholipids is essential for cilia formation and stability. INPP5E is a cilia-localized inositol 5-phosphatase; its deletion alters the phosphoinositide composition in the ciliary membrane, disrupting ciliary function., Methods: The EGFP-2xP4M SidM , PH PLCδ1 -EGFP, and SMO-tRFP plasmids were constructed by the Gateway system to establish a stable RPE1 cell line. The INPP5E KO RPE1 cell line was constructed with the CRISPR/Cas9 system. The localization of INPP5E and the distribution of PI(4,5)P 2 and PI4P were examined by immunofluorescence microscopy. The fluorescence intensity co-localized with cilia was quantified by ImageJ., Results: In RPE1 cells, PI4P is localized at the ciliary membrane, whereas PI(4,5)P 2 is localized at the base of cilia. Knocking down or knocking out INPP5E alters this distribution, resulting in the distribution of PI(4,5)P 2 along the ciliary membrane and the disappearance of PI4P from the cilia. Meanwhile, PI(4,5)P 2 is located in the ciliary membrane labeled by SMO-tRFP., Conclusions: INPP5E regulates the distribution of phosphoinositide on cilia. PI(4,5)P 2 localizes at the ciliary membrane labeled with SMO-tRFP, indicating that ciliary pocket membrane contains PI(4,5)P 2 , and phosphoinositide composition in early membrane structures may differ from that in mature ciliary membrane., (© 2024 The Authors. Journal of Clinical Laboratory Analysis published by Wiley Periodicals LLC.)

Phosphatidylinositol 3-phosphate (PI3P) levels govern membrane trafficking in Candida glabrata. Here, we present a confocal imaging-based protocol for PI3P localization analysis using the GFP-FYVE (found in Fab1, YOTB, Vac1, and EEA1) fusion protein. We describe steps for cloning the FYVE domain into the GFP-containing vector backbone, transforming FYVE-GFP into C. glabrata, and preparing slides with FYVE-GFP-expressing C. glabrata cells. We then detail procedures for acquiring and analyzing images and quantifying signal data. This protocol is adaptable to subcellular localization analysis of other low-abundant lipid and protein molecules. For complete details on the use and execution of this protocol, please refer to Askari et al. (2023). 1 ., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2023 The Author(s). Published by Elsevier Inc. All rights reserved.)

Oxysterol-binding protein-related proteins (ORPs) play key roles in the distribution of lipids in eukaryotic cells by exchanging sterol or phosphatidylserine for PI4P between the endoplasmic reticulum (ER) and other cell regions. However, it is unclear how their exchange capacity is coupled to PI4P metabolism. To address this question quantitatively, we analyze the activity of a representative ORP, Osh4p, in an ER/Golgi interface reconstituted with ER- and Golgi-mimetic membranes functionalized with PI4P phosphatase Sac1p and phosphatidylinositol (PI) 4-kinase, respectively. Using real-time assays, we demonstrate that upon adenosine triphosphate (ATP) addition, Osh4p creates a sterol gradient between these membranes, relying on the spatially distant synthesis and hydrolysis of PI4P, and quantify how much PI4P is needed for this process. Then, we develop a quantitatively accurate kinetic model, validated by our data, and extrapolate this to estimate to what extent PI4P metabolism can drive ORP-mediated sterol transfer in cells. Finally, we show that Sec14p can support PI4P metabolism and Osh4p activity by transferring PI between membranes. This study establishes that PI4P synthesis drives ORP-mediated lipid exchange and that ATP energy is needed to generate intermembrane lipid gradients. Furthermore, it defines to what extent ORPs can distribute lipids in the cell and reassesses the role of PI-transfer proteins in PI4P metabolism., Competing Interests: Competing interests statement:The authors declare no competing interest.

Phosphatidylinositol 4-phosphate 5-kinase (PIP5K) is a key enzyme producing the signaling lipid phosphatidylinositol 4,5-bisphosphate [PtdIns(4,5)P 2 ] in eukaryotes. Although PIP5K genes are reported to be involved in pollen tube germination and growth, the essential roles of PIP5K in these processes remain unclear. Here, we performed a comprehensive genetic analysis of the Arabidopsis thaliana PIP5K4, PIP5K5, and PIP5K6 genes and revealed that their redundant function is essential for pollen germination. Pollen with the pip5k4pip5k5pip5k6 triple mutation was sterile, while pollen germination efficiency and pollen tube growth were reduced in the pip5k6 single mutant and further reduced in the pip5k4pip5k6 and pip5k5pip5k6 double mutants. YFP-fusion proteins, PIP5K4-YFP, PIP5K5-YFP, and PIP5K6-YFP, which could rescue the sterility of the triple mutant pollen, preferentially localized to the tricolpate aperture area and the future germination site on the plasma membrane prior to germination. Triple mutant pollen grains under the germination condition, in which spatiotemporal localization of the PtdIns(4,5)P 2 fluorescent marker protein 2xmCHERRY-2xPH PLC as seen in the wild type was abolished, exhibited swelling and rupture of the pollen wall, but neither the conspicuous protruding site nor site-specific deposition of cell wall materials for germination. These data indicate that PIP5K4-6 and their product PtdIns(4,5)P 2 are essential for pollen germination, possibly through the establishment of the germination polarity in a pollen grain., (© 2023 Society for Experimental Biology and John Wiley & Sons Ltd.)

Vacuolar H + -ATPases (V-ATPases) are highly conserved multisubunit enzymes that maintain the distinct pH of eukaryotic organelles. The integral membrane a-subunit is encoded by tissue- and organelle-specific isoforms, and its cytosolic N-terminal domain (aNT) modulates organelle-specific regulation and targeting of V-ATPases. Organelle membranes have specific phosphatidylinositol phosphate (PIP) lipid enrichment linked to maintenance of organelle pH. In yeast, the aNT domains of the two a-subunit isoforms bind PIP lipids enriched in the organelle membranes where they reside; these interactions affect activity and regulatory properties of the V-ATPases containing each isoform. Humans have four a-subunit isoforms, and we hypothesize that the aNT domains of these isoforms will also bind to specific PIP lipids. The a1 and a2 isoforms of human V-ATPase a-subunits are localized to endolysosomes and Golgi, respectively. We determined that bacterially expressed Hua1NT and Hua2NT bind specifically to endolysosomal PIP lipids PI(3)P and PI(3,5)P 2 and Golgi enriched PI(4)P, respectively. Despite the lack of canonical PIP-binding sites, we identified potential binding sites in the HuaNT domains by sequence comparisons and existing subunit structures and models. We found that mutations at a similar location in the distal loops of both HuaNT isoforms compromise binding to their cognate PIP lipids, suggesting that these loops encode PIP specificity of the a-subunit isoforms. These data suggest a mechanism through which PIP lipid binding could stabilize and activate V-ATPases in distinct organelles., Competing Interests: Conflict of interest The authors declare that they have no conflicts of interest with the contents of this article., (Copyright © 2023 The Authors. Published by Elsevier Inc. All rights reserved.)

The involvement of the mGlu5 receptors in the pathophysiology of several forms of monogenic autism has been supported by numerous studies following the seminal observation that mGlu5 receptor-dependent long-term depression was enhanced in the hippocampus of mice modeling the fragile-X syndrome (FXS). Surprisingly, there are no studies examining the canonical signal transduction pathway activated by mGlu5 receptors (i.e. polyphosphoinositide - PI - hydrolysis) in mouse models of autism. We have developed a method for in vivo assessment of PI hydrolysis based on systemic injection of lithium chloride followed by treatment with the selective mGlu5 receptor PAM, VU0360172, and measurement of endogenous inositolmonophosphate (InsP) in brain tissue. Here, we report that mGlu5 receptor-mediated PI hydrolysis was blunted in the cerebral cortex, hippocampus, and corpus striatum of Ube3a m-/p+  mice modeling Angelman syndrome (AS), and in the cerebral cortex and hippocampus of Fmr1 knockout mice modeling FXS. In vivo mGlu5 receptor-mediated stimulation of Akt on threonine 308 was also blunted in the hippocampus of FXS mice. These changes were associated with a significant increase in cortical and striatal Homer1 levels and striatal mGlu5 receptor and Gαq levels in AS mice, and with a reduction in cortical mGlu5 receptor and hippocampal Gαq levels, and an increase in cortical phospholipase-Cβ and hippocampal Homer1 levels in FXS mice. This is the first evidence that the canonical transduction pathway activated by mGlu5 receptors is down-regulated in brain regions of mice modeling monogenic autism., Competing Interests: Declaration of competing interest None., (Copyright © 2023 Elsevier Ltd. All rights reserved.)

Myo-inositol belongs to one of the sugar alcohol groups known as cyclitols. Phosphatidylinositols are one of the derivatives of Myo-inositol, and constitute important mediators in many intracellular processes such as cell growth, cell differentiation, receptor recycling, cytoskeletal organization, and membrane fusion. They also have even more functions that are essential for cell survival. Mutations in genes encoding phosphatidylinositols and their derivatives can lead to many disorders. This review aims to perform an in-depth analysis of these connections. Many authors emphasize the significant influence of phosphatidylinositols and phosphatidylinositols' phosphates in the pathogenesis of myotubular myopathies, neurodegenerative disorders, carcinogenesis, and other less frequently observed diseases. In our review, we have focused on three of the most often mentioned groups of disorders. Inositols are the topic of many studies, and yet, there are no clear results of successful clinical trials. Analysis of the available literature gives promising results and shows that further research is still needed.

Phosphatidylinositol 3,5-bisphosphate enables transport of proteins to synaptic sites.

Neurons relay information via specialized presynaptic compartments for neurotransmission. Unlike conventional organelles, the specialized apparatus characterizing the neuronal presynapse must form de novo. How the components for presynaptic neurotransmission are transported and assembled is poorly understood. Our results show that the rare late endosomal signaling lipid phosphatidylinositol 3,5-bisphosphate [PI(3,5)P 2 ] directs the axonal cotransport of synaptic vesicle and active zone proteins in precursor vesicles in human neurons. Precursor vesicles are distinct from conventional secretory organelles, endosomes, and degradative lysosomes and are transported by coincident detection of PI(3,5)P 2 and active ARL8 via kinesin KIF1A to the presynaptic compartment. Our findings identify a crucial mechanism that mediates the delivery of synaptic vesicle and active zone proteins to developing synapses.