Molecular mechanism of ß-arrestin-2 pre-activation by phosphatidylinositol 4,5-bisphosphate.

Phosphorylated residues of G protein-coupled receptors bind to the N-domain of arrestin, resulting in the release of its C-terminus. This induces further allosteric conformational changes, such as polar core disruption, alteration of interdomain loops, and domain rotation, which transform arrestins into the receptor-activated state. It is widely accepted that arrestin activation occurs by conformational changes propagated from the N- to the C-domain. However, recent studies have revealed that binding of phosphatidylinositol 4,5-bisphosphate (PIP2) to the C-domain transforms arrestins into a pre-active state. Here, we aimed to elucidate the mechanisms underlying PIP2-induced arrestin pre-activation. We compare the conformational changes of ß-arrestin-2 upon binding of PIP2 or phosphorylated C-tail peptide of vasopressin receptor type 2 using hydrogen/deuterium exchange mass spectrometry (HDX-MS). Introducing point mutations on the potential routes of the allosteric conformational changes and analyzing these mutant constructs with HDX-MS reveals that PIP2-binding at the C-domain affects the back loop, which destabilizes the gate loop and ßXX to transform ß-arrestin-2 into the pre-active state.

PIP2 Is An Electrostatic Catalyst for Vesicle Fusion by Lowering the Hydration Energy: Arresting Vesicle Fusion by Masking PIP2.

Lipids are key factors in regulating membrane fusion. Lipids are not only structural components to form membranes but also active catalysts for vesicle fusion and neurotransmitter release, which are driven by soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) proteins. SNARE proteins seem to be partially assembled before fusion, but the mechanisms that arrest vesicle fusion before Ca2+ influx are still not clear. Here, we show that phosphatidylinositol 4,5-bisphosphate (PIP2) electrostatically triggers vesicle fusion as an electrostatic catalyst by lowering the hydration energy and that a myristoylated alanine-rich C-kinase substrate (MARCKS), a PIP2-binding protein, arrests vesicle fusion in a vesicle docking state where the SNARE complex is partially assembled. Vesicle-mimicking liposomes fail to reproduce vesicle fusion arrest by masking PIP2, indicating that native vesicles are essential for the reconstitution of physiological vesicle fusion. PIP2 attracts cations to repel water molecules from membranes, thus lowering the hydration energy barrier.

Minor electrostatic changes robustly increase VP40 membrane binding, assembly, and budding of Ebola virus matrix protein derived virus-like particles.

Ebola virus (EBOV) is a filamentous negative-sense RNA virus, which causes severe hemorrhagic fever. There are limited vaccines or therapeutics for prevention and treatment of EBOV, so it is important to get a detailed understanding of the virus lifecycle to illuminate new drug targets. EBOV encodes for the matrix protein, VP40, which regulates assembly and budding of new virions from the inner leaflet of the host cell plasma membrane (PM). In this work, we determine the effects of VP40 mutations altering electrostatics on PM interactions and subsequent budding. VP40 mutations that modify surface electrostatics affect viral assembly and budding by altering VP40 membrane-binding capabilities. Mutations that increase VP40 net positive charge by one (e.g., Gly to Arg or Asp to Ala) increase VP40 affinity for phosphatidylserine and phosphatidylinositol 4,5-bisphosphate in the host cell PM. This increased affinity enhances PM association and budding efficiency leading to more effective formation of virus-like particles. In contrast, mutations that decrease net positive charge by one (e.g., Gly to Asp) lead to a decrease in assembly and budding because of decreased interactions with the anionic PM. Taken together, our results highlight the sensitivity of slight electrostatic changes on the VP40 surface for assembly and budding. Understanding the effects of single amino acid substitutions on viral budding and assembly will be useful for explaining changes in the infectivity and virulence of different EBOV strains, VP40 variants that occur in nature, and for long-term drug discovery endeavors aimed at EBOV assembly and budding.

Regulatory role of cholesterol in modulating actin dynamics and cell adhesive interactions in the trabecular meshwork.

The trabecular meshwork (TM) tissue plays a crucial role in maintaining intraocular pressure (IOP) homeostasis. Increased TM contractility and stiffness are directly correlated with elevated IOP. Although cholesterol is known to be a determinant of glaucoma occurrence and elevated IOP, the underlying mechanisms remain elusive. In this study, we used human TM (HTM) cells to unravel the effects of cholesterol on TM stiffness. We achieved this by performing acute cholesterol depletion with Methyl-ß-cyclodextrin (MßCD) and cholesterol enrichment/replenishment with MßCD cholesterol complex (CHOL). Interestingly, cholesterol depletion triggered notable actin depolymerization and decreased focal adhesion formation, while enrichment/replenishment promoted actin polymerization, requiring the presence of actin monomers. Using a specific reporter of phosphatidylinositol 4,5-bisphosphate (PIP2), we demonstrated that cholesterol depletion decreases PIP2 levels on the cell membrane, whereas enrichment increases them. Given the critical role of PIP2 in actin remodeling and focal adhesion formation, we postulate that cholesterol regulates actin dynamics by modulating PIP2 levels on the membrane. Furthermore, we showed that cholesterol levels regulate integrin α5ß1 and αVß3 distribution and activation, subsequently altering cell-extracellular matrix (ECM) interactions. Notably, the depletion of cholesterol, as a major lipid constituent of the cell membrane, led to a decrease in HTM cell membrane tension, which was reversed upon cholesterol replenishment. Overall, our systematic exploration of cholesterol modulation on TM stiffness highlights the critical importance of maintaining appropriate membrane and cellular cholesterol levels for achieving IOP homeostasis.

Redundant function of the Arabidopsis phosphatidylinositol 4-phosphate 5-kinase genes PIP5K4-6 is essential for pollen germination.

Phosphatidylinositol 4-phosphate 5-kinase (PIP5K) is a key enzyme producing the signaling lipid phosphatidylinositol 4,5-bisphosphate [PtdIns(4,5)P2 ] 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)P2 fluorescent marker protein 2xmCHERRY-2xPHPLC 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)P2 are essential for pollen germination, possibly through the establishment of the germination polarity in a pollen grain.

Epidermal Growth Factor Receptors in Vascular Endothelial Cells Contribute to Functional Hyperemia in the Brain.

Functional hyperemia-activity-dependent increases in local blood perfusion-underlies the on-demand delivery of blood to regions of enhanced neuronal activity, a process that is crucial for brain health. Importantly, functional hyperemia deficits have been linked to multiple dementia risk factors, including aging, chronic hypertension, and cerebral small vessel disease (cSVD). We previously reported crippled functional hyperemia in a mouse model of genetic cSVD that was likely caused by depletion of phosphatidylinositol 4,5-bisphosphate (PIP2) in capillary endothelial cells (EC) downstream of impaired epidermal growth factor receptor (EGFR) signaling. Here, using EC-specific EGFR-knockout (KO) mice, we directly examined the role of endothelial EGFR signaling in functional hyperemia, assessed by measuring increases in cerebral blood flow in response to contralateral whisker stimulation using laser Doppler flowmetry. Molecular characterizations showed that EGFR expression was dramatically decreased in freshly isolated capillaries from EC-EGFR-KO mice, as expected. Notably, whisker stimulation-induced functional hyperemia was significantly impaired in these mice, an effect that was rescued by administration of PIP2, but not by the EGFR ligand, HB-EGF. These data suggest that the deletion of the EGFR specifically in ECs attenuates functional hyperemia, likely via depleting PIP2 and subsequently incapacitating Kir2.1 channel functionality in capillary ECs. Thus, our study underscores the role of endothelial EGFR signaling in functional hyperemia of the brain.

Epidermal growth factor receptors in vascular endothelial cells contribute to functional hyperemia in the brain.

Functional hyperemia - activity-dependent increases in local blood perfusion - underlies the on-demand delivery of blood to regions of enhanced neuronal activity, a process that is crucial for brain health. Importantly, functional hyperemia deficits have been linked to multiple dementia risk factors, including aging, chronic hypertension, and cerebral small vessel disease (cSVD). We previously reported crippled functional hyperemia in a mouse model of genetic cSVD that was likely caused by depletion of phosphatidylinositol 4,5-bisphosphate (PIP2) in capillary endothelial cells (EC) downstream of impaired epidermal growth factor receptor (EGFR) signaling. Here, using EC-specific EGFR-knockout (KO) mice, we directly examined the role of endothelial EGFR signaling in functional hyperemia, assessed by measuring increases in cerebral blood flow in response to contralateral whisker stimulation using laser Doppler flowmetry. Molecular characterizations showed that EGFR expression was dramatically decreased in freshly isolated capillaries from EC-EGFR-KO mice, as expected. Notably, whisker stimulation-induced functional hyperemia was significantly impaired in these mice, an effect that was rescued by exogenous administration of PIP2, but not by the EGFR ligand, HB-EGF. These data suggest that the deletion of the EGFR specifically in ECs depletes PIP2 and attenuates functional hyperemia, underscoring the central role of the endothelial EGFR signaling in cerebral blood flow regulation.

Synaptotagmin 1-triggered lipid signaling facilitates coupling of exo- and endocytosis.

Exocytosis and endocytosis are essential physiological processes and are of prime importance for brain function. Neurotransmission depends on the Ca2+-triggered exocytosis of synaptic vesicles (SVs). In neurons, exocytosis is spatiotemporally coupled to the retrieval of an equal amount of membrane and SV proteins by compensatory endocytosis. How exocytosis and endocytosis are balanced to maintain presynaptic membrane homeostasis and, thereby, sustain brain function is essentially unknown. We combine mouse genetics with optical imaging to show that the SV calcium sensor Synaptotagmin 1 couples exocytic SV fusion to the endocytic retrieval of SV membranes by promoting the local activity-dependent formation of the signaling lipid phosphatidylinositol 4,5-bisphosphate (PI(4,5)P2) at presynaptic sites. Interference with these mechanisms impairs PI(4,5)P2-triggered SV membrane retrieval but not exocytic SV fusion. Our findings demonstrate that the coupling of SV exocytosis and endocytosis involves local Synaptotagmin 1-induced lipid signaling to maintain presynaptic membrane homeostasis in central nervous system neurons.

Regulation of Presynaptic Calcium Channels.

Voltage-gated calcium channels (VGCCs), especially Cav2.1 and Cav2.2, are the major mediators of Ca2+ influx at the presynaptic membrane in response to neuron excitation, thereby exerting a predominant control on synaptic transmission. To guarantee the timely and precise release of neurotransmitters at synapses, the activity of presynaptic VGCCs is tightly regulated by a variety of factors, including auxiliary subunits, membrane potential, G protein-coupled receptors (GPCRs), calmodulin (CaM), Ca2+-binding proteins (CaBP), protein kinases, various interacting proteins, alternative splicing events, and genetic variations.

Quantitative super-resolution microscopy reveals the differences in the nanoscale distribution of nuclear phosphatidylinositol 4,5-bisphosphate in human healthy skin and skin warts.

Introduction: Imaging of human clinical formalin-fixed paraffin-embedded (FFPE) tissue sections provides insights into healthy and diseased states and therefore represents a valuable resource for basic research, as well as for diagnostic and clinical purposes. However, conventional light microscopy does not allow to observe the molecular details of tissue and cell architecture due to the diffraction limit of light. Super-resolution microscopy overcomes this limitation and provides access to the nanoscale details of tissue and cell organization. Methods: Here, we used quantitative multicolor stimulated emission depletion (STED) nanoscopy to study the nanoscale distribution of the nuclear phosphatidylinositol 4,5-bisphosphate (nPI(4,5)P2) with respect to the nuclear speckles (NS) marker SON. Results: Increased nPI(4,5)P2 signals were previously linked to human papillomavirus (HPV)-mediated carcinogenesis, while NS-associated PI(4,5)P2 represents the largest pool of nPI(4,5)P2 visualized by staining and microscopy. The implementation of multicolor STED nanoscopy in human clinical FFPE skin and wart sections allowed us to provide here the quantitative evidence for higher levels of NS-associated PI(4,5)P2 in HPV-induced warts compared to control skin. Discussion: These data expand the previous reports of HPV-induced increase of nPI(4,5)P2 levels and reveal for the first time the functional, tissue-specific localization of nPI(4,5)P2 within NS in clinically relevant samples. Moreover, our approach is widely applicable to other human clinical FFPE tissues as an informative addition to the classical histochemistry.

A Plethora of Functions Condensed into Tiny Phospholipids: The Story of PI4P and PI(4,5)P2.

Phosphoinositides (PIs) are small, phosphorylated lipids that serve many functions in the cell. They regulate endo- and exocytosis, vesicular trafficking, actin reorganization, and cell mobility, and they act as signaling molecules. The most abundant PIs in the cell are phosphatidylinositol-4-monophosphate (PI4P) and phosphatidylinositol-4,5-bisphosphate [PI(4,5)P2]. PI4P is mostly localized at the Golgi apparatus where it regulates the anterograde trafficking from the Golgi apparatus to the plasma membrane (PM), but it also localizes at the PM. On the other hand, the main localization site of PI(4,5)P2 is the PM where it regulates the formation of endocytic vesicles. The levels of PIs are regulated by many kinases and phosphatases. Four main kinases phosphorylate the precursor molecule phosphatidylinositol into PI4P, divided into two classes (PI4KIIα, PI4KIIß, PI4KIIIα, and PI4KIIIß), and three main kinases phosphorylate PI4P to form PI(4,5)P2 (PI4P5KIα, PI4P5KIß, and PI4P5KIγ). In this review, we discuss the localization and function of the kinases that produce PI4P and PI(4,5)P2, as well as the localization and function of their product molecules with an overview of tools for the detection of these PIs.

De novo missense variants in phosphatidylinositol kinase PIP5KIγ underlie a neurodevelopmental syndrome associated with altered phosphoinositide signaling.

Phosphoinositides (PIs) are membrane phospholipids produced through the local activity of PI kinases and phosphatases that selectively add or remove phosphate groups from the inositol head group. PIs control membrane composition and play key roles in many cellular processes including actin dynamics, endosomal trafficking, autophagy, and nuclear functions. Mutations in phosphatidylinositol 4,5 bisphosphate [PI(4,5)P2] phosphatases cause a broad spectrum of neurodevelopmental disorders such as Lowe and Joubert syndromes and congenital muscular dystrophy with cataracts and intellectual disability, which are thus associated with increased levels of PI(4,5)P2. Here, we describe a neurodevelopmental disorder associated with an increase in the production of PI(4,5)P2 and with PI-signaling dysfunction. We identified three de novo heterozygous missense variants in PIP5K1C, which encodes an isoform of the phosphatidylinositol 4-phosphate 5-kinase (PIP5KIγ), in nine unrelated children exhibiting intellectual disability, developmental delay, acquired microcephaly, seizures, visual abnormalities, and dysmorphic features. We provide evidence that the PIP5K1C variants result in an increase of the endosomal PI(4,5)P2 pool, giving rise to ectopic recruitment of filamentous actin at early endosomes (EEs) that in turn causes dysfunction in EE trafficking. In addition, we generated an in vivo zebrafish model that recapitulates the disorder we describe with developmental defects affecting the forebrain, including the eyes, as well as craniofacial abnormalities, further demonstrating the pathogenic effect of the PIP5K1C variants.

Plakophilin-3 Binds the Membrane and Filamentous Actin without Bundling F-Actin.

Plakophilin-3 is a ubiquitously expressed protein found widely in epithelial cells and is a critical component of desmosomes. The plakophilin-3 carboxy-terminal domain harbors nine armadillo repeat motifs with largely unknown functions. Here, we report the 5 Å cryogenic electron microscopy (cryoEM) structure of the armadillo repeat motif domain of plakophilin-3, one of the smaller cryoEM structures reported to date. We find that this domain is a monomer or homodimer in solution. In addition, using an in vitro actin co-sedimentation assay, we show that the armadillo repeat domain of plakophilin-3 directly interacts with F-actin. This feature, through direct interactions with actin filaments, could be responsible for the observed association of extra-desmosomal plakophilin-3 with the actin cytoskeleton directly attached to the adherens junctions in A431 epithelial cells. Further, we demonstrate, through lipid binding analyses, that plakophilin-3 can effectively be recruited to the plasma membrane through phosphatidylinositol-4,5-bisphosphate-mediated interactions. Collectively, we report on novel properties of plakophilin-3, which may be conserved throughout the plakophilin protein family and may be behind the roles of these proteins in cell-cell adhesion.

Activation Mechanisms and Diverse Functions of Mammalian Phospholipase C.

Phospholipase C (PLC) plays pivotal roles in regulating various cellular functions by metabolizing phosphatidylinositol 4,5-bisphosphate in the plasma membrane. This process generates two second messengers, inositol 1,4,5-trisphosphate and diacylglycerol, which respectively regulate the intracellular Ca2+ levels and protein kinase C activation. In mammals, six classes of typical PLC have been identified and classified based on their structure and activation mechanisms. They all share X and Y domains, which are responsible for enzymatic activity, as well as subtype-specific domains. Furthermore, in addition to typical PLC, atypical PLC with unique structures solely harboring an X domain has been recently discovered. Collectively, seven classes and 16 isozymes of mammalian PLC are known to date. Dysregulation of PLC activity has been implicated in several pathophysiological conditions, including cancer, cardiovascular diseases, and neurological disorders. Therefore, identification of new drug targets that can selectively modulate PLC activity is important. The present review focuses on the structures, activation mechanisms, and physiological functions of mammalian PLC.

Oculocerebrorenal syndrome of Lowe (OCRL) controls leukemic T-cell survival by preventing excessive PI(4,5)P2 hydrolysis in the plasma membrane.

T-cell acute lymphoblastic leukemia (T-ALL) is one of the deadliest and most aggressive hematological malignancies, but its pathological mechanism in controlling cell survival is not fully understood. Oculocerebrorenal syndrome of Lowe is a rare X-linked recessive disorder characterized by cataracts, intellectual disability, and proteinuria. This disease has been shown to be caused by mutation of oculocerebrorenal syndrome of Lowe 1 (OCRL1; OCRL), encoding a phosphatidylinositol 4,5-bisphosphate [PI(4,5)P2] 5-phosphatase involved in regulating membrane trafficking; however, its function in cancer cells is unclear. Here, we uncovered that OCRL1 is overexpressed in T-ALL cells, and knockdown of OCRL1 results in cell death, indicating the essential role of OCRL in controlling T-ALL cell survival. We show OCRL is primarily localized in the Golgi and can translocate to plasma membrane (PM) upon ligand stimulation. We found OCRL interacts with oxysterol-binding protein-related protein 4L, which facilitates OCRL translocation from the Golgi to the PM upon cluster of differentiation 3 stimulation. Thus, OCRL represses the activity of oxysterol-binding protein-related protein 4L to prevent excessive PI(4,5)P2 hydrolysis by phosphoinositide phospholipase C ß3 and uncontrolled Ca2+ release from the endoplasmic reticulum. We propose OCRL1 deletion leads to accumulation of PI(4,5)P2 in the PM, disrupting the normal Ca2+ oscillation pattern in the cytosol and leading to mitochondrial Ca2+ overloading, ultimately causing T-ALL cell mitochondrial dysfunction and cell death. These results highlight a critical role for OCRL in maintaining moderate PI(4,5)P2 availability in T-ALL cells. Our findings also raise the possibility of targeting OCRL1 to treat T-ALL disease.

Familial CCM Genes Might Not Be Main Drivers for Pathogenesis of Sporadic CCMs-Genetic Similarity between Cancers and Vascular Malformations.

Cerebral cavernous malformations (CCMs) are abnormally dilated intracranial capillaries that form cerebrovascular lesions with a high risk of hemorrhagic stroke. Recently, several somatic "activating" gain-of-function (GOF) point mutations in PIK3CA (phosphatidylinositol-4, 5-bisphosphate 3-kinase catalytic subunit p110α) were discovered as a dominant mutation in the lesions of sporadic forms of cerebral cavernous malformation (sCCM), raising the possibility that CCMs, like other types of vascular malformations, fall in the PIK3CA-related overgrowth spectrum (PROS). However, this possibility has been challenged with different interpretations. In this review, we will continue our efforts to expound the phenomenon of the coexistence of gain-of-function (GOF) point mutations in the PIK3CA gene and loss-of-function (LOF) mutations in CCM genes in the CCM lesions of sCCM and try to delineate the relationship between mutagenic events with CCM lesions in a temporospatial manner. Since GOF PIK3CA point mutations have been well studied in reproductive cancers, especially breast cancer as a driver oncogene, we will perform a comparative meta-analysis for GOF PIK3CA point mutations in an attempt to demonstrate the genetic similarities shared by both cancers and vascular anomalies.

Knockdown of MEF2D inhibits the development and progression of B-cell acute lymphoblastic leukemia.

Background: Myocyte enhancer factor 2D (MEF2D) is involved in the progression of various malignant tumors. However, its impact on B-cell acute lymphoblastic leukemia (B-ALL) has not been elucidated. Methods: In this study, the expression level of MEF2D in B-ALL patients was validated through the Gene Expression Omnibus (GEO) database and clinical specimens. MEF2D-knockdown B-ALL cell lines were constructed by lentivirus transfection, and the effects of MEF2D on the viability, apoptosis, cycle progression, and drug sensitivity of B-ALL cells were verified by Cell Counting Kit-8 (CCK-8) and flow cytometry (FCM). The effect of MEF2D on the proliferation of B-ALL cells in vivo was verified via the construction of a xenograft mouse model. The mechanism of MEF2D regulating B-ALL cells was explored by RNA sequencing analysis, quantitative reverse transcription polymerase chain reaction (qRT-PCR), western blotting, and immunohistochemical (IHC). Results: In this study, overexpression of MEF2D was observed in B-ALL patients and was remarkably correlated to disease progression in ALL patients. The knockdown of MEF2D expression suppressed cell viability, induced cell apoptosis, blockaded cell cycle progression, enhanced drug sensitivity of B-ALL cells in vitro, and reduced the tumor load in vivo. Furthermore, mechanistic studies revealed that MEF2D knockdown downregulated the expression of the phosphatidylinositol-4,5-bisphosphate 3-kinase (PI3K)-protein kinase B (AKT) signaling pathway. Conclusions: Our research demonstrated that MEF2D was markedly expressed in B-ALL. MEF2D knockdown inhibited cancer progression of B-ALL both in vitro and in vivo, which may be related to the downregulation of the PI3K-AKT signaling pathway. The data suggest that MEF2D plays a vital role in the process of tumorigenesis and may be a potential novel target for B-ALL therapy.

Role of PI(4,5)P2 and Cholesterol in Unconventional Protein Secretion.

Besides its protective role in the maintenance of cell homeostasis, the plasma membrane is the site of exchanges between the cell interior and the extracellular medium. To circumvent the hydrophobic barrier formed by the acyl chains of the lipid bilayer, protein channels and transporters are key players in the exchange of small hydrophilic compounds such as ions or nutrients, but they hardly account for the transport of larger biological molecules. Exchange of proteins usually relies on membrane-fusion events between vesicles and the plasma membrane. In recent years, several alternative unconventional protein secretion (UPS) pathways across the plasma membrane have been characterised for a specific set of secreted substrates, some of them excluding any membrane-fusion events (Dimou and Nickel, Curr Biol 28:R406-R410, 2018). One of thesbe pathways, referred as type I UPS, relies on the direct translocation of the protein across the plasma membrane and not surprisingly, lipids are essential players in this process. In this chapter, we discuss the roles of phosphatidylinositol(4,5)bisphosphate (PI(4,5)P2) and cholesterol in unconventional pathways involving Engrailed-2 homeoprotein and fibroblast growth factor 2.

Roles of Phosphatidylinositol 4-Phosphorylation in Non-vesicular Cholesterol Trafficking.

Cholesterol (Chol) is an essential component of all eukaryotic cell membranes that affects the function of numerous peripheral as well as integral membrane proteins. Chol is synthesized in the ER, but it is selectively enriched within the plasma membrane (PM) and other endomembranes, which requires Chol to cross the aqueous phase of the cytoplasm. In addition to the classical vesicular trafficking pathways that are known to facilitate the bulk transport of membrane intermediates, Chol is also transported via non-vesicular lipid transfer proteins that work primarily within specialized membrane contact sites. Some of these transport pathways work against established concentration gradients and hence require energy. Recent studies highlight the unique role of phosphoinositides (PPIns), and phosphatidylinositol 4-phosphate (PI4P) in particular, for the control of non-vesicular Chol transport. In this chapter, we will review the emerging connection between Chol, PPIns, and lipid transfer proteins that include the important family of oxysterol-binding protein related proteins, or ORPs.

PI(4,5)P2 and Cholesterol: Synthesis, Regulation, and Functions.

Phosphatidylinositol 4,5-bisphosphate (PI(4,5)P2) is the most abundant membrane phosphoinositide and cholesterol is an essential component of the plasma membrane (PM). Both lipids play key roles in a variety of cellular functions including as signaling molecules and major regulators of protein function. This chapter provides an overview of these two important lipids. Starting from a brief description of their structure, synthesis, and regulation, the chapter continues to describe the primary functions and signaling processes in which PI(4,5)P2 and cholesterol are involved. While PI(4,5)P2 and cholesterol can act independently, they often act in concert or affect each other's impact. The chapters in this volume on "Cholesterol and PI(4,5)P2 in Vital Biological Functions: From Coexistence to Crosstalk" focus on the emerging relationship between cholesterol and PI(4,5)P2 in a variety of biological systems and processes. In this chapter, the next section provides examples from the ion channel field demonstrating that PI(4,5)P2 and cholesterol can act via common mechanisms. The chapter ends with a discussion of future directions.