Lipid rafts and plasma membrane microorganization: insights from Ras

The spatial organization of plasma membrane components in discrete microdomains is thought to be a key factor in the generation of distinct signal outputs. A detailed characterization of plasma membrane microdomains, including descriptions of their size, dynamics and abundance, has proved to be a taxing problem for cell biologists and biophysicists. The use of novel techniques is providing exciting new insights into the challenging problem of plasma membrane microstructure and has allowed the visualization of domains with the characteristics expected of lipid rafts - microdomains of the plasma membrane enriched in cholesterol and sphingolipids. This review focuses on some of these recent advances and uses Ras signaling as a paradigm for understanding inner plasma membrane organization and the role of lipid rafts in cellular function.

Three separable domains regulate GTP-dependent association of H-ras with the plasma membrane

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.

The origin and evolution of the surfactant system in fish: Insights into the evolution of lungs and swim bladders

Several times throughout their radiation fish have evolved either lungs or swim bladders as gas-holding structures. Lungs and swim bladders have different ontogenetic origins and can be used either for buoyancy or as an accessory respiratory organ. Therefore, the presence of air-filled bladders or lungs in different groups of fishes is an example of convergent evolution. We propose that air breathing could not occur without the presence of a surfactant system and suggest that this system may have originated in epithelial cells lining the pharynx. Here we present new data on the surfactant system in swim bladders of three teleost fish ( the air-breathing pirarucu Arapaima gigas and tarpon Megalops cyprinoides and the non-air-breathing New Zealand snapper Pagrus auratus). We determined the presence of surfactant using biochemical, biophysical, and morphological analyses and determined homology using immunohistochemical analysis of the surfactant proteins (SPs). We relate the presence and structure of the surfactant system to those previously described in the swim bladders of another teleost, the goldfish, and those of the air-breathing organs of the other members of the Osteichthyes, the more primitive air-breathing Actinopterygii and the Sarcopterygii. Snapper and tarpon swim bladders are lined with squamous and cuboidal epithelial cells, respectively, containing membrane-bound lamellar bodies. Phosphatidylcholine dominates the phospholipid (PL) profile of lavage material from all fish analyzed to date. The presence of the characteristic surfactant lipids in pirarucu and tarpon, lamellar bodies in tarpon and snapper, SP-B in tarpon and pirarucu lavage, and SPs ( A, B, and D) in swim bladder tissue of the tarpon provide strong evidence that the surfactant system of teleosts is homologous with that of other fish and of tetrapods. This study is the first demonstration of the presence of SP-D in the air-breathing organs of nonmammalian species and SP-B in actinopterygian fishes. The extremely high cholesterol/disaturated PL and cholesterol/PL ratios of surfactant extracted from tarpon and pirarucu bladders and the poor surface activity of tarpon surfactant are characteristics of the surfactant system in other fishes. Despite the paraphyletic phylogeny of the Osteichthyes, their surfactant is uniform in composition and may represent the vertebrate protosurfactant.

H-ras, K-ras, and inner plasma membrane raft proteins operate in nanoclusters with differential dependence on the actin cytoskeleton

Plasma membrane compartmentalization imposes lateral segregation on membrane proteins that is important for regulating signal transduction. We use computational modeling of immunogold spatial point patterns on intact plasma membrane sheets to test different models of inner plasma membrane organization. We find compartmentalization at the nanoscale level but show that a classical raft model of preexisting stable domains into which lipid raft proteins partition is incompatible with the spatial point patterns generated by the immunogold labeling of a palmitoylated raft marker protein. Rather, approximate to 30% of the raft protein exists in cholesterol-dependent nanoclusters, with approximate to 70% distributed as monomers. The cluster/monomer ratio (number of proteins in clusters/number of proteins outside clusters) is independent of expression level. H-rasG12V and K-rasG12V proteins also operate in nanoclusters with fixed cluster/monomer ratios that are independent of expression level. Detailed calibration of the immunogold imaging protocol suggests that radii of raft and RasG12V protein nanoclusters may be as small as 11 and 6 nm, respectively, and shows that the nanoclusters contain small numbers (6.0-7.7) of proteins. Raft nanoclusters do not form if the actin cytoskeleton is disassembled. The formation of K-rasG12V but not H-rasG12V nanoclusters also is actin-dependent. K-rasG12V but not H-rasG12V signaling is abrogated by actin cytoskeleton disassembly, which shows that nanoclustering is critical for Ras function. These findings argue against stable preexisting domains on the inner plasma membrane in favor of dynamic actively regulated nanoclusters similar to those proposed for the outer plasma membrane. RasG12V nanoclusters may facilitate the assembly of essential signal transduction complexes.

Individual Palmitoyl Residues Serve Distinct Roles in H-ras Trafficking, Microlocalization, and Signaling

H-ras is anchored to the plasma membrane by two palmitoylated cysteine residues, Cys181 and Cys184, operating in concert with a C-terminal S-farnesyl cysteine carboxymethylester. Here we demonstrate that the two palmitates serve distinct biological roles. Monopalmitoylation of Cys181 is required and sufficient for efficient trafficking of H-ras to the plasma membrane, whereas monopallmitoylation of Cys184 does not permit efficient trafficking beyond the Golgi apparatus. However, once at the plasma membrane, monopalmitoylation of Cys184 supports correct GTP-regulated lateral segregation of H-ras between cbolesterol-dependent and cholesterol-independent microdomains. In contrast, monopallmitoylation of Cys181 dramatically reverses H-ras lateral segregation, driving GTP-loaded H-ras into cholesterol-dependent microdomains. Intriguingly, the Cys181 monopalmitoylated H-ras anchor emulates the GTP-regulated microdomain interactions of N-ras. These results identify N-ras as the Ras isoform that normally signals from lipid rafts but also reveal that spacing between palmitate and prenyl groups influences anchor interactions with the lipid bilayer. This concept is further supported by the different plasma membrane affinities of the monopalmitoylated anchors: Cys181-palmitate is equivalent to the dually palmitoylated wild-type anchor, whereas Cys184-pahnitate is weaker. Thus, membrane affinity of a pallmitoylated anchor is a function both of the hydrophobicity of the lipid moieties and their spatial organization. Finally we show that the plasma membrane affinity of monopahnitoylated anchors is absolutely dependent on cholesterol, identifying a new role for cholesterol in promoting interactions with the raft and nonraft plasma membrane.

W/O microemulsions for ocular delivery: Evaluation of ocular irritation and precorneal retention

Water-in-oil microemulsions (w/o ME) capable of undergoing a phase-transition to lamellar liquid crystals (LC) or bicontinuous ME upon aqueous dilution were formulated using Crodarnol EO, Crill 1 and Crillet 4, an alkanol or alkanediol as cosurfactant and water. The hypothesis that phase-transition of ME to LC may be induced by tears and serve to prolong precomeal retention was tested. The ocular irritation potential of components and formulations was assessed using a modified hen's egg chorioallantoic membrane test (HET-CAM) and the preocular retention of selected formulations was investigated in rabbit eye using gamma scintigraphy. Results showed that Crill 1, Crillet 4 and Crodamol EO were non-irritant. However, all other cosurfactants investigated were irritant and their irritation was dependent on their carbon chain length. A w/o ME formulated without cosurfactant showed a protective effect when a strong irritant (0.1 M NaOH) was used as the aqueous phase. Precorneal clearance studies revealed that the retention of colloidal and coarse dispersed systems was significantly greater than an aqueous solution with no significant difference between ME systems (containing 5% and 10% water) as well as o/w emulsion containing 85% water. Conversely, a LC system formulated without cosurfactant displayed a significantly greater retention compared to other formulations. (c) 2005 Elsevier B.V. All rights reserved.

Identifying optimal lipid raft characteristics required to promote nanoscale protein-protein interactions on the plasma membrane

The dynamic lateral segregation of signaling proteins into microdomains is proposed to facilitate signal transduction, but the constraints on microdomain size, mobility, and diffusion that might realize this function are undefined. Here we interrogate a stochastic spatial model of the plasma membrane to determine how microdomains affect protein dynamics. Taking lipid rafts as representative microdomains, we show that reduced protein mobility in rafts segregates dynamically partitioning proteins, but the equilibrium concentration is largely independent of raft size and mobility. Rafts weakly impede small-scale protein diffusion but more strongly impede long-range protein mobility. The long-range mobility of raft-partitioning and raft-excluded proteins, however, is reduced to a similar extent. Dynamic partitioning into rafts increases specific interprotein collision rates, but to maximize this critical, biologically relevant function, rafts must be small (diameter, 6 to 14 nm) and mobile. Intermolecular collisions can also be favored by the selective capture and exclusion of proteins by rafts, although this mechanism is generally less efficient than simple dynamic partitioning. Generalizing these results, we conclude that microdomains can readily operate as protein concentrators or isolators but there appear to be significant constraints on size and mobility if microdomains are also required to function as reaction chambers that facilitate nanoscale protein-protein interactions. These results may have significant implications for the many signaling cascades that are scaffolded or assembled in plasma membrane microdomains.

Histopathology of oligofructose-induced acute laminitis in heifers

Histopathology of the dermo-epidermal junction in the lamellar region of front claws was examined in 6 dairy heifers given an alimentary oligofructose overload and compared with sections from a control group of 6 heifers. Four of the 6 heifers administered oligofructose developed clinical signs of acute laminitis before they were euthanized. Postmortem samples from front claws were processed for histology. Eleven histopathologic characteristics were selected from the existing literature and used in a blinded evaluation of sections. In total, 104 front claw samples, including 8 samples from 2 cows having spontaneously occurring acute laminitis, were evaluated histologically using hematoxylin and eosin as well as periodic acid-Schiff staining. The major morphological features associated with oligofructose-induced acute clinical laminitis were stretching of lamellae, dermal edema, hemorrhage, changes in basal cell morphology, presence of white blood cells in dermis, and signs of basement membrane detachment. Changes at the lamellar junction of claw tissue affected by oligofructose-induced clinical laminitis resembled tissue from the 2 cows suffering from spontaneous acute clinical laminitis, and generally were consistent with existing descriptions of laminitis histopathology. Important exceptions to existing descriptions in the literature were stretching of lamellae and basement membrane changes. Not previously described, we considered these early signs of acute laminitis. In conclusion, this study documents that oligofructose-induced clinical laminitis is associated with histopathological changes at the lamellar interface. A weakened dermo-epidermal junction is a possible intermediate stage in the pathophysiology of bovine sole ulceration at the typical site.

Submicron dispersions of hexosomes based on novel glycerate surfactants

Glycerate-based surfactants are a new class of swelling amphiphiles which swell to a finite degree with water. Among this class of surfactants, oleyl (cis-octadec-9-enyl) glycerate is very similar in structure to a well characterized mesophase-forming lipid, glyceryl monooleate (GMO). Despite the similar structural characteristics, a subtle change in connectivity of the ester bond substantially alters the binary surfactant-water phase behaviour. Whereas the phase behaviour of GMO is diverse and dominated by cubic phases, the phase behaviour of oleyl glycerate and a terpenoid analogue phytanyl (3,7,11,15-tetramethyl-hexadecane) glycerate is much simplified. Both exhibit an inverse hexagonal phase (H-II), which is stable to dilution with excess water, and an inverse micellar phase (L-II) at ambient temperatures. The inverse hexagonal phases formed by oleyl glycerate and phytanyl glycerate have been characterized using SAXS. Analogous to GMO cubosomes, the inverse hexagonal phase of phytanyl glycerate has been dispersed to form hexagonally facetted particles, termed hexosomes, whose structure has been verified using cryo-TEM.

Flotillins and the PHB domain protein family: Rafts, worms and anaesthetics

While our understanding of lipid microdomains has advanced in recent years, many aspects of their formation and dynamics are still unclear. In particular, the molecular determinants that facilitate the partitioning of integral membrane proteins into lipid raft domains are yet to be clarified. This review focuses on a family of raft-associated integral membrane proteins, termed flotillins, which belongs to a larger class of integral membrane proteins that carry an evolutionarily conserved domain called the prohibitin homology (PHB) domain. A number of studies now suggest that eucaryotic proteins carrying this domain have affinity for lipid raft domains. The PHB domain is carried by a diverse array of proteins including stomatin, podocin, the archetypal PHB protein, prohibitin, lower eucaryotic proteins such as the Dictyostelium discoideum proteins vacuolin A and vacuolin B and the Caenorhabditis elegans proteins unc-1, unc-24 and mec-2. The presence of this domain in some procaryotic proteins suggests that the PHB domain may constitute a primordial lipid recognition motif. Recent work has provided new insights into the trafficking and targeting of flotillin and other PHB domain proteins. While the function of this large family of proteins remains unclear, studies of the C. elegans PHB proteins suggest possible links to a class of volatile anaesthetics raising the possibility that these lipophilic agents could influence lipid raft domains. This review will discuss recent insights into the cell biology of flotillins and the large family of evolutionarily conserved PHB domain proteins.

Cholesterol and fatty acids regulate dynamic caveolin trafficking through the golgi complex and between the cell surface and lipid bodies

Caveolins are a crucial component of plasma membrane (PM) caveolae but have also been localized to intracellular compartments, including the Golgi complex and lipid bodies. Mutant caveolins associated with human disease show aberrant trafficking to the PM and Golgi accumulation. We now show that the Golgi pool of mainly newly synthesized protein is detergent-soluble and predominantly in a monomeric state, in contrast to the surface pool. Caveolin at the PM is not recognized by specific caveolin antibodies unless PM cholesterol is depleted. Exit from the Golgi complex of wild-type caveolin-1 or -3, but not vesicular stomatitis virus-G protein, is modulated by changing cellular cholesterol levels. In contrast, a muscular dystrophy-associated mutant of caveolin-3, Cav3P104L, showed increased accumulation in the Golgi complex upon cholesterol treatment. In addition, we demonstrate that in response to fatty acid treatment caveolin can follow a previously undescribed pathway from the PM to lipid bodies and can move from lipid bodies to the PM in response to removal of fatty acids. The results suggest that cholesterol is a rate-limiting component for caveolin trafficking. Changes in caveolin flux through the exocytic pathway can therefore be an indicator of cellular cholesterol and fatty acid levels.

Surface-active phospholipid (surfactant) in equine tendon and tendon sheath fluid

AIM: To investigate the presence of surface-active phospholipid (SAPL, or surfactant) in equine tendon and tendon sheath fluid. METHODS: The left front flexor tendon and sheath were removed from five Thoroughbred horses. Phospholipid was extracted from tendon sheath fluid using Folch reagent and quantified using spectroscopy. Transmission electron microscopy (TEM) was used to observe the tendon surfaces. RESULTS: The presence of phospholipid (90.6 (SD 4.3) mu g/ml) in tendon sheath fluid, plus the appearance of oligolamellar layers and lamellar bodies on the tendon surface were indicative of SAPL. CONCLUSIONS: Evidence of SAPL was found in equine tendon, and may have a similar lubricating function as reported for synovial joints. CLINICAL RELEVANCE: These findings may have important implications for normal tendon function and possible therapeutic adjuncts for tendon and tendon sheath injuries.

Magnetic resonance microscopy of the equine hoof wall: a study of resolution and potential

Reasons for performing study: Obtaining magnetic resonance images of the inner hoof wall tissue at the microscopic level would enable early accurate diagnosis of laminitis and therefore more effective therapy. Objectives: To optimise magnetic resonance imaging (MRI) parameters in order to obtain the highest possible resolution of the structures beneath the equine hoof wall. Methods: Magnetic resonance microscopy (MRM) was performed in front feet from 6 cadaver horses using T-2-weighted fast spin echo (FSE-T-2), and T-1-weighted gradient echo (GRE-T-1) sequences. Results: In T-2 weighted FSE images most of the stratum medium showed no signal, however the coronary, terminal and sole papillae were visible. The stratum lamellatum was clearly visible and primary epidermal lamellae could be differentiated from dermal lamellae. Conclusion: Most structures beneath the hoof wall were differentiated. Conventional scanners for diagnostic MRI in horses are low or high field. However this study used ultra-high field scanners currently not available for clinical use. Signal-to-noise ratio (SIN) increases as a function of field strength. An increase of spatial resolution of the image results in a decreased SIN. SIN can also be improved with better coils and the resolution of high field MRI scanners will increase as technology develops and surface array coils become more readily available. Potential relevance: Although MR images with microscopic resolution were obtained ex vivo, this study demonstrates the potential for detection of lamellar pathology as it occurs. Early recognition of the development of laminitis to instigate effective therapy at an earlier stage and may improve the outcome for laminitic horses. Clinical MR is now readily available at 3 T, while 4 T, 7 T and 9 T systems are being used for human whole body applications.

Phase behavior of a phospholipid/fatty acid/water mixture studied in atomic detail

Molecular dynamics simulations have been used to study the phase behavior of a dipalmitoylphosphatidylcholine (DPPC)/palmitic acid (PA)/water 1:2:20 mixture in atomic detail. Starting from a random solution of DPPC and PA in water, the system adopts either a gel phase at temperatures below similar to 330 K or an inverted hexagonal phase above similar to 330 K in good agreement with experiment. It has also been possible to observe the direct transformation from a gel to an inverted hexagonal phase at elevated temperature (similar to 390 K). During this transformation, a metastable fluid lamellar intermediate is observed. Interlamellar connections or stalks form spontaneously on a nanosecond time scale and subsequently elongate, leading to the formation of an inverted hexagonal phase. This work opens the possibility of studying in detail how the formation of nonlamellar phases is affected by lipid composition and (fusion) peptides and, thus, is an important step toward understanding related biological processes, such as membrane fusion.

Measuring the dynamic mechanical response of hydrated mouse bone by nanoindentation

This study demonstrates a novel approach to characterizing hydrated bone's viscoelastic behavior at lamellar length scales using dynamic indentation techniques. We studied the submicron-level viscoelastic response of bone tissue from two different inbred mouse strains, A/J and B6, with known differences in whole bone and tissue-level mechanical properties. Our results show that bone having a higher collagen content or a lower mineral-to-matrix ratio demonstrates a trend towards a larger viscoelastic response. When normalized for anatomical location relative to biological growth patterns in the antero-medial (AM) cortex, bone tissue from B6 femora, known to have a lower mineral-to-matrix ratio, is shown to exhibit a significantly higher viscoelastic response compared to A/J tissue. Newer bone regions with a higher collagen content (closer to the endosteal edge of the AM cortex) showed a trend towards a larger viscoelastic response. Our study demonstrates the feasibility of this technique for analyzing local composition-property relationships in bone. Further, this technique of viscoelastic nanoindentation mapping of the bone surface at these submicron length scales is shown to be highly advantageous in studying subsurface features, such as porosity, of wet hydrated biological specimens, which are difficult to identify using other methods. © 2010 Elsevier Ltd.