Structure and cholesterol domain dynamics of an enriched caveolae/raft isolate

Despite the importance of cholesterol in the formation and function of caveolar microdomains in plasma membranes, almost nothing is known regarding the structural properties, cholesterol dynamics or intracellular factors affecting caveolar cholesterol dynamics. A non-detergent method was employed to isolate caveolae/raft domains from purified plasma membranes of murine fibroblasts. A series of fluorescent lipid probe molecules or a fluorescent cholesterol analogue, dehydroergosterol, were then incorporated into the caveolae/raft domains to show that: (i) fluorescence polarization of the multiple probe molecules {diphenylhexatriene analogues, DiI18 (1,1′-dioctadecyl-3,3,3′,3′-tetramethylindocarbocyanine perchlorate), parinaric acids and NBD-stearic acid {12-(N-methyl)-N-[(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino]-octadecanoic acid} indicated that acyl chains in caveolae/raft domains were significantly less ‘fluid’ (i.e. more rigid) and the transbilayer ‘fluidity gradient’ was 4.4-fold greater than in plasma membranes; (ii) although sterol was more ordered in caveolae/raft domains than plasma membranes, spontaneous sterol transfer from caveolae/raft domains was faster (initial rate, 32%; half-time, t1/2, 57%) than from the plasma membrane; (iii) although kinetic analysis showed similar proportions of exchangeable and non-exchangeable sterol pools in caveolae/raft domains and plasma membranes, addition of SCP-2 (sterol carrier protein-2) 1.3-fold more selectively increased sterol transfer from caveolae/raft domains by decreasing the t1/2 (50%) and increasing the initial rate (5-fold); (iv) SCP-2 was also 2-fold more selective in decreasing the amount of non-exchangeable sterol in caveolae/raft domains compared with plasma membranes, such that nearly 80% of caveolar/raft sterol became exchangeable. In summary, although caveolae/raft lipids were less fluid than those of plasma membranes, sterol domains in caveolae/rafts were more spontaneously exchangeable and more affected by SCP-2 than those of the bulk plasma membranes. Thus caveolae/raft domains isolated without the use of detergents display unique structure, cholesterol domain kinetics and responsiveness to SCP-2 as compared with the parent plasma membrane.

Download Full-text

myo-Inositol 1,4,5-trisphosphate mobilizes Ca2+ from isolated adipocyte endoplasmic reticulum but not from plasma membranes

Biochemical Journal ◽

10.1042/bj2360037 ◽

1986 ◽

Vol 236 (1) ◽

pp. 37-44 ◽

Cited By ~ 42

Author(s):

D M Delfert ◽

S Hill ◽

H A Pershadsingh ◽

W R Sherman ◽

J M McDonald

Keyword(s):

Endoplasmic Reticulum ◽

Plasma Membrane ◽

Plasma Membranes ◽

Initial Rate ◽

Membrane Vesicles ◽

Heavy Fraction ◽

Differential Centrifugation ◽

Plasma Membrane Vesicles ◽

Inositol 1,4,5 Trisphosphate ◽

Uptake And Release

The effects of myo-inositol 1,4,5-trisphosphate (IP3) on Ca2+ uptake and release from isolated adipocyte endoplasmic reticulum and plasma membrane vesicles were investigated. Effects of IP3 were initially characterized using an endoplasmic reticulum preparation with cytosol present (S1-ER). Maximal and half-maximal effects of IP3 on Ca2+ release from S1-ER vesicles occurred at 20 microM- and 7 microM-IP3, respectively, in the presence of vanadate which prevents the re-uptake of released Ca2+ via the endoplasmic reticulum Ca2+ pump. At saturating IP3 concentrations, Ca2+ release in the presence of vanadate was 20% of the exchangeable Ca2+ pool. IP3-induced release of Ca2+ from S1-ER was dependent on extravesicular free Ca2+ concentration with maximal release occurring at 0.13 microM free Ca2+. At 20 microM-IP3 there was no effect on the initial rate of Ca2+ uptake by S1-ER. IP3 promoted Ca2+ release from isolated endoplasmic reticulum vesicles (cytosol not present) to a similar level as compared with S1-ER. Addition of cytosol to isolated endoplasmic reticulum vesicles did not affect IP3-induced Ca2+ release. The endoplasmic reticulum preparation was further fractionated into heavy and light vesicles by differential centrifugation. Interestingly, the heavy fraction, but not the light fraction, released Ca2+ when challenged with IP3. IP3 (20 microM) did not promote Ca2+ release from plasma membrane vesicles and had no effect on the (Ca2+ + Mg2+)-ATPase activity or on the initial rate of ATP-dependent Ca2+ uptake by these vesicles. These results support the concept that IP3 acts exclusively at the endoplasmic reticulum to promote Ca2+ release.

Download Full-text

Sphingolipids: membrane microdomains in brain development, function and neurological diseases

Open Biology ◽

10.1098/rsob.170069 ◽

2017 ◽

Vol 7 (5) ◽

pp. 170069 ◽

Cited By ~ 81

Author(s):

Anne S. B. Olsen ◽

Nils J. Færgeman

Keyword(s):

Plasma Membrane ◽

Brain Development ◽

Plasma Membranes ◽

Neurological Diseases ◽

Sphingolipid Metabolism ◽

Membrane Microdomains ◽

Vast Number ◽

Cellular Events ◽

Long Time ◽

And Function

Sphingolipids are highly enriched in the nervous system where they are pivotal constituents of the plasma membranes and are important for proper brain development and functions. Sphingolipids are not merely structural elements, but are also recognized as regulators of cellular events by their ability to form microdomains in the plasma membrane. The significance of such compartmentalization spans broadly from being involved in differentiation of neurons and synaptic transmission to neuronal–glial interactions and myelin stability. Thus, perturbations of the sphingolipid metabolism can lead to rearrangements in the plasma membrane, which has been linked to the development of various neurological diseases. Studying microdomains and their functions has for a long time been synonymous with studying the role of cholesterol. However, it is becoming increasingly clear that microdomains are very heterogeneous, which among others can be ascribed to the vast number of sphingolipids. In this review, we discuss the importance of microdomains with emphasis on sphingolipids in brain development and function as well as how disruption of the sphingolipid metabolism (and hence microdomains) contributes to the pathogenesis of several neurological diseases.

Download Full-text

Abstract 5385: Transport of Free Fatty Acids in Cardiac Myocytes is Mediated by a Plasma Membrane Pump as Revealed by Monitoring Cytoplasmic Unbound Free Fatty Acids

Circulation ◽

10.1161/circ.118.suppl_18.s_539-d ◽

2008 ◽

Vol 118 (suppl_18) ◽

Author(s):

Andrew N Carley ◽

J P Kampf ◽

Alan M Kleinfeld

Keyword(s):

Fatty Acids ◽

Plasma Membrane ◽

Steady State ◽

Free Fatty Acids ◽

Cardiac Myocytes ◽

Time Course ◽

Plasma Membranes ◽

Initial Rate ◽

Ffa Metabolism ◽

Ffa Transport

The transport of FFA across the plasma membrane represents one of the earliest points at which FFA metabolism can be controlled by cardiac myocytes. Using novel methods to measure the intracellular unbound concentration of FFA ([FFA i ]), the first direct measurements of FFA transport across cardiac plasma membranes have been performed in freshly isolated cardiac myoctyes. Measurements of the unbound concentrations of FFA (FFA u ) in the aqueous phase were performed using the fluorescent ratio probe ADIFAB. Cardiac myocytes were microinjected with ADIFAB, and the transport of oleate and palmitate was determined by monitoring [FFA i ] using fluorescence ratio microscopy. FFA influx was initiated by rapidly increasing the extracellular concentration of FFA u ([FFA o ]) using FFA-BSA complexes, which clamped [FFA o ] at fixed values. The time course of influx was monitored from the change in [FFA i ], which rose exponentially to a steady state level (k influx ~ 0.01 s −1 ). Once steady state was achieved, efflux was initiated by changing the extracellular media back to zero [FFA o ]. Efflux was monitored by the decrease in [FFA i ] which, like influx, revealed exponential behavior (k efflux ~ 0.02 s −1 ). At steady state [FFA i ] was greater than [FFA o ] by a factor of ~3.5, indicating that during influx FFA are pumped up a concentration gradient. Both the initial rate of transport and the gradient ([FFA i ] > [FFA o ]) revealed saturation with increasing [FFA o ]. The initial rate of influx saturated at [FFA o ] > 200 nM, while the [FFA i ] > [FFA o ] gradient was relatively constant (~ 3.5) but began to decrease and approached 1 at [FFA o ] > 200 nM. The efflux rate constant decreased for [FFA o ] > zero, suggesting that efflux may be regulated by a mechanism that senses the level of circulating FFA u . Our results indicate that the mechanism of FFA transport across cardiac myocytes is regulated by the plasma membrane and allows for the efficient storage and release of FFA from cardiac myocytes. We suggest that this mechanism involves an as yet unknown membrane protein pump which enables the cells to accumulate surprisingly high concentrations of FFA. The ability to measure [FFA i ] and the demonstration of efflux are significant steps in understanding cardiac FFA metabolism. This research has received full or partial funding support from the American Heart Association, AHA Western States Affiliate (California, Nevada & Utah).

Download Full-text

Structure and Function of the Plasma Membrane

The Journal of General Physiology ◽

10.1085/jgp.52.1.257 ◽

1968 ◽

Vol 52 (1) ◽

pp. 257-278 ◽

Cited By ~ 54

Author(s):

Edward D. Korn

Keyword(s):

Plasma Membrane ◽

Plasma Membranes ◽

Current Knowledge ◽

Structure And Function ◽

Attractive Alternative ◽

Membrane Function ◽

Classical Models ◽

And Function ◽

Direct Attack ◽

Membrane Biosynthesis

The paucimolecular unit membrane model of the structure of the plasma membrane is critically reviewed in relation to current knowledge of the chemical and enzymatic composition of isolated plasma membranes, the properties of phospholipids, the chemistry of fixation for electron microscopy, the conformation of membrane proteins, the nature of the lipid-protein bonds in membranes, and possible mechanisms of transmembrane transport and membrane biosynthesis. It is concluded that the classical models, although not disproven, are not well supported by, and are difficult to reconcile with, the data now available. On the other hand, although a model based on lipoprotein subunits is, from a biochemical perspective, an attractive alternative, it too is far from proven. Many of the questions may be resolved by studies of membrane function and membrane biosynthesis rather than by a direct attack on membrane structure.

Download Full-text

Raft-based sphingomyelin interactions revealed by new fluorescent sphingomyelin analogs

The Journal of Cell Biology ◽

10.1083/jcb.201607086 ◽

2017 ◽

Vol 216 (4) ◽

pp. 1183-1204 ◽

Cited By ~ 57

Author(s):

Masanao Kinoshita ◽

Kenichi G.N. Suzuki ◽

Nobuaki Matsumori ◽

Misa Takada ◽

Hikaru Ano ◽

...

Keyword(s):

Plasma Membrane ◽

Positive Charge ◽

Live Cell ◽

Fluorescent Molecule ◽

Raft Domains ◽

And Function

Sphingomyelin (SM) has been proposed to form cholesterol-dependent raft domains and sphingolipid domains in the plasma membrane (PM). How SM contributes to the formation and function of these domains remains unknown, primarily because of the scarcity of suitable fluorescent SM analogs. We developed new fluorescent SM analogs by conjugating a hydrophilic fluorophore to the SM choline headgroup without eliminating its positive charge, via a hydrophilic nonaethylene glycol linker. The new analogs behaved similarly to the native SM in terms of their partitioning behaviors in artificial liquid order-disorder phase-separated membranes and detergent-resistant PM preparations. Single fluorescent molecule tracking in the live-cell PM revealed that they indirectly interact with each other in cholesterol- and sphingosine backbone–dependent manners, and that, for ∼10–50 ms, they undergo transient colocalization-codiffusion with a glycosylphosphatidylinositol (GPI)-anchored protein, CD59 (in monomers, transient-dimer rafts, and clusters), in CD59-oligomer size–, cholesterol-, and GPI anchoring–dependent manners. These results suggest that SM continually and rapidly exchanges between CD59-associated raft domains and the bulk PM.

Download Full-text

Chronic alcohol feeding and its withdrawal on the structure and function of the rat liver plasma membrane: a study with 125I-labelled glucagon binding as a metabolic probe

Canadian Journal of Physiology and Pharmacology ◽

10.1139/y82-170 ◽

1982 ◽

Vol 60 (9) ◽

pp. 1171-1176 ◽

Cited By ~ 9

Author(s):

Hung Lee ◽

E. A. Hosein

Keyword(s):

Plasma Membrane ◽

Rat Liver ◽

Plasma Membranes ◽

Structure And Function ◽

Scatchard Analysis ◽

Chronic Alcohol ◽

Alcohol Administration ◽

Liver Plasma Membranes ◽

And Function ◽

Chronic Alcohol Administration

The effect of chronic alcohol administration on the structure and function of the rat liver plasma membranes has been investigated. Chronic alcohol administration did not affect the yield of these membranes using conventional isolation procedures. The extent of plasma membrane enrichment or contamination with other interior membranes was identical in the control and alcoholic preparations. The binding of 125I-labelled glucagon to these experimental liver plasma membranes was significantly decreased. Scatchard analysis of the high affinity sites showed a significant reduction [Formula: see text] in receptor number rather than binding affinity, which was not altered. This anomaly persisted through 72-h withdrawal of alcohol. These data suggest that very stable changes were induced in these liver plasma membranes after prolonged alcohol ingestion.

Download Full-text

Plasma membrane organization and function: moving past lipid rafts

Molecular Biology of the Cell ◽

10.1091/mbc.e13-03-0165 ◽

2013 ◽

Vol 24 (18) ◽

pp. 2765-2768 ◽

Cited By ~ 107

Author(s):

Mary L. Kraft

Keyword(s):

Plasma Membrane ◽

Lipid Rafts ◽

Protein Interactions ◽

Lipid Raft ◽

Mammalian Cells ◽

Plasma Membranes ◽

Imaging Techniques ◽

Cellular Function ◽

Membrane Organization ◽

And Function

“Lipid raft” is the name given to the tiny, dynamic, and ordered domains of cholesterol and sphingolipids that are hypothesized to exist in the plasma membranes of eukaryotic cells. According to the lipid raft hypothesis, these cholesterol- and sphingolipid-enriched domains modulate the protein–protein interactions that are essential for cellular function. Indeed, many studies have shown that cellular levels of cholesterol and sphingolipids influence plasma membrane organization, cell signaling, and other important biological processes. Despite 15 years of research and the application of highly advanced imaging techniques, data that unambiguously demonstrate the existence of lipid rafts in mammalian cells are still lacking. This Perspective summarizes the results that challenge the lipid raft hypothesis and discusses alternative hypothetical models of plasma membrane organization and lipid-mediated cellular function.

Download Full-text

Membrane microdomains: lessons from microscopy

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s0424820100140257 ◽

1995 ◽

Vol 53 ◽

pp. 776-777

Author(s):

J.M. Robinson ◽

J.M Oliver

Keyword(s):

Spatial Distribution ◽

Plasma Membrane ◽

Epithelial Cells ◽

Plasma Membranes ◽

Cell Types ◽

Imaging Modalities ◽

Membrane Microdomains ◽

Membrane Domains ◽

Lateral Heterogeneity ◽

Functional Importance

Specialized regions of plasma membranes displaying lateral heterogeneity are the focus of this Symposium. Specialized membrane domains are known for certain cell types such as differentiated epithelial cells where lateral heterogeneity in lipids and proteins exists between the apical and basolateral portions of the plasma membrane. Lateral heterogeneity and the presence of microdomains in membranes that are uniform in appearance have been more difficult to establish. Nonetheless a number of studies have provided evidence for membrane microdomains and indicated a functional importance for these structures.This symposium will focus on the use of various imaging modalities and related approaches to define membrane microdomains in a number of cell types. The importance of existing as well as emerging imaging technologies for use in the elucidation of membrane microdomains will be highlighted. The organization of membrane microdomains in terms of dimensions and spatial distribution is of considerable interest and will be addressed in this Symposium.

Download Full-text

Caveolin-1, galectin-3 and lipid raft domains in cancer cell signalling

Essays in Biochemistry ◽

10.1042/bse0570189 ◽

2015 ◽

Vol 57 ◽

pp. 189-201 ◽

Cited By ~ 13

Author(s):

Jay Shankar ◽

Cecile Boscher ◽

Ivan R. Nabi

Keyword(s):

Plasma Membrane ◽

Lipid Rafts ◽

Cancer Cell ◽

Cellular Response ◽

Spatial Organization ◽

Essential Feature ◽

Cell Signalling ◽

Coated Pits ◽

Signalling Molecules ◽

Raft Domains

Spatial organization of the plasma membrane is an essential feature of the cellular response to external stimuli. Receptor organization at the cell surface mediates transmission of extracellular stimuli to intracellular signalling molecules and effectors that impact various cellular processes including cell differentiation, metabolism, growth, migration and apoptosis. Membrane domains include morphologically distinct plasma membrane invaginations such as clathrin-coated pits and caveolae, but also less well-defined domains such as lipid rafts and the galectin lattice. In the present chapter, we will discuss interaction between caveolae, lipid rafts and the galectin lattice in the control of cancer cell signalling.

Download Full-text