Phosphatidylserine enrichment in the nuclear membrane regulates key enzymes of phosphatidylcholine synthesis.

Phosphatidylserine (PS) is an important anionic phospholipid that is synthesized within the endoplasmic reticulum (ER). While PS shows the highest enrichment and serves important functional roles in the plasma membrane (PM) but its role in the nucleus is poorly explored. Using three orthogonal approaches, we found that PS is also uniquely enriched in the inner nuclear membrane (INM) and the nuclear reticulum (NR). Nuclear PS is critical for supporting the translocation of CCTα and Lipin1α, two key enzymes important for phosphatidylcholine (PC) biosynthesis, from the nuclear matrix to the INM and NR in response to oleic acid treatment. We identified the PS-interacting regions within the M-domain of CCTα and M-Lip domain of Lipin1α, and show that lipid droplet formation is altered by manipulations of nuclear PS availability. Our studies reveal an unrecognized regulatory role of nuclear PS levels in the regulation of key PC synthesizing enzymes within the nucleus.

Molecular basis for plasma membrane recruitment of PI4KA by EFR3.

The lipid kinase phosphatidylinositol 4 kinase III alpha (PI4KIIIα/PI4KA) is a master regulator of the lipid composition and asymmetry of the plasma membrane. PI4KA exists primarily in a heterotrimeric complex with its regulatory proteins TTC7 and FAM126. Fundamental to PI4KA activity is its targeted recruitment to the plasma membrane by the lipidated proteins EFR3A and EFR3B. Here, we report a cryo-EM structure of the C-terminus of EFR3A bound to the PI4KA-TTC7B-FAM126A complex, with extensive validation using both hydrogen deuterium exchange mass spectrometry (HDX-MS), and mutational analysis. The EFR3A C-terminus undergoes a disorder-order transition upon binding to the PI4KA complex, with an unexpected direct interaction with both TTC7B and FAM126A. Complex disrupting mutations in TTC7B, FAM126A, and EFR3 decrease PI4KA recruitment to the plasma membrane. Multiple post-translational modifications and disease linked mutations map to this site, providing insight into how PI4KA membrane recruitment can be regulated and disrupted in human disease.

Non-vesicular phosphatidylinositol transfer plays critical roles in defining organelle lipid composition.

Phosphatidylinositol (PI) is the precursor lipid for the minor phosphoinositides (PPIns), which are critical for multiple functions in all eukaryotic cells. It is poorly understood how phosphatidylinositol, which is synthesized in the ER, reaches those membranes where PPIns are formed. Here, we used VT01454, a recently identified inhibitor of class I PI transfer proteins (PITPs), to unravel their roles in lipid metabolism, and solved the structure of inhibitor-bound PITPNA to gain insight into the mode of inhibition. We found that class I PITPs not only distribute PI for PPIns production in various organelles such as the plasma membrane (PM) and late endosomes/lysosomes, but that their inhibition also significantly reduced the levels of phosphatidylserine, di- and triacylglycerols, and other lipids, and caused prominent increases in phosphatidic acid. While VT01454 did not inhibit Golgi PI4P formation nor reduce resting PM PI(4,5)P2 levels, the recovery of the PM pool of PI(4,5)P2 after receptor-mediated hydrolysis required both class I and class II PITPs. Overall, these studies show that class I PITPs differentially regulate phosphoinositide pools and affect the overall cellular lipid landscape.

Defining the EM-signature of successful cell-transfection.

In this report, we describe the architecture of Lipofectamine 2000 and 3000 transfection- reagents, as they appear inside of transfected cells, using classical transmission electron microscopy (EM). We also demonstrate that they provoke consistent structural changes after they have entered cells, changes that not only provide new insights into the mechanism of action of these particular transfection-reagents, but also provide a convenient and robust method for identifying by EM which cells in any culture have been successfully transfected. This also provides clues to the mechanism(s) of their toxic effects, when they are applied in excess. We demonstrate that after being bulk-endocytosed by cells, the cationic spheroids of Lipofectamine remain intact throughout the entire time of culturing, but escape from their endosomes and penetrate directly into the cytoplasm of the cell. In so doing, they provoke a stereotypical recruitment and rearrangement of endoplasmic reticulum (ER), and they ultimately end up escaping into the cytoplasm and forming unique 'inclusion-bodies.' Once free in the cytoplasm, they also invariably develop dense and uniform coatings of cytoplasmic ribosomes on their surfaces, and finally, they become surrounded by 'annulate' lamellae' of the ER. In the end, these annulate-lamellar enclosures become the ultrastructural 'signatures' of these inclusion-bodies, and serve to positively and definitively identify all cells that have been effectively transfected. Importantly, these new EM-observations define several new and unique properties of these classical Lipofectamines, and allow them to be discriminated from other lipoidal or particulate transfection-reagents, which we find do not physically break out of endosomes or end up in inclusion bodies, and in fact, provoke absolutely none of these 'signature' cytoplasmic reactions.

Nir1-LNS2 is a novel phosphatidic acid biosensor that reveals mechanisms of lipid production.

Despite various roles of phosphatidic acid (PA) in cellular functions such as lipid homeostasis and vesicular trafficking, there is a lack of high-affinity tools to study PA in live cells. After analysis of the predicted structure of the LNS2 domain in the lipid transfer protein Nir1, we suspected that this domain could serve as a novel PA biosensor. We created a fluorescently tagged Nir1-LNS2 construct and then performed liposome binding assays as well as pharmacological and genetic manipulations of HEK293A cells to determine how specific lipids affect the interaction of Nir1-LNS2 with membranes. We found that Nir1-LNS2 bound to both PA and PIP2 in vitro. Interestingly, only PA was necessary and sufficient to localize Nir1-LNS2 to membranes in cells. Nir1-LNS2 also showed a heightened responsiveness to PA when compared to biosensors using the Spo20 PA binding domain (PABD). Nir1-LNS2's high sensitivity revealed a modest but discernible contribution of PLD to PA production downstream of muscarinic receptors, which has not been visualized with previous Spo20-based probes. In summary, Nir1-LNS2 emerges as a versatile and sensitive biosensor, offering researchers a new powerful tool for real-time investigation of PA dynamics in live cells.

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.

PI4K2A deficiency causes innate error in intracellular trafficking with developmental and epileptic-dyskinetic encephalopathy.

OBJECTIVE: Intracellular signaling networks rely on proper membrane organization to control an array of cellular processes such as metabolism, proliferation, apoptosis, and macroautophagy in eukaryotic cells and organisms. Phosphatidylinositol 4-phosphate (PI4P) emerged as an essential regulatory lipid within organelle membranes that defines their lipid composition and signaling properties. PI4P is generated by four distinct phosphatidylinositol 4-kinases (PI4K) in mammalian cells: PI4KA, PI4KB, PI4K2A, PI4K2B. Animal models and human genetic studies suggest vital roles of PI4K enzymes in development and function of various organs, including the nervous system. Bi-allelic variants in PI4KA were recently associated with neurodevelopmental disorders (NDD), brain malformations, leukodystrophy, primary immunodeficiency, and inflammatory bowel disease. Here, we describe patients from two unrelated consanguineous families with PI4K2A deficiency and functionally explored the pathogenic mechanism. METHODS: Two patients with PI4K2A deficiency were identified by exome sequencing, presenting with developmental and epileptic-dyskinetic encephalopathy. Neuroimaging showed corpus callosum dysgenesis, diffuse white matter volume loss, and hypoplastic vermis. In addition to NDD, we observed recurrent infections and death at toddler age. We further explored identified variants with cellular assays. RESULTS: This clinical presentation overlaps with what was previously reported in two affected siblings with homozygous nonsense PI4K2A variant. Cellular studies analyzing these human variants confirmed their deleterious effect on PI4K2A activity and, together with the central role of PI4K2A in Rab7-associated vesicular trafficking, establish a link between late endosome-lysosome defects and NDD. INTERPRETATION: Our study establishes the genotype-phenotype spectrum of PI4K-associated NDD and highlights several commonalities with other innate errors of intracellular trafficking.

Metabolic routing maintains the unique fatty acid composition of phosphoinositides.

Phosphoinositide lipids (PPIn) are enriched in stearic- and arachidonic acids (38:4) but how this enrichment is established and maintained during phospholipase C (PLC) activation is unknown. Here we show that the metabolic fate of newly synthesized phosphatidic acid (PA), the lipid precursor of phosphatidylinositol (PI), is influenced by the fatty acyl-CoA used with preferential routing of the arachidonoyl-enriched species toward PI synthesis. Furthermore, during agonist stimulation the unsaturated forms of PI(4,5P)2 are replenished significantly faster than the more saturated ones, suggesting a favored recycling of the unsaturated forms of the PLC-generated hydrolytic products. Cytidine diphosphate diacylglycerol synthase 2 (CDS2) but not CDS1 was found to contribute to increased PI resynthesis during PLC activation. Lastly, while the lipid transfer protein, Nir2 is found to contribute to rapid PPIn resynthesis during PLC activation, the faster re-synthesis of the 38:4 species does not depend on Nir2. Therefore, the fatty acid side-chain composition of the lipid precursors used for PI synthesis is an important determinant of their metabolic fates, which also contributes to the maintenance of the unique fatty acid profile of PPIn lipids.

De novo loss-of-function variant in PTDSS1 is associated with developmental delay.

Heterozygous de novo missense pathogenic variants in PTDSS1 that result in gain-of-function of phosphatidylserine synthase 1 are associated with Lenz-Majewski hyperostotic dwarfism (LMHD). We identified the novel heterozygous de novo variant p.(Leu137Phe) in PTDSS1 in a child with mild-to-moderate developmental delay. Skeletal survey revealed no evidence of LMHD in this patient. Functional assessment of the p.Leu137Phe variant was performed by overexpressing the mutant protein into HEK293 cells. Following C14 -serine labeling and TLC analysis of lipids, we observed that the p.(Leu137Phe) variant displayed no catalytic activity compared to the wild-type enzyme. We conclude that p.(Leu137Phe) variant has decreased enzymatic activity and that is likely to be the etiology of the patient's symptoms given the gene's constraint in the population. This is the first report of the clinical phenotype seen in an individual with a heterozygous loss-of-function variant in PTDSS1. This phenotype is distinct from LMHD, which results from gain-of-function pathogenic variants in the same gene. Evaluation of the neurodevelopmental phenotype of additional individuals with loss-of-function variants in PTDSS1 is indicated to determine the spectrum of associated phenotypes.

Nesfatin-1 stimulates the hypothalamus-pituitary-interrenal axis hormones in goldfish.

Stress in vertebrates is mediated by the hypothalamus-pituitary-adrenal (in mammals)/interrenal (in fish) (HPA/I) axis, which produces the corticotropin-releasing factor (CRF), adrenocorticotropic hormone (ACTH), and corticosteroids, respectively. Nesfatin-1, a novel anorexigenic peptide encoded in the precursor nucleobindin-2 (NUCB2), is increasingly acknowledged as a peptide that influences the stress axis in mammals. The primary aim of this study was to characterize the putative effects of nesfatin-1 on the fish HPI axis, using goldfish (Carassius auratus) as an animal model. Our results demonstrated that nucb2/nesfatin-1 transcript abundance was detected in the HPI tissues of goldfish, with most abundant expression in the pituitary. NUCB2/nesfatin-1-like immunoreactivity was found in the goldfish hypothalamus, pituitary, and interrenal cells of the head kidney. GPCR12, a putative receptor for nesfatin-1, was also detected in the pituitary and interrenal cells. NUCB2/nesfatin-1-like immunoreactivity was observed in ACTH-expressing pituitary corticotrophs. Acute netting and restraint stress upregulated nucb2/nesfatin-1 mRNA levels in the forebrain, hypothalamus, and pituitary, as well as crf and crf-r1 expression in the forebrain and hypothalamus. Intraperitoneal and intracerebroventricular administration of nesfatin-1 increased cortisol release and hypothalamic crf mRNA levels, respectively. Finally, we found that nesfatin-1 significantly stimulated ACTH secretion from dispersed pituitary cells in vitro. Collectively, our data provide the first evidence showing that nesfatin-1 is a stress responsive peptide, which modulates the stress axis hormones in fish.

Nesfatin-1 is an inhibitor of the growth hormone-insulin-like growth factor axis in goldfish (Carassius auratus).

Nesfatin-1, an 82 amino acid peptide cleaved from the N-terminal of its precursor nucleobindin-2 (NUCB2), is emerging as a multifunctional peptide in fish. The present study aimed to determine whether nesfatin-1 plays a role in fish somatic growth by modulating the growth hormone (GH)/insulin-like growth factor (IGF) axis, using a representative teleost model, the goldfish (Carassius auratus). The results demonstrated that a single i.p. injection of synthetic goldfish nesfatin-1 significantly decreased the expression of hypothalamic pacap (approximately 90%) and pituitary Gh (approximately 90%) mRNAs at 15 minutes post-injection. Serum GH levels were also reduced as a result of nesfatin-1 administration, by approximately 45% and 55% at 15 and 30 minutes post-injection, respectively. Likewise, in vitro treatment of goldfish dispersed pituitary cells with nesfatin-1 reduced Gh secretion, suggesting that nesfatin-1 acts directly on pituitary somatotrophs to inhibit Gh release. Exposure of cultured liver fragments to nesfatin-1 (0.1, 1 and 10 nmol L-1 ) led to a significant reduction in igf-1 mRNA at 120 minutes and of igf-II mRNA at 30 and 60 minutes post-incubation. Collectively, these results indicate a suppressive role for nesfatin-1 on the goldfish GH/IGF axis. Immunohistochemical studies demonstrated that NUCB2/nesfatin-1-like immunoreactivity, although present in the goldfish pituitary, is not colocalised with GH in goldfish somatotrophs. Thus, nesfatin-1 does not appear to act in an autocrine manner to regulate GH secretion. Taken together, this research found that the pituitary gland is an important source of endogenous NUCB2/nesfatin-1 and also that nesfatin-1 directly suppresses the Gh/IGF axis in goldfish.

PHOSPHOINOSITIDES AND CALCIUM SIGNALING. A MARRIAGE ARRANGED IN ER-PM CONTACT SITES.

Calcium (Ca2+) ions are critically important in orchestrating countless regulatory processes in eukaryotic cells. Consequently, cells tightly control cytoplasmic Ca2+ concentrations using a complex array of Ca2+-selective ion channels, transporters, and signaling effectors. Ca2+ transport through various cellular membranes is highly dependent on the intrinsic properties of specific membrane compartments and conversely, local Ca2+ changes have profound effects on the membrane lipid composition of such membrane sub-domains. In particular, inositol phospholipids are a minor class of phospholipids that play pivotal roles in the control of Ca2+-dependent signaling pathways. In this review, we will highlight some of the recent advances in this field as well as their impact in defining future research directions.

Ribosome-associated vesicles: A dynamic subcompartment of the endoplasmic reticulum in secretory cells.

The endoplasmic reticulum (ER) is a highly dynamic network of membranes. Here, we combine live-cell microscopy with in situ cryo-electron tomography to directly visualize ER dynamics in several secretory cell types including pancreatic ß-cells and neurons under near-native conditions. Using these imaging approaches, we identify a novel, mobile form of ER, ribosome-associated vesicles (RAVs), found primarily in the cell periphery, which is conserved across different cell types and species. We show that RAVs exist as distinct, highly dynamic structures separate from the intact ER reticular architecture that interact with mitochondria via direct intermembrane contacts. These findings describe a new ER subcompartment within cells.

Defining the subcellular distribution and metabolic channeling of phosphatidylinositol.

Phosphatidylinositol (PI) is an essential structural component of eukaryotic membranes that also serves as the common precursor for polyphosphoinositide (PPIn) lipids. Despite the recognized importance of PPIn species for signal transduction and membrane homeostasis, there is still a limited understanding of the relationship between PI availability and the turnover of subcellular PPIn pools. To address these shortcomings, we established a molecular toolbox for investigations of PI distribution within intact cells by exploiting the properties of a bacterial enzyme, PI-specific PLC (PI-PLC). Using these tools, we find a minor presence of PI in membranes of the ER, as well as a general enrichment within the cytosolic leaflets of the Golgi complex, peroxisomes, and outer mitochondrial membrane, but only detect very low steady-state levels of PI within the plasma membrane (PM) and endosomes. Kinetic studies also demonstrate the requirement for sustained PI supply from the ER for the maintenance of monophosphorylated PPIn species within the PM, Golgi complex, and endosomal compartments.

Characterization of the c10orf76-PI4KB complex and its necessity for Golgi PI4P levels and enterovirus replication.

The lipid kinase PI4KB, which generates phosphatidylinositol 4-phosphate (PI4P), is a key enzyme in regulating membrane transport and is also hijacked by multiple picornaviruses to mediate viral replication. PI4KB can interact with multiple protein binding partners, which are differentially manipulated by picornaviruses to facilitate replication. The protein c10orf76 is a PI4KB-associated protein that increases PI4P levels at the Golgi and is essential for the viral replication of specific enteroviruses. We used hydrogen-deuterium exchange mass spectrometry to characterize the c10orf76-PI4KB complex and reveal that binding is mediated by the kinase linker of PI4KB, with formation of the heterodimeric complex modulated by PKA-dependent phosphorylation. Complex-disrupting mutations demonstrate that PI4KB is required for membrane recruitment of c10orf76 to the Golgi, and that an intact c10orf76-PI4KB complex is required for the replication of c10orf76-dependent enteroviruses. Intriguingly, c10orf76 also contributed to proper Arf1 activation at the Golgi, providing a putative mechanism for the c10orf76-dependent increase in PI4P levels at the Golgi.

Selective recruitment of Nck and Syk contribute to distinct leukocyte immune-type receptor-initiated target interactions.

The ability of phagocytes to recognize, immobilize, and engulf extracellular targets are fundamental immune cell processes that allow for the destruction of a variety of microbial intruders. The phagocytic process depends onsignalling events that initiate dynamic changes in the plasma membrane architecture that are required to accommodate the internalization of large particulate targets. To better understand fundamental molecular mechanisms responsible for facilitating phagocytic receptor-mediated regulation of cytoskeletal networks, our research has focused on investigating representative immunoregulatory proteins from the channel catfish (Ictalurus punctatus) leukocyte immune-type receptor family (IpLITRs). Specifically, we have shown that a specific IpLITR-type can regulate the constitutive deployment of filopodial-like structures to actively capture and secure targets to the phagocyte surface, which is followed by F-actin mediated membrane dynamics that are associated with the formation of phagocytic cup-like structures that precede target engulfment. In the present study, we use confocal imaging to examine the recruitment of mediators of the F-actin cytoskeleton during IpLITR-mediated regulation of membrane dynamics. Our results provide novel details regarding the dynamic recruitment of the signaling effectors Nck and Syk during classical as well as atypical IpLITR-induced phagocytic processes.

Integrated regulation of the phosphatidylinositol cycle and phosphoinositide-driven lipid transport at ER-PM contact sites.

Among the structural phospholipids that form the bulk of eukaryotic cell membranes, phosphatidylinositol (PtdIns) is unique in that it also serves as the common precursor for low-abundance regulatory lipids, collectively referred to as polyphosphoinositides (PPIn). The metabolic turnover of PPIn species has received immense attention because of the essential functions of these lipids as universal regulators of membrane biology and their dysregulation in numerous human pathologies. The diverse functions of PPIn lipids occur, in part, by orchestrating the spatial organization and conformational dynamics of peripheral or integral membrane proteins within defined subcellular compartments. The emerging role of stable contact sites between adjacent membranes as specialized platforms for the coordinate control of ion exchange, cytoskeletal dynamics, and lipid transport has also revealed important new roles for PPIn species. In this review, we highlight the importance of membrane contact sites formed between the endoplasmic reticulum (ER) and plasma membrane (PM) for the integrated regulation of PPIn metabolism within the PM. Special emphasis will be placed on non-vesicular lipid transport during control of the PtdIns biosynthetic cycle as well as toward balancing the turnover of the signaling PPIn species that define PM identity.

Lipid Dynamics at Contact Sites Between the Endoplasmic Reticulum and Other Organelles.

Phospholipids are synthesized primarily within the endoplasmic reticulum and are subsequently distributed to various subcellular membranes to maintain the unique lipid composition of specific organelles. As a result, in most cases, the steady-state localization of membrane phospholipids does not match their site of synthesis. This raises the question of how diverse lipid species reach their final membrane destinations and what molecular processes provide the energy to maintain the lipid gradients that exist between various membrane compartments. Recent studies have highlighted the role of inositol phospholipids in the nonvesicular transport of lipids at membrane contact sites. This review attempts to summarize our current understanding of these complex lipid dynamics and highlights their implications for defining future research directions.

Correction to: Polyphosphoinositide-Binding Domains: Insights from Peripheral Membrane and Lipid-Transfer Proteins.

This chapter was inadvertently published with an incorrect copyright holder. It has now been updated as below.

Polyphosphoinositide-Binding Domains: Insights from Peripheral Membrane and Lipid-Transfer Proteins.

Within eukaryotic cells, biochemical reactions need to be organized on the surface of membrane compartments that use distinct lipid constituents to dynamically modulate the functions of integral proteins or influence the selective recruitment of peripheral membrane effectors. As a result of these complex interactions, a variety of human pathologies can be traced back to improper communication between proteins and membrane surfaces; either due to mutations that directly alter protein structure or as a result of changes in membrane lipid composition. Among the known structural lipids found in cellular membranes, phosphatidylinositol (PtdIns) is unique in that it also serves as the membrane-anchored precursor of low-abundance regulatory lipids, the polyphosphoinositides (PPIn), which have restricted distributions within specific subcellular compartments. The ability of PPIn lipids to function as signaling platforms relies on both non-specific electrostatic interactions and the selective stereospecific recognition of PPIn headgroups by specialized protein folds. In this chapter, we will attempt to summarize the structural diversity of modular PPIn-interacting domains that facilitate the reversible recruitment and conformational regulation of peripheral membrane proteins. Outside of protein folds capable of capturing PPIn headgroups at the membrane interface, recent studies detailing the selective binding and bilayer extraction of PPIn species by unique functional domains within specific families of lipid-transfer proteins will also be highlighted. Overall, this overview will help to outline the fundamental physiochemical mechanisms that facilitate localized interactions between PPIn lipids and the wide-variety of PPIn-binding proteins that are essential for the coordinate regulation of cellular metabolism and membrane dynamics.