Measuring Membrane Lipid Turnover with the pH-sensitive Fluorescent Lipid Analog ND6

10.3791/62717 ◽  
2021 ◽  
Author(s):  
Shahriar Alamgir ◽  
Oliver B. Pelletier ◽  
Deborah Thomas ◽  
Vicente Rubio ◽  
Maciej J. Stawikowski ◽  
...  
Keyword(s):  
Membrane Lipid ◽  
Ph Sensitive ◽  
Lipid Turnover ◽  
Fluorescent Lipid

Sphingolipids related to apoptosis from the point of view of membrane structure and topology

2001 ◽  
Vol 29 (6) ◽  
pp. 819-824 ◽  
Author(s):  
W. J. van Blitterswijk ◽  
A. H. van der Luit ◽  
W. Caan ◽  
M. Verheij ◽  
J. Borst
Keyword(s):  
Membrane Structure ◽  
De Novo ◽  
Cell Signalling ◽  
Membrane Lipid ◽  
Point Of View ◽  
Sphingolipid Metabolism ◽  
Membrane Microdomains ◽  
And Topology ◽  
Lipid Turnover ◽  
Ceramide Accumulation

Sphingolipids and their metabolites are implicated in signal transduction, but the mechanisms are still poorly understood. In particular, the presumed function of ceramide as a second messenger remains controversial. Here, we emphasize the importance of both ceramide and sphingomyelin for membrane structure. The effects of sphingo-lipid turnover in the induction and effector phases of apoptosis are explained by their impact on membrane microdomains that are relevant for cell signalling or changes in morphology. The topology of sphingolipid metabolism is important because of their limited transbilayer and inter-membrane movement. For instance, glycosylceramide synthase converts de novo synthesized ceramide to glycosylceramide, but it is neither a general attenuator of ceramide accumulation at the plasma membrane, nor of the apoptotic process. Synthetic alkyl-lysophospholipids modulate membrane-lipid composition and, therefore, apoptosis sensitivity.

scholarly journals Lipid recycling between the plasma membrane and intracellular compartments: transport and metabolism of fluorescent sphingomyelin analogues in cultured fibroblasts.

1989 ◽  
Vol 108 (6) ◽  
pp. 2169-2181 ◽  
Author(s):  
M Koval ◽  
R E Pagano
Keyword(s):  
Plasma Membrane ◽  
Intracellular Transport ◽  
Chinese Hamster ◽  
Membrane Lipid ◽  
Cell Periphery ◽  
Perinuclear Region ◽  
Cultured Fibroblasts ◽  
Plasma Membrane Lipid ◽  
Intracellular Compartments ◽  
Fluorescent Lipid

We examined the metabolism and intracellular transport of the D-erythro and L-threo stereoisomers of a fluorescent analogue of sphingomyelin, N-(N-[6-[(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino] caproyl])-sphingosylphosphorylcholine (C6-NBD-SM), in Chinese hamster ovary (CHO-K1) fibroblast monolayers. C6-NBD-SM was integrated into the plasma membrane bilayer by transfer of C6-NBD-SM monomers from liposomes to cells at 7 degrees C. The cells were washed, and within 10-15 min of being warmed to 37 degrees C, C6-NBD-SM was internalized from the plasma membrane to a perinuclear location that colocalized with the centriole and was distinct from the lysosomes and the Golgi apparatus. This perinuclear region was also labeled by internalized rhodamine-conjugated transferrin. C6-NBD-SM endocytosis was not inhibited when the microtubules were disrupted with nocodazole; rather, the fluorescent lipid was distributed in vesicles throughout the cell periphery instead of being internalized to the perinuclear region of the cell. The metabolism of C6-NBD-SM to other fluorescent sphingolipids at 37 degrees C and its effect on C6-NBD-SM transport was also examined. To study plasma membrane lipid recycling, C6-NBD-SM was first inserted into the plasma membrane of CHO-K1 cells and then allowed to be internalized by the cells at 37 degrees C. Any C6-NBD-SM remaining at the plasma membrane was then removed by incubation with nonfluorescent liposomes at 7 degrees C, leaving cells containing only internalized fluorescent lipid. The return of C6-NBD-SM to the plasma membrane from intracellular compartments upon further 37 degrees C incubation was then observed. The half-time for a complete round C6-NBD-SM recycling between the plasma membrane and intracellular compartments was approximately 40 min. Pretreatment of cells with either monensin or nocodazole did not inhibit C6-NBD-SM recycling.

scholarly journals Ten steps to investigate a cellular system with mathematical modeling

2021 ◽  
Vol 17 (5) ◽  
pp. e1008921
Author(s):  
Jasia King ◽  
Kerbaï Saïd Eroumé ◽  
Roman Truckenmüller ◽  
Stefan Giselbrecht ◽  
Ann E. Cowan ◽  
...  
Keyword(s):  
Mathematical Modeling ◽  
In Silico ◽  
Membrane Lipid ◽  
Cellular System ◽  
Spatiotemporal Resolution ◽  
Number Of Components ◽  
Modeling Process ◽  
Lipid Turnover ◽  
Generic Modeling ◽  
Spatiotemporal Scales

Cellular and intracellular processes are inherently complex due to the large number of components and interactions, which are often nonlinear and occur at different spatiotemporal scales. Because of this complexity, mathematical modeling is increasingly used to simulate such systems and perform experiments in silico, many orders of magnitude faster than real experiments and often at a higher spatiotemporal resolution. In this article, we will focus on the generic modeling process and illustrate it with an example model of membrane lipid turnover.

scholarly journals Transport of a fluorescent phosphatidylcholine analog from the plasma membrane to the Golgi apparatus.

1984 ◽  
Vol 99 (2) ◽  
pp. 742-751 ◽  
Author(s):  
R G Sleight ◽  
R E Pagano
Keyword(s):  
Plasma Membrane ◽  
Golgi Apparatus ◽  
Cell Surface ◽  
Fluorescent Protein ◽  
Chinese Hamster ◽  
Membrane Lipid ◽  
Lipid Transfer ◽  
Outer Leaflet ◽  
Membrane Lipid Bilayer ◽  
Fluorescent Lipid

We have examined the internalization and degradation of a fluorescent analog of phosphatidylcholine after its insertion into the plasma membrane of cultured Chinese hamster fibroblasts. 1-acyl-2-(N-4-nitrobenzo-2-oxa-1,3-diazole)-aminocaproyl phosphatidylcholine (C6-NBD-PC) was incorporated into the cell surface by liposome-cell lipid transfer at 2 degrees C. The fluorescent lipid remained localized at the plasma membrane as long as the cells were kept at 2 degrees C; however, when the cells were warmed to 37 degrees C, internalization of some of the fluorescent lipid occurred. Most of the internalized C6-NBD-PC accumulated in the Golgi apparatus although a small amount was found randomly distributed throughout the cytoplasm in punctate fluorescent structures. Internalization of the fluorescent lipid at 37 degrees C was blocked by the presence of inhibitors of endocytosis. Incubation of cells containing C6-NBD-PC at 37 degrees C resulted in a rapid degradation of the fluorescent lipid. This degradation occurred predominantly at the plasma membrane. The degradation of C6-NBD-PC resulted in the release of NBD-fatty acid into the medium. We have compared the internalization of the fluorescent lipid with that of a fluorescent protein bound to the cell surface. Both fluorescent lipid and protein remained at the plasma membrane at 2 degrees C and neither were internalized at 37 degrees C in the presence of inhibitors of endocytosis. However, when incubated at 37 degrees C under conditions that permit endocytosis, the two fluorescent species appeared at different intracellular sites. Our data suggest that there is no transmembrane movement of C6-NBD-PC and that the fluorescent probe reflects the internalization of the outer leaflet of the plasma membrane lipid bilayer. The results are consistent with the Golgi apparatus as being the primary delivery site of phospholipid by bulk membrane movement from the plasma membrane.

Inhibition of Platelet Aggregation by Ethanol in Vitro Shows Specificity for Aggregating Agent Used and Is Influenced by Platelet Lipid Composition

1982 ◽  
Vol 48 (01) ◽  
pp. 049-053 ◽  
Author(s):  
C G Fenn ◽  
J M Littleton
Keyword(s):  
Platelet Aggregation ◽  
Lipid Composition ◽  
Saturated Fatty Acids ◽  
Calcium Ionophore ◽  
Cholesterol Content ◽  
Membrane Lipid ◽  
Calcium Ionophore A23187 ◽  
Ionophore A23187 ◽  
Increased Sensitivity

SummaryEthanol at physiologically tolerable concentrations inhibited platelet aggregation in vitro in a relatively specific way, which may be influenced by platelet membrane lipid composition. Aggregation to collagen, calcium ionophore A23187 and thrombin (low doses) were often markedly inhibited by ethanol, adrenaline and ADP responses were little affected, and aggregation to exogenous arachidonic acid was actually potentiated by ethanol. Aggregation to collagen, thrombin and A23187 was inhibited more by ethanol in platelets enriched with saturated fatty acids than in those enriched with unsaturated fats. Platelets enriched with cholesterol showed increased sensitivity to ADP, arachidonate and adrenaline but this increase in cholesterol content did not appear to influence the inhibition by ethanol of platelet responses. The results suggest that ethanol may inhibit aggregation by an effect on membrane fluidity and/or calcium mobilization resulting in decreased activity of a membrane-bound phospholipase.

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