RESUMEN
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.
RESUMEN
Membrane polyphosphoinositides (PPIs) are lipid-signaling molecules that undergo metabolic turnover and influence a diverse range of cellular functions. PPIs regulate the activity and/or spatial localization of a number of actin-binding proteins (ABPs) through direct interactions; however, it is much less clear whether ABPs could also be an integral part in regulating PPI signaling. In this study, we show that ABP profilin1 (Pfn1) is an important molecular determinant of the cellular content of PI(4,5)P2 (the most abundant PPI in cells). In growth factor (EGF) stimulation setting, Pfn1 depletion does not impact PI(4,5)P2 hydrolysis but enhances plasma membrane (PM) enrichment of PPIs that are produced downstream of activated PI3-kinase, including PI(3,4,5)P3 and PI(3,4)P2, the latter consistent with increased PM recruitment of SH2-containing inositol 5' phosphatase (SHIP2) (a key enzyme for PI(3,4)P2 biosynthesis). Although Pfn1 binds to PPIs in vitro, our data suggest that Pfn1's affinity to PPIs and PM presence in actual cells, if at all, is negligible, suggesting that Pfn1 is unlikely to directly compete with SHIP2 for binding to PM PPIs. Additionally, we provide evidence for Pfn1's interaction with SHIP2 in cells and modulation of this interaction upon EGF stimulation, raising an alternative possibility of Pfn1 binding as a potential restrictive mechanism for PM recruitment of SHIP2. In conclusion, our findings challenge the dogma of Pfn1's binding to PM by PPI interaction, uncover a previously unrecognized role of Pfn1 in PI(4,5)P2 homeostasis and provide a new mechanistic avenue of how an ABP could potentially impact PI3K signaling byproducts in cells through lipid phosphatase control.
Asunto(s)
Fosfatidilinositoles , Profilinas , Factor de Crecimiento Epidérmico/metabolismo , Fosfatidilinositol 3-Quinasas/metabolismo , Fosfatidilinositol-3,4,5-Trifosfato 5-Fosfatasas/metabolismo , Fosfatidilinositoles/metabolismo , Humanos , Células HEK293 , Profilinas/metabolismoRESUMEN
The lipid molecule phosphatidylinositol (4,5)-bisphosphate [PI(4,5)P2] controls all aspects of plasma membrane (PM) function in animal cells, from its selective permeability to the attachment of the cytoskeleton. Although disruption of PI(4,5)P2 is associated with a wide range of diseases, it remains unclear how cells sense and maintain PI(4,5)P2 levels to support various cell functions. Here, we show that the PIP4K family of enzymes, which synthesize PI(4,5)P2 via a minor pathway, also function as sensors of tonic PI(4,5)P2 levels. PIP4Ks are recruited to the PM by elevated PI(4,5)P2 levels, where they inhibit the major PI(4,5)P2-synthesizing PIP5Ks. Perturbation of this simple homeostatic mechanism reveals differential sensitivity of PI(4,5)P2-dependent signaling to elevated PI(4,5)P2 levels. These findings reveal that a subset of PI(4,5)P2-driven functions might drive disease associated with disrupted PI(4,5)P2 homeostasis.
Asunto(s)
Fosfatidilinositol 4,5-Difosfato , Transducción de Señal , Animales , Fosfatidilinositol 4,5-Difosfato/metabolismo , Transducción de Señal/fisiología , Membrana Celular/metabolismo , Fosfatidilinositoles/metabolismo , HomeostasisRESUMEN
The lipid phosphatidyl-D-myo-inositol-4,5-bisphosphate [PI(4,5)P2] is a master regulator of plasma membrane (PM) function. Its effector proteins regulate transport, signaling, and cytoskeletal processes that define PM structure and function. How a single type of lipid regulates so many parallel processes is unclear. We tested the hypothesis that spatially separate PI(4,5)P2 pools associate with different PM complexes. The mobility of PI(4,5)P2 was measured using biosensors by single-particle tracking. We found that PM lipids including PI(4,5)P2 diffuse rapidly (â¼0.3 µm2/s) with Brownian motion, although they spend one third of their time diffusing more slowly. Surprisingly, areas of the PM occupied by PI(4,5)P2-dependent complexes did not slow PI(4,5)P2 lateral mobility. Only the spectrin and septin cytoskeletons showed reduced PI(4,5)P2 diffusion. We conclude that even structures with high densities of PI(4,5)P2 effector proteins, such as clathrin-coated pits and focal adhesions, do not corral unbound PI(4,5)P2, questioning a role for spatially segregated PI(4,5)P2 pools in organizing and regulating PM functions.
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Membrana Celular , Lípidos de la Membrana , Fosfatidilinositoles , Citoesqueleto de Actina , Difusión , EspectrinaRESUMEN
In the almost 70 years since the first hints of its existence, the phosphoinositide, phosphatidyl-D-myo-inositol 4,5-bisphosphate has been found to be central in the biological regulation of plasma membrane (PM) function. Here, we provide an overview of the signaling, transport and structural roles the lipid plays at the cell surface in animal cells. These include being substrate for second messenger generation, direct modulation of receptors, control of membrane traffic, regulation of ion channels and transporters, and modulation of the cytoskeleton and cell polarity. We conclude by re-evaluating PI(4,5)P2's designation as a signaling molecule, instead proposing a cofactor role, enabling PM-selective function for many proteins.
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Fosfatidilinositoles , Transducción de Señal , Animales , Membrana Celular/metabolismo , Fosfatidilinositoles/metabolismo , Canales Iónicos/metabolismo , Fosfatidilinositol 4,5-Difosfato/metabolismoRESUMEN
Specific lipid species play central roles in cell biology. Their presence or enrichment in individual membranes can control properties or direct protein localization and/or activity. Therefore, probes to detect and observe these lipids in intact cells are essential tools in the cell biologist's freezer box. Herein, we discuss genetically encoded lipid biosensors, which can be expressed as fluorescent protein fusions to track lipids in living cells. We provide a state-of-the-art list of the most widely available and reliable biosensors and highlight new probes (circa 2018-2021). Notably, we focus on advances in biosensors for phosphatidylinositol, phosphatidic acid, and PI 3-kinase lipid products.
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Técnicas Biosensibles , Colorantes Fluorescentes/metabolismo , Fosfatidilinositol 3-Quinasas/metabolismo , Fosfatidilinositoles , Transporte de ProteínasRESUMEN
Lipids, like phosphoinositides, can be visualized in living cells in real time using genetically encoded biosensors and fluorescence microscopy. Sensor localization can be quantified by determining the fluorescence intensity of each fluorophore. Enrichment of lipids at membranes can be determined by generating and applying an organelle-specific binary mask. In this chapter, we provide a detailed list of reagents and methods to visualize and quantify relative lipid levels. Applying this approach, changes in lipid levels can be assessed in cases when lipid metabolizing enzymes are mutated or otherwise altered.
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Lípidos/análisis , Microscopía Fluorescente/métodos , Fosfatidilinositoles/análisis , Animales , Técnicas Biosensibles/métodos , Colorantes Fluorescentes/química , Humanos , Metabolismo de los Lípidos/fisiología , Lípidos/químicaRESUMEN
Chemical dimerization systems have been used to drive acute depletion of polyphosphoinsitides (PPIns). They do so by inducing subcellular localization of enzymes that catabolize PPIns. By using this approach, all seven PPIns can be depleted in living cells and in real time. The rapid permeation of dimerizer agents and the specific expression of recruiter proteins confer great spatial and temporal resolution with minimal cell perturbation. In this chapter, we provide detailed instructions to monitor and induce depletion of PPIns in live cells.
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Microscopía Fluorescente/métodos , Fosfatos de Fosfatidilinositol/análisis , Fosfatos de Fosfatidilinositol/química , Animales , Membrana Celular/metabolismo , Dimerización , Células HEK293 , Humanos , Fosfatos de Fosfatidilinositol/metabolismo , Transporte de ProteínasRESUMEN
The polyphosphoinositides (PPIn) are central regulatory lipids that direct membrane function in eukaryotic cells. Understanding how their synthesis is regulated is crucial to revealing these lipids' role in health and disease. PPIn are derived from the major structural lipid, phosphatidylinositol (PI). However, although the distribution of most PPIn has been characterized, the subcellular localization of PI available for PPIn synthesis is not known. Here, we used several orthogonal approaches to map the subcellular distribution of PI, including localizing exogenous fluorescent PI, as well as detecting lipid conversion products of endogenous PI after acute chemogenetic activation of PI-specific phospholipase and 4-kinase. We report that PI is broadly distributed throughout intracellular membrane compartments. However, there is a surprising lack of PI in the plasma membrane compared with the PPIn. These experiments implicate regulation of PI supply to the plasma membrane, as opposed to regulation of PPIn-kinases, as crucial to the control of PPIn synthesis and function at the PM.
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Membrana Celular/metabolismo , Membranas Intracelulares/metabolismo , Fosfatos de Fosfatidilinositol/metabolismo , Fosfatidilinositoles/metabolismo , Animales , Células COS , Chlorocebus aethiops , Diglicéridos/metabolismo , Cinética , Proteínas Luminiscentes/genética , Proteínas Luminiscentes/metabolismo , Microscopía Confocal , Antígenos de Histocompatibilidad Menor/genética , Antígenos de Histocompatibilidad Menor/metabolismo , Fosfatidilinositol 4,5-Difosfato/metabolismo , Fosfotransferasas (Aceptor de Grupo Alcohol)/genética , Fosfotransferasas (Aceptor de Grupo Alcohol)/metabolismo , Fosfolipasas de Tipo C/genética , Fosfolipasas de Tipo C/metabolismoRESUMEN
Lipids convey both structural and functional properties to eukaryotic membranes. Understanding the basic lipid composition and the dynamics of these important molecules, in the context of cellular membranes, can shed light on signaling, metabolism, trafficking, and even membrane identity. The development of genetically encoded lipid biosensors has allowed for the visualization of specific lipids inside individual, living cells. However, a number of caveats and considerations have emerged with the overexpression of these biosensors. In this Technical Perspective, we provide a current list of available genetically encoded lipid biosensors, together with criteria that determine their veracity. We also provide some suggestions for the optimal utilization of these biosensors when both designing experiments and interpreting results.
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Técnicas Biosensibles/métodos , Técnicas Genéticas , Lípidos/química , Animales , Células HeLa , Humanos , Procesamiento de Imagen Asistido por ComputadorRESUMEN
Phosphatidylinositol 4,5-bisphosphate (PI(4,5)P2) is a critically important regulatory lipid of the plasma membrane (PM); however, little is known about how cells regulate PM PI(4,5)P2 levels. Here, we show that the phosphatidylinositol 4-phosphate (PI4P)/phosphatidylserine (PS) transfer activity of the endoplasmic reticulum (ER)-resident ORP5 and ORP8 is regulated by both PM PI4P and PI(4,5)P2 Dynamic control of ORP5/8 recruitment to the PM occurs through interactions with the N-terminal Pleckstrin homology domains and adjacent basic residues of ORP5/8 with both PI4P and PI(4,5)P2 Although ORP5 activity requires normal levels of these inositides, ORP8 is called on only when PI(4,5)P2 levels are increased. Regulation of the ORP5/8 attachment to the PM by both phosphoinositides provides a powerful means to determine the relative flux of PI4P toward the ER for PS transport and Sac1-mediated dephosphorylation and PIP 5-kinase-mediated conversion to PI(4,5)P2 Using this rheostat, cells can maintain PI(4,5)P2 levels by adjusting the availability of PI4P in the PM.
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Membrana Celular/metabolismo , Retículo Endoplásmico/metabolismo , Fosfatidilinositol 4,5-Difosfato/metabolismo , Fosfatos de Fosfatidilinositol/metabolismo , Fosfatidilserinas/metabolismo , Animales , Transporte Biológico , Células HEK293 , Humanos , Fosfotransferasas (Aceptor de Grupo Alcohol)/metabolismo , Dominios Proteicos , Ratas , Receptores de Esteroides/química , Receptores de Esteroides/metabolismo , Especificidad por SustratoRESUMEN
Gradients of PtdIns4P between organelle membranes and the endoplasmic reticulum (ER) are thought to drive counter-transport of other lipids via non-vesicular traffic. This novel pathway requires the SAC1 phosphatase to degrade PtdIns4P in a 'cis' configuration at the ER to maintain the gradient. However, SAC1 has also been proposed to act in 'trans' at membrane contact sites, which could oppose lipid traffic. It is therefore crucial to determine which mode SAC1 uses in living cells. We report that acute inhibition of SAC1 causes accumulation of PtdIns4P in the ER, that SAC1 does not enrich at membrane contact sites, and that SAC1 has little activity in 'trans', unless a linker is added between its ER-anchored and catalytic domains. The data reveal an obligate 'cis' activity of SAC1, supporting its role in non-vesicular lipid traffic and implicating lipid traffic more broadly in inositol lipid homeostasis and function.
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Retículo Endoplásmico/enzimología , Retículo Endoplásmico/metabolismo , Fosfatos de Fosfatidilinositol/metabolismo , Monoéster Fosfórico Hidrolasas/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Animales , Células COS , Chlorocebus aethiops , Células HEK293 , HumanosRESUMEN
Emerging evidence suggests that impaired regulation of adipocyte lipolysis contributes to the proinflammatory immune cell infiltration of metabolic tissues in obesity, a process that is proposed to contribute to the development and exacerbation of insulin resistance. To test this hypothesis in vivo, we generated mice with adipocyte-specific deletion of adipose triglyceride lipase (ATGL), the rate-limiting enzyme catalyzing triacylglycerol hydrolysis. In contrast to previous models, adiponectin-driven Cre expression was used for targeted ATGL deletion. The resulting adipocyte-specific ATGL knockout (AAKO) mice were then characterized for metabolic and immune phenotypes. Lean and diet-induced obese AAKO mice had reduced adipocyte lipolysis, serum lipids, systemic lipid oxidation, and expression of peroxisome proliferator-activated receptor alpha target genes in adipose tissue (AT) and liver. These changes did not increase overall body weight or fat mass in AAKO mice by 24 weeks of age, in part due to reduced expression of genes involved in lipid uptake, synthesis, and adipogenesis. Systemic glucose and insulin tolerance were improved in AAKO mice, primarily due to enhanced hepatic insulin signaling, which was accompanied by marked reduction in diet-induced hepatic steatosis as well as hepatic immune cell infiltration and activation. In contrast, although adipocyte ATGL deletion reduced AT immune cell infiltration in response to an acute lipolytic stimulus, it was not sufficient to ameliorate, and may even exacerbate, chronic inflammatory changes that occur in AT in response to diet-induced obesity.
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Adipocitos/metabolismo , Inflamación/genética , Resistencia a la Insulina/genética , Lipasa/genética , Obesidad/genética , Tejido Adiposo/metabolismo , Animales , Antígenos de Diferenciación/genética , Antígenos de Diferenciación/metabolismo , Antígeno CD11c/genética , Antígeno CD11c/metabolismo , Células Dendríticas/metabolismo , Dieta Alta en Grasa/efectos adversos , Expresión Génica , Immunoblotting , Inflamación/sangre , Inflamación/metabolismo , Lipasa/metabolismo , Metabolismo de los Lípidos/genética , Lípidos/sangre , Lipólisis/genética , Hígado/metabolismo , Macrófagos/metabolismo , Ratones de la Cepa 129 , Ratones Endogámicos C57BL , Ratones Noqueados , Obesidad/etiología , Obesidad/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa InversaRESUMEN
OBJECTIVE: Autotaxin (ATX) is an adipocyte-derived lysophospholipase that generates the lipid signaling molecule lysophosphatidic acid (LPA). The aim of this study was to determine the relationship between serum ATX and nonalcoholic fatty liver disease (NAFLD) in females with obesity. METHODS: 101 nondiabetic women with obesity (age: 31.5-55.8 years; BMI: 35.0-64.5 kg/m2) were classified as having NAFLD (36.3%) or not having NAFLD (63.7%) based on the degree of hepatic steatosis on abdominal CT. Subjects were characterized for metabolic phenotype including measures of energy, glucose, and lipid homeostasis. Fasting serum adipokines and inflammatory markers were determined by ELISA. Linear regression analysis was used to determine features independently associated with NAFLD. RESULTS: Subjects with and without NAFLD differed in several key features of metabolic phenotype including BMI, waist circumference, fasting glucose and insulin, HOMA-IR, VLDL, triglycerides, and ALT. Serum adipokines, including ATX and leptin, were higher in subjects with NAFLD. Serum ATX was significantly correlated with alkaline phosphatase, fasting glucose, fasting insulin, and HOMA-IR. Linear regression analysis revealed that serum triglycerides and log-transformed ATX were independently associated with hepatic steatosis. CONCLUSIONS: Serum ATX may be a potential pathogenic factor and/or biomarker for NAFLD in nondiabetic women with obesity.
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Enfermedad del Hígado Graso no Alcohólico/sangre , Enfermedad del Hígado Graso no Alcohólico/diagnóstico , Obesidad Mórbida/sangre , Hidrolasas Diéster Fosfóricas/sangre , Adipoquinas/sangre , Adulto , Biomarcadores/sangre , Índice de Masa Corporal , Femenino , Humanos , Insulina/sangre , Leptina/sangre , Modelos Lineales , Persona de Mediana Edad , Obesidad Mórbida/complicaciones , Estudios Retrospectivos , Índice de Severidad de la Enfermedad , Triglicéridos/sangre , Circunferencia de la CinturaRESUMEN
Adipose triglyceride lipase (ATGL) is the rate-limiting enzyme mediating triacylglycerol hydrolysis in virtually all cells, including adipocytes and skeletal myocytes, and hence, plays a critical role in mobilizing fatty acids. Global ATGL deficiency promotes skeletal myopathy and exercise intolerance in mice and humans, and yet the tissue-specific contributions to these phenotypes remain unknown. The goal of this study was to determine the relative contribution of ATGL-mediated triacylglycerol hydrolysis in adipocytes vs. skeletal myocytes to acute exercise performance. To achieve this goal, we generated murine models with adipocyte- and skeletal myocyte-specific targeted deletion of ATGL. We then subjected untrained mice to acute peak and submaximal exercise interventions and assessed exercise performance and energy substrate metabolism. Impaired ATGL-mediated lipolysis within adipocytes reduced peak and submaximal exercise performance, reduced peripheral energy substrate availability, shifted energy substrate preference toward carbohydrate oxidation, and decreased HSL Ser(660) phosphorylation and mitochondrial respiration within skeletal muscle. In contrast, impaired ATGL-mediated lipolysis within skeletal myocytes was not sufficient to reduce peak and submaximal exercise performance or peripheral energy substrate availability and instead tended to enhance metabolic flexibility during peak exercise. Furthermore, the expanded intramyocellular triacylglycerol pool in these mice was reduced following exercise in association with preserved HSL phosphorylation, suggesting that HSL may compensate for impaired ATGL action in skeletal muscle during exercise. These data suggest that adipocyte rather than skeletal myocyte ATGL-mediated lipolysis plays a greater role during acute exercise in part because of compensatory mechanisms that maintain lipolysis in muscle, but not adipose tissue, when ATGL is absent.
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Adipocitos/metabolismo , Lipasa/genética , Fibras Musculares Esqueléticas/metabolismo , Condicionamiento Físico Animal/fisiología , Esfuerzo Físico/genética , Animales , Rendimiento Atlético , Tolerancia al Ejercicio/genética , Femenino , Eliminación de Gen , Lipasa/metabolismo , Lipólisis/genética , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones NoqueadosRESUMEN
OBJECTIVE: Autotaxin (ATX) is an adipocyte-derived lysophospholipase D that generates the lipid signaling molecule lysophosphatidic acid (LPA). The ATX/LPA pathway in adipose tissue has recently been implicated in obesity and insulin resistance in animal models, but the role of circulating ATX in humans remains unclear. The aim of the present study was to determine the relationship between serum ATX and insulin resistance. METHODS: Older (60-75 years), nondiabetic human participants with overweight or obesity (BMI 25-37 kg m(-2) ) were characterized for metabolic phenotype including measures of energy, glucose, and lipid homeostasis. The relationship between serum ATX and metabolic parameters was then determined using correlative and predictive statistics. RESULTS: Serum ATX was higher in females than in males. After controlling for sex, serum ATX correlated with multiple measures of adiposity and glucose homeostasis/insulin action. Serum ATX and BMI also independently predicted glucose infusion rate during a hyperinsulinemic euglycemic clamp and homeostatic model assessment of insulin resistance after controlling for sex and medication use. CONCLUSIONS: Serum ATX correlates with and predicts measures of glucose homeostasis and insulin sensitivity in older humans, suggesting that it may be a potential pathogenic factor and/or diagnostic/therapeutic target for insulin resistance in this population.