Impacts of the Covid-19 pandemic on the health of university students.

The Covid-19 pandemic caused by the novel Sars-CoV-2 coronavirus, has resulted in millions of deaths and disruption to daily life across the globe. University students have been additionally affected by a sudden move to online learning, the closure of campuses and dramatic societal changes that have upended their experiences of higher education. Here we focus on the physical and mental health consequences of the pandemic for this population sector during 2020, and the interdependencies of these impacts. We survey the challenges for infection control on campuses and for monitoring the disease dynamics in student communities. Finally, we explore the psychological and mental health problems that have been exacerbated by the pandemic and evaluate the underlying factors that are most relevant to students.

Novel defect in phosphatidylinositol 4-kinase type 2-alpha (PI4K2A) at the membrane-enzyme interface is associated with metabolic cutis laxa.

Inherited cutis laxa, or inelastic, sagging skin is a genetic condition of premature and generalised connective tissue ageing, affecting various elastic components of the extracellular matrix. Several cutis laxa syndromes are inborn errors of metabolism and lead to severe neurological symptoms. In a patient with cutis laxa, a choreoathetoid movement disorder, dysmorphic features and intellectual disability we performed exome sequencing to elucidate the underlying genetic defect. We identified the amino acid substitution R275W in phosphatidylinositol 4-kinase type IIα, caused by a homozygous missense mutation in the PI4K2A gene. We used lipidomics, complexome profiling and functional studies to measure phosphatidylinositol 4-phosphate synthesis in the patient and evaluated PI4K2A deficient mice to define a novel metabolic disorder. The R275W residue, located on the surface of the protein, is involved in forming electrostatic interactions with the membrane. The catalytic activity of PI4K2A in patient fibroblasts was severely reduced and lipid mass spectrometry showed that particular acyl-chain pools of PI4P and PI(4,5)P2 were decreased. Phosphoinositide lipids play a major role in intracellular signalling and trafficking and regulate the balance between proliferation and apoptosis. Phosphatidylinositol 4-kinases such as PI4K2A mediate the first step in the main metabolic pathway that generates PI4P, PI(4,5)P2 and PI(3,4,5)P3 . Although neurologic involvement is common, cutis laxa has not been reported previously in metabolic defects affecting signalling. Here we describe a patient with a complex neurological phenotype, premature ageing and a mutation in PI4K2A, illustrating the importance of this enzyme in the generation of inositol lipids with particular acylation characteristics.

Immunohistochemical staining reveals differential expression of ACSL3 and ACSL4 in hepatocellular carcinoma and hepatic gastrointestinal metastases.

Long-chain fatty acyl CoA synthetases (ACSLs) activate fatty acids by CoA addition thus facilitating their intracellular metabolism. Dysregulated ACSL expression features in several cancers and can affect processes such as ferroptosis, fatty acid ß-oxidation, prostaglandin biosynthesis, steroidogenesis and phospholipid acyl chain remodelling. Here we investigate long chain acyl-CoA synthetase 3 (ACSL3) and long chain acyl-CoA synthetase 4 (ACSL4) expression in liver malignancies. The expression and subcellular localisations of the ACSL3 and ACSL4 isoforms in hepatocellular carcinoma (HCC), cholangiocarcinoma (CCA) and hepatic metastases were assessed by immunohistochemical analyses of multiple tumour tissue arrays and by subcellular fractionation of cultured HepG2 cells. The expression of both enzymes was increased in HCC compared with normal liver. Expression of ACSL3 was similar in HCC and hepatic metastases but lower in healthy tissue. Increased ACSL3 expression distinguished HCC from CCA with a sensitivity of 87.2% and a specificity of 75%. ACSL4 expression was significantly greater in HCC than in all other tumours and distinguished HCC from normal liver tissue with a sensitivity of 93.8% and specificity of 93.6%. Combined ACSL3 and ACSL4 staining scores distinguished HCC from hepatic metastases with 80.1% sensitivity and 77.1% specificity. These enzymes had partially overlapping intracellular distributions, ACSL4 localised to the plasma membrane and both isoforms associated with lipid droplets and the endoplasmic reticulum (ER). In conclusion, analysis of ACSL3 and ACSL4 expression can distinguish different classes of hepatic tumours.

The Great Escape: how phosphatidylinositol 4-kinases and PI4P promote vesicle exit from the Golgi (and drive cancer).

Phosphatidylinositol 4-phosphate (PI4P) is a membrane glycerophospholipid and a major regulator of the characteristic appearance of the Golgi complex as well as its vesicular trafficking, signalling and metabolic functions. Phosphatidylinositol 4-kinases, and in particular the PI4KIIIß isoform, act in concert with PI4P to recruit macromolecular complexes to initiate the biogenesis of trafficking vesicles for several Golgi exit routes. Dysregulation of Golgi PI4P metabolism and the PI4P protein interactome features in many cancers and is often associated with tumour progression and a poor prognosis. Increased expression of PI4P-binding proteins, such as GOLPH3 or PITPNC1, induces a malignant secretory phenotype and the release of proteins that can remodel the extracellular matrix, promote angiogenesis and enhance cell motility. Aberrant Golgi PI4P metabolism can also result in the impaired post-translational modification of proteins required for focal adhesion formation and cell-matrix interactions, thereby potentiating the development of aggressive metastatic and invasive tumours. Altered expression of the Golgi-targeted PI 4-kinases, PI4KIIIß, PI4KIIα and PI4KIIß, or the PI4P phosphate Sac1, can also modulate oncogenic signalling through effects on TGN-endosomal trafficking. A Golgi trafficking role for a PIP 5-kinase has been recently described, which indicates that PI4P is not the only functionally important phosphoinositide at this subcellular location. This review charts new developments in our understanding of phosphatidylinositol 4-kinase function at the Golgi and how PI4P-dependent trafficking can be deregulated in malignant disease.

The endogenous subcellular localisations of the long chain fatty acid-activating enzymes ACSL3 and ACSL4 in sarcoma and breast cancer cells.

Fatty acid uptake and metabolism are often dysregulated in cancer cells. Fatty acid activation is a critical step that allows these biomolecules to enter cellular metabolic pathways such as mitochondrial ß-oxidation for ATP generation or the lipogenic routes that generate bioactive lipids such as the inositol phospholipids. Fatty acid activation by the addition of coenzyme A is catalysed by a family of enzymes called the acyl CoA synthetase ligases (ACSL). Furthermore, enhanced expression of particular ACSL isoforms, such as ACSL4, is a feature of some more aggressive cancers and may contribute to the oncogenic phenotype. This study focuses on ACSL3 and ACSL4, closely related structural homologues that preferentially activate palmitate and arachidonate fatty acids, respectively. In this study, immunohistochemical screening of multiple soft tissue tumour arrays revealed that ACSL3 and ACSL4 were highly, but differentially, expressed in a subset of leiomyosarcomas, fibrosarcomas and rhabdomyosarcomas, with consistent cytoplasmic and granular stainings of tumour cells. The intracellular localisations of endogenously expressed ACSL3 and ACSL4 were further investigated by detailed subcellular fractionation analyses of HT1080 fibrosarcoma and MCF-7 breast cancer cells. ACSL3 distribution closely overlapped with proteins involved in trafficking from the trans-Golgi network and endosomes. In contrast, the ACSL4 localisation pattern more closely followed that of calnexin which is an  endoplasmic reticulum resident chaperone. Confocal immunofluorescence imaging of MCF-7 cells confirmed the intracellular localisations of both enzymes. These observations reveal new information regarding the compartmentation of fatty acid metabolism in cancer cells.

Phosphatidylinositol 4-kinase IIß negatively regulates invadopodia formation and suppresses an invasive cellular phenotype.

The type II phosphatidylinositol 4-kinase (PI4KII) enzymes synthesize the lipid phosphatidylinositol 4-phosphate (PI(4)P), which has been detected at the Golgi complex and endosomal compartments and recruits clathrin adaptors. Despite common mechanistic similarities between the isoforms, the extent of their redundancy is unclear. We found that depletion of PI4KIIα and PI4KIIß using small interfering RNA led to actin remodeling. Depletion of PI4KIIß also induced the formation of invadopodia containing membrane type I matrix metalloproteinase (MT1-MMP). Depletion of PI4KII isoforms also differentially affected trans-Golgi network (TGN) pools of PI(4)P and post-TGN traffic. PI4KIIß depletion caused increased MT1-MMP trafficking to invasive structures at the plasma membrane and was accompanied by reduced colocalization of MT1-MMP with membranes containing the endosomal markers Rab5 and Rab7 but increased localization with the exocytic Rab8. Depletion of PI4KIIß was sufficient to confer an aggressive invasive phenotype on minimally invasive HeLa and MCF-7 cell lines. Mining oncogenomic databases revealed that loss of the PI4K2B allele and underexpression of PI4KIIß mRNA are associated with human cancers. This finding supports the cell data and suggests that PI4KIIß may be a clinically significant suppressor of invasion. We propose that PI4KIIß synthesizes a pool of PI(4)P that maintains MT1-MMP traffic in the degradative pathway and suppresses the formation of invadopodia.

Assay for CDP-Diacylglycerol Generation by CDS in Membrane Fractions.

CDP-DAG is a liponucleotide formed by the condensation of CTP with the phospholipid phosphatidic acid in a reaction catalyzed by CDP-DAG synthase (CDS). CDP-DAG is required for the synthesis of phosphatidylinositol; the parent molecule whence all seven phosphoinositides including the signaling molecules PI4P, PI(4,5)P2, and PI(3,4,5)P3 are derived. This protocol describes a highly sensitive radiometric assay to detect the generation of CDP-DAG on isolated biological membrane fractions.

Chromosomal Instability and Phosphoinositide Pathway Gene Signatures in Glioblastoma Multiforme.

Structural rearrangements of chromosome 10 are frequently observed in glioblastoma multiforme and over 80 % of tumour samples archived in the catalogue of somatic mutations in cancer database had gene copy number loss for PI4K2A which encodes phosphatidylinositol 4-kinase type IIalpha. PI4K2A loss of heterozygosity mirrored that of PTEN, another enzyme that regulates phosphoinositide levels and also PIK3AP1, MINPP1, INPP5A and INPP5F. These results indicated a reduction in copy number for a set of phosphoinositide signalling genes that co-localise to chromosome 10q. This analysis was extended to a panel of phosphoinositide pathway genes on other chromosomes and revealed a number of previously unreported associations with glioblastoma multiforme. Of particular note were highly penetrant copy number losses for a group of X-linked phosphoinositide phosphatase genes OCRL, MTM1 and MTMR8; copy number amplifications for the chromosome 19 genes PIP5K1C, AKT2 and PIK3R2, and also for the phospholipase C genes PLCB1, PLCB4 and PLCG1 on chromosome 20. These mutations are likely to affect signalling and trafficking functions dependent on the PI(4,5)P2, PI(3,4,5)P3 and PI(3,5)P2 lipids as well as the inositol phosphates IP3, IP5 and IP6. Analysis of flanking genes with functionally unrelated products indicated that chromosomal instability as opposed to a phosphoinositide-specific process underlay this pattern of copy number variation. This in silico study suggests that in glioblastoma multiforme, karyotypic changes have the potential to cause multiple abnormalities in sets of genes involved in phosphoinositide metabolism and this may be important for understanding drug resistance and phosphoinositide pathway redundancy in the advanced disease state.

Modeling the effects of cyclodextrin on intracellular membrane vesicles from Cos-7 cells prepared by sonication and carbonate treatment.

Cholesterol has important functions in the organization of membrane structure and this may be mediated via the formation of cholesterol-rich, liquid-ordered membrane microdomains often referred to as lipid rafts. Methyl-beta-cyclodextrin (cyclodextrin) is commonly used in cell biology studies to extract cholesterol and therefore disrupt lipid rafts. However, in this study we reassessed this experimental strategy and investigated the effects of cyclodextrin on the physical properties of sonicated and carbonate-treated intracellular membrane vesicles isolated from Cos-7 fibroblasts. We treated these membranes, which mainly originate from the trans-Golgi network and endosomes, with cyclodextrin and measured the effects on their equilibrium buoyant density, protein content, represented by the palmitoylated protein phosphatidylinositol 4-kinase type IIα, and cholesterol. Despite the reduction in mass stemming from cholesterol removal, the vesicles became denser, indicating a possible large volumetric decrease, and this was confirmed by measurements of hydrodynamic vesicle size. Subsequent mathematical analyses demonstrated that only half of this change in membrane size was attributable to cholesterol loss. Hence, the non-selective desorption properties of cyclodextrin are also involved in membrane size and density changes. These findings may have implications for preceding studies that interpreted cyclodextrin-induced changes to membrane biochemistry in the context of lipid raft disruption without taking into account our finding that cyclodextrin treatment also reduces membrane size.

PIPs in neurological diseases.

Phosphoinositide (PIP) lipids regulate many aspects of cell function in the nervous system including receptor signalling, secretion, endocytosis, migration and survival. Levels of PIPs such as PI4P, PI(4,5)P2 and PI(3,4,5)P3 are normally tightly regulated by phosphoinositide kinases and phosphatases. Deregulation of these biochemical pathways leads to lipid imbalances, usually on intracellular endosomal membranes, and these changes have been linked to a number of major neurological diseases including Alzheimer's, Parkinson's, epilepsy, stroke, cancer and a range of rarer inherited disorders including brain overgrowth syndromes, Charcot-Marie-Tooth neuropathies and neurodevelopmental conditions such as Lowe's syndrome. This article analyses recent progress in this area and explains how PIP lipids are involved, to varying degrees, in almost every class of neurological disease. This article is part of a Special Issue entitled Brain Lipids.

Amplification of Chromosome 1q Genes Encoding the Phosphoinositide Signalling Enzymes PI4KB, AKT3, PIP5K1A and PI3KC2B in Breast Cancer.

Little is known about the possible oncogenic roles of genes encoding for the phosphatidylinositol 4-kinases, a family of enzymes that regulate an early step in phosphoinositide signalling. To address this issue, the mutational status of all four human phosphatidylinositol 4-kinases genes was analyzed across 852 breast cancer samples using the COSMIC data resource. Point mutations in the phosphatidylinositol 4-kinase genes were uncommon and appeared in less than 1% of the patient samples however, 62% of the tumours had increases in gene copy number for PI4KB which encodes the phosphatidylinositol 4-kinase IIIbeta isozyme. Extending this analysis to subsequent enzymes in the phosphoinositide signalling cascades revealed that the only PIP5K1A, PI3KC2B and AKT3 genes exhibited similar patterns of gene copy number variation. By comparison, gene copy number increases for established oncogenes such as EGFR and HER2/Neu were only evident in 20% of the samples. The PI4KB, PIP5K1A, PI3KC2B and AKT3 genes are related in that they all localize to chromosome 1q which is often structurally and numerically abnormal in breast cancer. These results demonstrate that a gene quartet encoding a potential phosphoinositide signalling pathway is amplified in a subset of breast cancers.

Raft-like membranes from the trans-Golgi network and endosomal compartments.

This article describes a procedure to prepare a raft-like intracellular membrane fraction enriched for the trans-Golgi network (TGN) and endosomal compartments. The initial step in this technique involves cell disruption by homogenization, followed by clearance of the plasma membrane, late endosomes, mitochondria and the endoplasmic reticulum by differential sedimentation. Carbonate treatment, sonication and sucrose density-gradient ultracentrifugation are subsequently used to isolate the target membranes. The isolated subcellular fraction contains less than 1% of the total cellular proteins, but it is highly enriched for syntaxin-6 and Rab11. Typically, 40-60% of the cellular pool of GM1 glycosphingolipid and 10-20% of the total cellular cholesterol cofractionate with this buoyant membrane fraction. Given the role of GM1 as a cell-surface receptor for the cholera toxin and that levels of both GM1 and cholesterol in the TGN-endosomal compartment are upregulated in some inherited diseases, this protocol can potentially be applied to the analysis of disease-associated changes to GM1-enriched intracellular membranes. The isolated membranes are very well separated from caveolin-rich domains of the plasma membrane, the TGN and recycling endosomes. The entire protocol can be completed in as little as 1 d.

Mammalian phosphatidylinositol 4-kinases as modulators of membrane trafficking and lipid signaling networks.

The four mammalian phosphatidylinositol 4-kinases modulate inter-organelle lipid trafficking, phosphoinositide signalling and intracellular vesicle trafficking. In addition to catalytic domains required for the synthesis of PI4P, the phosphatidylinositol 4-kinases also contain isoform-specific structural motifs that mediate interactions with proteins such as AP-3 and the E3 ubiquitin ligase Itch, and such structural differences determine isoform-specific roles in membrane trafficking. Moreover, different permutations of phosphatidylinositol 4-kinase isozymes may be required for a single cellular function such as occurs during distinct stages of GPCR signalling and in Golgi to lysosome trafficking. Phosphatidylinositol 4-kinases have recently been implicated in human disease. Emerging paradigms include increased phosphatidylinositol 4-kinase expression in some cancers, impaired functioning associated with neurological pathologies, the subversion of PI4P trafficking functions in bacterial infection and the activation of lipid kinase activity in viral disease. We discuss how the diverse and sometimes overlapping functions of the phosphatidylinositol 4-kinases present challenges for the design of isoform-specific inhibitors in a therapeutic context.

Phosphatidylinositol 4-kinases and PI4P metabolism in the nervous system: roles in psychiatric and neurological diseases.

The four mammalian phosphatidylinositol 4-kinases, together with the PI(4,5)P(2) depleting 5-phosphatases of the oculocerebrorenal syndrome of Lowe and synaptojanin families, modulate neuronal pools of PI4P lipid and regulate intracellular membrane trafficking in the endocytic and secretory pathways. Dysfunctions in these enzymes have been associated with a broad spectrum of disorders including schizophrenia, bipolar disorder, Lowe syndrome, age-related neurodegeneration, Alzheimer's disease and Down syndrome. Recent work has shown that reduced expression of individual phosphatidylinositol 4-kinase isozymes is associated with impaired survival of specific neuronal populations within the CNS. Furthermore, alterations to the concentrations of different phosphoinositide lipid species in the brain and, in particular, the ratio of PI4P to PI(4,5)P(2) can have deleterious effects on clathrin-dependent membrane trafficking both in the Golgi-endosomal pathway and at the plasma membrane. In this article, we focus on the cell biology, biochemistry and neuronal functions of the phosphatidylinositol 4-kinases and their emerging roles in psychiatric and neurological pathologies.

Phosphatidylinositol 4-kinases, phosphatidylinositol 4-phosphate and cancer.

This article focuses on the emerging roles for phosphatidylinositol 4-phosphate and the phosphatidylinositol 4-kinases in cancer. Phosphatidylinositol 4-phosphate is a common substrate for both the phosphatidylinositol 3-kinase and phospholipase C pathways, and has been implicated in the membrane targeting of proteins such as Girdin/GIV and OSBP. Alterations to phosphatidylinositol 4-kinase expression levels can modulate MAP kinase and Akt signalling, and are important for chemoresistance, tumour angiogenesis and the suppression of apoptosis and metastases. Recent improvements in high-throughput screening assays, and the discoveries that some anti-viral molecules are isoform selective phosphatidylinositol 4-kinase inhibitors have advanced the drugability of these enzymes.

Lipid rafts, microdomain heterogeneity and inter-organelle contacts: impacts on membrane preparation for proteomic studies.

In recent years, there has been considerable interest in mapping the protein content of isolated organelles using mass spectrometry. However, many subcellular compartments are highly dynamic with diverse and intricate architectures that are not always preserved during membrane isolation procedures. Furthermore, lateral heterogeneities in intra-membrane lipid and protein concentrations underlie the formation of membrane microdomains, trafficking vesicles and inter-membrane contacts. These complexities in membrane organisation have important consequences for the design of membrane preparation strategies and test the very concept of organelle purity. We illustrate how some of these biological considerations are relevant to membrane preparation and assess the numerous potential pitfalls in attempting to purify organelles from mammalian cells.

The phosphatidylinositol 4-kinases: don't call it a comeback.

Phosphatidylinositol 4-phosphate (PtdIns4P) is a quantitatively minor membrane phospholipid which is the precursor of PtdIns(4,5)P (2) in the classical agonist-regulated phospholipase C signalling pathway. However, PtdIns4P also governs the recruitment and function of numerous trafficking molecules, principally in the Golgi complex. The majority of phosphoinositides (PIs) phosphorylated at the D4 position of the inositol headgroup are derived from PtdIns4P and play roles in a diverse array of fundamental cellular processes including secretion, cell migration, apoptosis and mitogenesis; therefore, PtdIns4P biosynthesis can be regarded as key point of regulation in many PI-dependent processes.Two structurally distinct sequence families, the type II and type III PtdIns 4-kinases, are responsible for PtdIns4P synthesis in eukaryotic organisms. These important proteins are differentially expressed, localised and regulated by distinct mechanisms, indicating that the enzymes perform non-redundant roles in trafficking and signalling. In recent years, major advances have been made in our understanding of PtdIns4K biology and here we summarise current knowledge of PtdIns4K structure, function and regulation.

CDP-diacylglycerol phospholipid synthesis in detergent-soluble, non-raft, membrane microdomains of the endoplasmic reticulum.

Phosphatidylinositol (PI) is essential for numerous cell functions and is generated by consecutive reactions catalyzed by CDP-diacylglycerol synthase (CDS) and PI synthase. In this study, we investigated the membrane organization of CDP-diacylglycerol synthesis. Separation of mildly disrupted A431 cell membranes on sucrose density gradients revealed cofractionation of CDS and PI synthase activities with cholesterol-poor, endoplasmic reticulum (ER) membranes and partial overlap with plasma membrane caveolae. Cofractionation of CDS activity with caveolae was also observed when low-buoyant density caveolin-enriched membranes were prepared using a carbonate-based method. However, immunoisolation studies determined that CDS activity localized to ER membrane fragments containing calnexin and type III inositol (1,4,5)-trisphosphate receptors but not to caveolae. Membrane fragmentation in neutral pH buffer established that CDP-diacylglycerol and PI syntheses were restricted to a subfraction of the calnexin-positive ER. In contrast to lipid rafts enriched for caveolin, cholesterol, and GM1 glycosphingolipids, the CDS-containing ER membranes were detergent soluble. In cell imaging studies, CDS and calnexin colocalized in microdomain-sized patches of the ER and also unexpectedly at the plasma membrane. These results demonstrate that key components of the PI pathway localize to nonraft, phospholipid-synthesizing microdomains of the ER that are also enriched for calnexin.

Detergent-free isolation and characterization of cholesterol-rich membrane domains from trans-Golgi network vesicles.

Cholesterol is an abundant lipid of the trans-Golgi network (TGN) and of certain endosomal membranes where cholesterol-rich microdomains are important in the organization and compartmentalization of vesicular trafficking. Here we describe the development of a rapid method to isolate a cholesterol-rich endomembrane fraction. We show that widely used subcellular fractionation techniques incompletely separate cholesterol-rich membranes, such as the TGN, from organelles, such as late endosomes and lysosomes. To address this issue, we devised a new subcellular fractionation scheme involving two rounds of velocity centrifugation, membrane sonication, and discontinuous sucrose density gradient centrifugation. This strategy resulted in the isolation of a cholesterol and GM1 glycosphingolipid-enriched membrane fraction that was completely cleared of plasma membrane, endoplasmic reticulum, and mitochondria. This buoyant fraction was enriched for the TGN and recycling endosome proteins Rab11 and syntaxin-6, and it was well resolved from cis-Golgi and early and late endosomal membranes. We demonstrate that this technique can give useful insights into the compartmentation of phosphoinositide synthesis, and it facilitates the isolation of cholesterol-rich membranes from a population of TGN-trafficking vesicles.

Relationship between phosphatidylinositol 4-phosphate synthesis, membrane organization, and lateral diffusion of PI4KIIalpha at the trans-Golgi network.

Type II phosphatidylinositol 4-kinase IIalpha (PI4KIIalpha) is the dominant phosphatidylinositol kinase activity measured in mammalian cells and has important functions in intracellular vesicular trafficking. Recently PI4KIIalpha has been shown to have important roles in neuronal survival and tumorigenesis. This study focuses on the relationship between membrane cholesterol levels, phosphatidylinositol 4-phosphate (PI4P) synthesis, and PI4KIIalpha mobility. Enzyme kinetic measurements, sterol substitution studies, and membrane fragmentation analyses all revealed that cholesterol regulates PI4KIIalpha activity indirectly through effects on membrane structure. In particular, we found that cholesterol levels determined the distribution of PI4KIIalpha to biophysically distinct membrane domains. Imaging studies on cells expressing enhanced green fluorescent protein (eGFP)-tagged PI4KIIalpha demonstrated that cholesterol depletion resulted in morphological changes to the juxtanuclear membrane pool of the enzyme. Lateral membrane diffusion of eGFP-PI4KIIalpha was assessed by fluorescence recovery after photobleaching (FRAP) experiments, which revealed the existence of both mobile and immobile pools of the enzyme. Sterol depletion decreased the size of the mobile pool of PI4KIIalpha. Further measurements revealed that the reduction in the mobile fraction of PI4KIIalpha correlated with a loss of trans-Golgi network (TGN) membrane connectivity. We conclude that cholesterol modulates PI4P synthesis through effects on membrane organization and enzyme diffusion.