scholarly journals C24 sphingolipids play a surprising and central role in governing cholesterol and lateral organization of the live cell plasma membrane

2017 ◽  
Author(s):  
K. C. Courtney ◽  
W Pezeshkian ◽  
R Raghupathy ◽  
C Zhang ◽  
A Darbyson ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Mammalian Cells ◽  
Acyl Chain ◽  
Live Cells ◽  
Membrane Microdomains ◽  
Giant Unilamellar Vesicles ◽  
Unilamellar Vesicles ◽  
Outer Leaflet ◽  
Acyl Chains ◽  
Lateral Organization

AbstractMammalian cell sphingolipids, primarily with C24 and C16 acyl chains, reside in the outer leaflet of the plasma membrane. Curiously, little is known how C24 sphingolipids impact cholesterol and membrane microdomains. Here, we generated giant unilamellar vesicles and live mammalian cells with C24 or C16 sphingomyelin exclusively in the outer leaflet and compared microdomain formation. In giant unilamellar vesicles, we observed that asymmetrically placed C24 sphingomyelin suppresses microdomains. Conversely, C16 sphingomyelin facilitates microdomains. Replacing endogenous sphingolipids with C24 or C16 sphingomyelin in live HeLa cells has a similar impact on microdomains, characterized by FRET between GPI-anchored proteins: C24, but not C16, sphingomyelin suppresses submicron domains in the plasma membrane. Molecular dynamics simulations indicated that, when in the outer leaflet, the acyl chain of C24 sphingomyelin interdigitates into the opposing leaflet, thereby favouring cholesterol in the inner leaflet. We indeed found that cholesterol prefers the inner over the outer leaflet of asymmetric unilamellar vesicles (80/20) when C24 sphingomyelin is in the outer leaflet. However, when C16 sphingomyelin is in the outer leaflet, cholesterol is evenly partitioned between leaflets (50/50). Interestingly, when a mixture of C24/C16 sphingomyelin is in the outer leaflet of unilamellar vesicles, cholesterol still prefers the inner leaflet (80/20). Indeed, in human erythrocyte plasma membrane, where a mixture of C24 and C16 sphingolipids are naturally in the outer leaflet, cholesterol prefers the cytoplasmic leaflet (80/20). Therefore, C24 sphingomyelin uniquely interacts with cholesterol and governs the lateral organization in asymmetric membranes, including the plasma membrane, potentially by generating cholesterol asymmetry.Statement of SignificanceThe plasma membrane bilayer of mammalian cells has distinct phospholipids between the outer and inner leaflet, with sphingolipids exclusively in the outer leaflet. A large portion of mammalian sphingolipids have very long acyl chains (C24). Little is known how C24 sphingolipids function in the outer leaflet. Mutations in the ceramide synthase 2 gene is found to decrease C24. This severely perturbs homeostasis in mice and humans. Here, we investigated unilamellar vesicles and mammalian cells with C24 sphingomyelin exclusively in the outer leaflet. We provide evidence that outer leaflet C24 sphingomyelin suppresses microdomains in model membranes and live cells by partitioning cholesterol into the inner leaflet. We propose that C24 sphingolipids are critical to the function of the plasma membrane.

scholarly journals Analysing plasma membrane asymmetry of lipid organisation by fluorescence lifetime and correlation spectroscopy

2019 ◽  
Author(s):  
Anjali Gupta ◽  
Thomas Korte ◽  
Andreas Herrmann ◽  
Thorsten Wohland
Keyword(s):  
Plasma Membrane ◽  
Fluorescence Lifetime ◽  
Mammalian Cells ◽  
Correlation Spectroscopy ◽  
Live Cells ◽  
Fluorescence Lifetime Imaging ◽  
Outer Leaflet ◽  
Liquid Ordered ◽  
Fluorescent Lipid Analogues ◽  
Fluorescent Lipid

ABSTRACTA fundamental feature of a eukaryotic cell membrane is the asymmetric arrangement of lipids in the two leaflets. A cell invests significant energy to maintain this asymmetry and utilizes it to regulate important biological processes such as apoptosis and vesiculation. Here, we employ fluorescence lifetime imaging microscopy (FLIM) and imaging total internal reflection fluorescence correlation spectroscopy (ITIR-FCS) to differentiate the dynamics and organization of the exofacial and cytoplasmic leaflet of live mammalian cells. We characterize the biophysical properties of fluorescent analogues of phosphatidylcholine (PC), sphingomyelin (SM) and phosphatidylserine (PS) in two mammalian cell membranes. Due to their specific transverse membrane distribution, these probes allow leaflet specific investigation of the plasma membrane. We compare the results with regard to the different temporal and spatial resolution of the methods. Fluorescence lifetimes of fluorescent lipid analogues were found to be in a characteristic range for the liquid ordered phase in the outer leaflet and liquid disordered phase in the inner leaflet. The observation of a more fluid inner leaflet is supported by free diffusion in the inner leaflet with high average diffusion coefficients. The liquid ordered phase in the outer leaflet is accompanied by slower diffusion and diffusion with intermittent transient trapping. Our results show that the combination of FLIM and ITIR-FCS with specific fluorescent lipid analogues provides a powerful tool to investigate lateral and trans-bilayer characteristics of plasma membrane in live cells.Abstract Figure

scholarly journals Long acyl chain ceramides govern cholesterol and cytoskeleton dependence of membrane outer leaflet dynamics

2019 ◽  
Author(s):  
Anjali Gupta ◽  
Sneha Muralidharan ◽  
Federico Torta ◽  
Markus R. Wenk ◽  
Thorsten Wohland
Keyword(s):  
Plasma Membrane ◽  
Immunoglobulin E ◽  
Acyl Chain ◽  
Membrane Dynamics ◽  
Correlation Spectroscopy ◽  
Membrane Composition ◽  
Outer Leaflet ◽  
Lipid Mass ◽  
Ceramide Content ◽  
Acyl Chains

ABSTRACTThe cellular plasma membrane composition and organization is crucial for the regulation of biological processes. Based on our earlier work showing that the same lipid probe, DiI, exhibits different dynamics in CHO-K1 and RBL-2H3 cells, we investigate the molecular factors that govern these differences. First, we determined that the cytoskeleton-interacting Immunoglobulin E receptor (FcεRI), which is abundant in RBL-2H3 but not in CHO-K1 cells, is not responsible for the DiI confinement found in RBL-2H3 cells. Second, lipid mass spectrometry of the plasma membrane of the two cells indicated differences in ceramide content, especially with long and very long acyl chains (C16 to C24). We, therefore, measure membrane dynamics by imaging total internal reflection fluorescence correlation spectroscopy in dependence on these ceramides. Our results show that C24 and C16 saturated ceramides uniquely alter the membrane dynamics by promoting the formation of cholesterol-independent domains and by elevating inter-leaflet coupling.

scholarly journals Three Separable Domains Regulate GTP-Dependent Association of H-ras with the Plasma Membrane

2004 ◽  
Vol 24 (15) ◽  
pp. 6799-6810 ◽  
Author(s):  
Barak Rotblat ◽  
Ian A. Prior ◽  
Cornelia Muncke ◽  
Robert G. Parton ◽  
Yoel Kloog ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Lipid Rafts ◽  
Catalytic Domain ◽  
Attractive Force ◽  
Live Cells ◽  
Membrane Microdomains ◽  
Ras Signaling ◽  
Signaling Specificity ◽  
Linker Domain ◽  
Lipid Anchor

ABSTRACT The microlocalization of Ras proteins to different microdomains of the plasma membrane is critical for signaling specificity. Here we examine the complex membrane interactions of H-ras with a combination of FRAP on live cells to measure membrane affinity and electron microscopy of intact plasma membrane sheets to spatially map microdomains. We show that three separable forces operate on H-ras at the plasma membrane. The lipid anchor, comprising a processed CAAX motif and two palmitic acid residues, generates one attractive force that provides a high-affinity interaction with lipid rafts. The adjacent hypervariable linker domain provides a second attractive force but for nonraft plasma membrane microdomains. Operating against the attractive interaction of the lipid anchor for lipid rafts is a repulsive force generated by the N-terminal catalytic domain that increases when H-ras is GTP loaded. These observations lead directly to a novel mechanism that explains how H-ras lateral segregation is regulated by activation state: GTP loading decreases H-ras affinity for lipid rafts and allows the hypervariable linker domain to target to nonraft microdomains, the primary site of H-ras signaling.

scholarly journals Lipid Lateral Organization on Giant Unilamellar Vesicles Containing Lipopolysaccharides

2011 ◽  
Vol 100 (3) ◽  
pp. 341a
Author(s):  
Jakub Kubiak ◽  
Jonathan Brewer ◽  
Søren Hansen ◽  
Luis A. Bagatolli
Keyword(s):  
Giant Unilamellar Vesicles ◽  
Unilamellar Vesicles ◽  
Lateral Organization

scholarly journals Phosphatidylinositol Acyl Chains Configure TLR-Dependent Priming and Activation of the NLRP3 Inflammasome

2020 ◽  
Author(s):  
Claire Hamilton ◽  
Gerald Larrouy-Maumus ◽  
Paras K. Anand
Keyword(s):  
Abc Transporter ◽  
Nlrp3 Inflammasome ◽  
Metabolic Regulation ◽  
Metabolic Diseases ◽  
Acyl Chain ◽  
Membrane Microdomains ◽  
Inflammasome Activation ◽  
Acyl Chains ◽  
Transporter Deficiency ◽  
Phosphatidylinositol Phosphates

AbstractLipids are important in establishing cellular homeostasis by conducting varied functions including relay of extracellular signals. Imbalance in lipid homeostasis results in metabolic diseases, and is tightly connected to discrepancies in immune signalling. The phosphorylation status of the lipid second messenger phosphatidylinositol phosphates is implicated in key physiological functions and pathologies. By contrast, little is known as to how phosphatidylinositol (PI) lipid acyl chains contribute to cellular processes. Here, by employing a mass-spectrometry-based method, we show a role for PI acyl group chains in regulating NLRP3 inflammasome activation in cells lacking ABC transporter ABCB1. In response to canonical stimuli, Abcb1-/- cells revealed defective priming and activation of the NLRP3 inflammasome owing to blunted TLR-dependent signalling. Cellular lipidomics demonstrated that ABC transporter deficiency shifted the total PI balance such that Abcb1-/- cells exhibited reduced ratio of the short-chain to long-chain acyl chain lipids. Changes in PI acyl chain configuration accompanied diminished levels of ganglioside GM1, a marker of cholesterol-rich membrane microdomains, in deficient cells. Strikingly, this was not due to differences in the expression of enzymes that either synthesize PI or are involved in acyl chain remodelling. Our study thus suggests an important role for PI lipid chains in priming and activation of the NLRP3 inflammasome thereby highlighting the metabolic regulation of immune responses.

scholarly journals Biosynthesis and biology of mammalian GPI-anchored proteins

Open Biology ◽  
2020 ◽  
Vol 10 (3) ◽  
pp. 190290 ◽  
Author(s):  
Taroh Kinoshita
Keyword(s):  
Mammalian Cells ◽  
Membrane Microdomains ◽  
Membrane Rafts ◽  
Outer Leaflet ◽  
C Terminus ◽  
Polarized Cells ◽  
Human Proteins ◽  
Membrane Anchoring ◽  
Conserved Core ◽  
Protein Moiety

At least 150 human proteins are glycosylphosphatidylinositol-anchored proteins (GPI-APs). The protein moiety of GPI-APs lacking transmembrane domains is anchored to the plasma membrane with GPI covalently attached to the C-terminus. The GPI consists of the conserved core glycan, phosphatidylinositol and glycan side chains. The entire GPI-AP is anchored to the outer leaflet of the lipid bilayer by insertion of fatty chains of phosphatidylinositol. Because of GPI-dependent membrane anchoring, GPI-APs have some unique characteristics. The most prominent feature of GPI-APs is their association with membrane microdomains or membrane rafts. In the polarized cells such as epithelial cells, many GPI-APs are exclusively expressed in the apical surfaces, whereas some GPI-APs are preferentially expressed in the basolateral surfaces. Several GPI-APs act as transcytotic transporters carrying their ligands from one compartment to another. Some GPI-APs are shed from the membrane after cleavage within the GPI by a GPI-specific phospholipase or a glycosidase. In this review, I will summarize the current understanding of GPI-AP biosynthesis in mammalian cells and discuss examples of GPI-dependent functions of mammalian GPI-APs.

scholarly journals Efficient replacement of plasma membrane outer leaflet phospholipids and sphingolipids in cells with exogenous lipids

2016 ◽  
Vol 113 (49) ◽  
pp. 14025-14030 ◽  
Author(s):  
Guangtao Li ◽  
JiHyun Kim ◽  
Zhen Huang ◽  
Johnna R. St. Clair ◽  
Deborah A. Brown ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Lipid Composition ◽  
Mammalian Cells ◽  
Cellular Membrane ◽  
Membrane Lipid ◽  
Exchange Method ◽  
Cellular Behavior ◽  
Outer Leaflet ◽  
Lipid Exchange ◽  
Cellular Lipids

Our understanding of membranes and membrane lipid function has lagged far behind that of nucleic acids and proteins, largely because it is difficult to manipulate cellular membrane lipid composition. To help solve this problem, we show that methyl-α-cyclodextrin (MαCD)-catalyzed lipid exchange can be used to maximally replace the sphingolipids and phospholipids in the outer leaflet of the plasma membrane of living mammalian cells with exogenous lipids, including unnatural lipids. In addition, lipid exchange experiments revealed that 70–80% of cell sphingomyelin resided in the plasma membrane outer leaflet; the asymmetry of metabolically active cells was similar to that previously defined for erythrocytes, as judged by outer leaflet lipid composition; and plasma membrane outer leaflet phosphatidylcholine had a significantly lower level of unsaturation than phosphatidylcholine in the remainder of the cell. The data also provided a rough estimate for the total cellular lipids residing in the plasma membrane (about half). In addition to such lipidomics applications, the exchange method should have wide potential for investigations of lipid function and modification of cellular behavior by modification of lipids.

scholarly journals Surfactant-free production of biomimetic giant unilamellar vesicles using PDMS-based microfluidics

2021 ◽  
Vol 4 (1) ◽  
Author(s):  
Naresh Yandrapalli ◽  
Julien Petit ◽  
Oliver Bäumchen ◽  
Tom Robinson
Keyword(s):  
High Throughput ◽  
Paradigm Shift ◽  
Mammalian Cells ◽  
Lipid Vesicles ◽  
Giant Unilamellar Vesicles ◽  
Unilamellar Vesicles ◽  
Artificial Cells ◽  
Membrane Function ◽  
Lipid Diffusion ◽  
Protein Incorporation

AbstractMicrofluidic production of giant lipid vesicles presents a paradigm-shift in the development of artificial cells. While production is high-throughput and the lipid vesicles are mono-disperse compared to bulk methods, current technologies rely heavily on the addition of additives such as surfactants, glycerol and even ethanol. Here we present a microfluidic method for producing biomimetic surfactant-free and additive-free giant unilamellar vesicles. The versatile design allows for the production of vesicle sizes ranging anywhere from ~10 to 130 µm with either neutral or charged lipids, and in physiological buffer conditions. Purity, functionality, and stability of the membranes are validated by lipid diffusion, protein incorporation, and leakage assays. Usability as artificial cells is demonstrated by increasing their complexity, i.e., by encapsulating plasmids, smaller liposomes, mammalian cells, and microspheres. This robust method capable of creating truly biomimetic artificial cells in high-throughput will prove valuable for bottom-up synthetic biology and the understanding of membrane function.

scholarly journals A novel mitochondrial Kv1.3–caveolin axis controls cell survival and apoptosis

eLife ◽  
2021 ◽  
Vol 10 ◽  
Author(s):  
Jesusa Capera ◽  
Mireia Pérez-Verdaguer ◽  
Roberta Peruzzo ◽  
María Navarro-Pérez ◽  
Juan Martínez-Pinna ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Cell Survival ◽  
Mammalian Cells ◽  
Physiological Role ◽  
Membrane Microdomains ◽  
Functional Link ◽  
Caveolin 1 ◽  
Molecular Determinants ◽  
Apoptotic Effects

The voltage-gated potassium channel Kv1.3 plays an apparent dual physiological role by participating in activation and proliferation of leukocytes as well as promoting apoptosis in several types of tumor cells. Therefore, Kv1.3 is considered a potential pharmacological target for immunodeficiency and cancer. Different cellular locations of Kv1.3, at the plasma membrane or the mitochondria, could be responsible for such duality. While plasma membrane Kv1.3 facilitates proliferation, the mitochondrial channel modulates apoptotic signaling. Several molecular determinants of Kv1.3 drive the channel to the cell surface, but no information is available about its mitochondrial targeting. Caveolins, which are able to modulate cell survival, participate in the plasma membrane targeting of Kv1.3. The channel, via a caveolin-binding domain (CDB), associates with caveolin 1 (Cav1), which localizes Kv1.3 to lipid raft membrane microdomains. The aim of our study was to understand the role of such interactions not only for channel targeting but also for cell survival in mammalian cells. By using a caveolin association-deficient channel (Kv1.3 CDBless), we demonstrate here that while the Kv1.3–Cav1 interaction is responsible for the channel localization in the plasma membrane, a lack of such interaction accumulates Kv1.3 in the mitochondria. Kv1.3 CDBless severely affects mitochondrial physiology and cell survival, indicating that a functional link of Kv1.3 with Cav1 within the mitochondria modulates the pro-apoptotic effects of the channel. Therefore, the balance exerted by these two complementary mechanisms fine-tune the physiological role of Kv1.3 during cell survival or apoptosis. Our data highlight an unexpected role for the mitochondrial caveolin–Kv1.3 axis during cell survival and apoptosis.

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