Subcellular Localization of Secondary Lipid Metabolites Including Fragrance Volatiles in Carnation Petals

Pulse-chase labeling of carnation (Dianthus caryophyllus L. cv Improved White Sim) petals with [14C]acetate has provided evidence for a hydrophobic subcompartment of lipid-protein particles within the cytosol that resemble oil bodies, are formed by blebbing from membranes, and are enriched in lipid metabolites (including fragrance volatiles) derived from membrane fatty acids. Fractionation of the petals during pulse-chase labeling revealed that radiolabeled fatty acids appear first in microsomal membranes and subsequently in cytosolic lipid-protein particles, indicating that the particles originate from membranes. This interpretation is supported by the finding that the cytosolic lipid-protein particles contain phospholipid as well as the same fatty acids found in microsomal membranes. Radiolabeled polar lipid metabolites (methanol/water-soluble) were detectable in both in situ lipid-protein particles isolated from the cytosol and those generated in vitro from isolated radiolabeled microsomal membranes. The lipid-protein particles were also enriched in hexanal, trans-2-hexenal, 1-hexanol, 3-hexen-1-ol, and 2-hexanol, volatiles of carnation flower fragrance that are derived from membrane fatty acids through the lipoxygenase pathway. Therefore, secondary lipid metabolites, including components of fragrance, appear to be formed within membranes of petal tissue and are subsequently released from the membrane bilayers into the cytosol by blebbing of lipid-protein particles.     

Lipid peroxidation increases the molecular order of microsomal membranes

The effect of enzymatic lipid peroxidation on the molecular order of microsomal membranes was evaluated by ESR spectroscopy using the spin probes 5-, 12-, and 16-doxyl-stearic acid. Rat liver microsomal membranes were peroxidized by the NADPH-dependent reaction in the presence of the chelate ADP-Fe3+. Peroxidation resulted in a preferential depletion of polyenoic fatty acids and an increase in the percentage composition of shorter fatty acyl chains. There was no change in the cholesterol/phospholipid ratio of the peroxidized microsomes. The molecular order of both control and peroxidized membranes decreased toward the central region of the bilayer, and the order parameter (S) of each probe was temperature dependent. Peroxidation of the microsomal membrane lipids resulted in an increase in the order parameter determined with the three stearic acid spin probes. Of the three probes, 12-doxylstearic acid was the most sensitive to the changes in membrane organization caused by peroxidation. These data indicate that ESR spectroscopy is a sensitive method of detecting changes in membrane order accompanying peroxidation of membrane lipids.     

Cytoplasmic membrane senescence in bean cotyledons

Distinguishable patterns of cytoplasmic membrane senescence in cotyledon tissue of Phaseolus vulgaris have been elucidated by examining the behavior of four microsomal enzymes—NADH-cytochrome C reductase, NADPH-cytochrome C reductase, glucose-6-phosphatase and 5′-nucleotidase during germination. For young cotyledon tissue, specific activities for the phosphatases were similar for rough and smooth microsomal fractions, but both cytochrome C reductases were 2–3 times more concentrated in the smooth fraction. These proportionalities changed with increasing age. As senescence becomes more intense the enzyme activities change independently of one another. These changes do not appear to be influenced by the presence or absence of ribosomes on the membranes. Parallel analyses of phospholipid levels in the isolated fractions revealed that loss of microsomal enzyme activity correlates with an ultimate dismantling of the membranes into their macromolecular constituents. The data have been interpreted as indicating that functionally distinct membranes or regions of the same membrane are differentially sensitive to senescence.     

Isolation of nonsedimentable lipid-protein particles from insect intestine

Nonsedimentable lipid-protein particles have been isolated from intestinal tissue of the American cockroach, Periplaneta americana. Most of the particles were within the range 30–50 nm in diameter and appear to originate from larger structures. Lipid analysis of the particles showed them to be enriched in neutral lipid components relative to microsomal membranes. Specifically, there is a decline in the amounts of phosphatidylcholine and phosphatidylethanolamine in the nonsedimentable particles compared with the microsomal membranes. Also, in contrast to microsomal membranes, the particles have a higher content of phosphatidic acid along with 1,2- and 1,3-diacyglycerols, free fatty acids and an unidentified lipid that co-migrates with sterol ester, wax ester and hydrocarbon standards in thin layer chromatograms. The cytosol, separated from the particles by ultrafiltration, contained phosphatidic acid, free fatty acids and the unidentified lipid. By contrast, the composition of neutral lipids in the cytosol resembles that of the particles. SDS—PAGE analysis of microsomal membranes, the particles and particle free cytosol shows an enrichment of low molecular weight proteins in the particles and cytosol. The particles and cytosol appear to possess proteolytic activity that is distinguishable from that of corresponding microsomal membranes since the incubation of these components with BSA resulted in the formation of distinct polypeptides. Many characteristics of these particles resemble those of the deteriosomes that have been isolated from plant tissue. © 1995 Wiley-Liss, Inc.     

Nonsedimentable microvesicles from senescing bean cotyledons contain gel phase-forming phospholipid degradation products

A mixture of liquid-crystalline and gel-phase lipid domains is detectable by wide angle x-ray diffraction in smooth microsomal membranes isolated from senescent 7-day-old cotyledons, whereas corresponding membranes from young 2-day-old cotyledons are exclusively liquid-crystalline. The gel-phase domains in the senescent membranes comprise phospholipid degradation products including diacylglycerols, free fatty acids, long-chain aldehydes, and long-chain hydrocarbons. The same complement of phospholipid degradation products is also present in nonsedimentable microvesicles isolated from senescent 7-day-old cotyledons by filtration of a 250,000g, 12-hour supernatant through a 300,000 dalton cut-off filter. The phospholipid degradation products in the microvesicles form gel-phase lipid domains when reconstituted into phospholipid liposomes. Nonsedimentable microvesicles of a similar size, which are again enriched in the same gel-phase-forming phospholipid degradation products, are also generated in vitro from smooth microsomal membranes isolated from 2-day-old cotyledons when Ca²⁺ is added to activate membrane-associated lipolytic enzymes. The Ca²⁺-treated membranes do not contain detectable gel-phase domains, suggesting that the phospholipid degradation products are completely removed by microvesiculation. The observations collectively indicate that these nonsedimentable microvesicles serve as a vehicle for moving phospholipid degradation products out of membrane bilayers into the cytosol. As noted previously (Yao K, Paliyath G, Humphrey RW, Hallett FR, Thompson JE [1991] Proc Natl Acad Sci USA 88: 2269-2273), the term “deteriosome” connotes this putative function and would serve to distinguish these microvesicles from other cytoplasmic microvesicles unrelated to deterioration.     

Phase Behavior of Chloroplast and Microsomal Membranes during Leaf Senescence

Wide angle x-ray diffraction of chloroplast and microsomal membranes from primary leaves of Phaseolus vulgaris has revealed that for both types of membrane, portions of the lipid become crystalline as the tissue senesces. For young leaves the transition temperature is about 23 C for microsomes and below −30 C for chloroplast membranes, indicating that at physiological temperature the lipid is entirely liquid-crystalline. Between 2 and 3 weeks after planting the transition temperature rises to 38 C for microsomes, but for chloroplasts this increase to a point above physiological temperature does not occur until between 3 and 4 weeks. Thereafter the transition temperature continues to rise for both types of membrane with advancing senescence, although the rate of increase is greater for chloroplasts than for microsomes. The appearance at physiological temperature of gel phase lipid in the microsomes coincides temporally with the initiation of a decline in total protein in the tissue, and the incidence of crystallinity in chloroplasts coincides with loss of chlorophyll. This change in phase behavior cannot be attributed to an alteration in fatty acid composition, but for both membrane systems it correlates with an increase of about 4-fold in the sterol to phospholipid ratio.     

The effect of phosphate deficiency on membrane phospholipid composition of bean (Phaseolus vulgaris L.) roots

Plasma membranes were isolated from roots of bean (Phaseolus vulgaris L.) plants cultured on phosphate sufficient or phosphate deficient medium. The phospholipid composition of plasma membranes was analyzed and compared with that of the microsomal fraction. Phosphate deficiency had no influence on lipid/protein ratio in microsomal as well as plasma membrane fraction. In phosphate deficient roots phospholipid content was lower in the plasma membrane, but did not change in the microsomal fraction. Phosphatidylcholine and phosphatidylethanolamine were two major phospholipids in plasmalemma and microsomal membranes (80 % of the total). After two weeks of phosphate starvation a considerable decrease (about 50 %) in phosphatidylcholine and phosphatidylethanolamine in microsomal membranes was observed. The decline in two major phospholipids was accompanied by an increase in phosphatidic acid and lysophosphatidylcholine content. The effect of alterations in plasma membrane phospholipids on membrane function e.g. nitrate uptake is discussed.     

Immunopurification of H+-ATPase-containing lipid-protein particles from the cytosol of carnation petals

Lipid-protein particles originating from the plasma membrane were immunopurified from the cytosol of carnation petal cells ( Dianthus caryophyllus L. cv. Improved White Sim) using antibodies raised against the central hydrophilic domain of the H⁺-ATPase. The immunopurified particles are enriched in lipid metabolites, in particular free fatty acids and steryl/wax esters, by comparison with corresponding microsomal membranes, and the lipids of the particles are more saturated than those of microsomal membranes. Proteolytic catabolites of the H⁺-ATPase, a protein associated with the plasma membrane, but not the native H⁺-ATPase protein, are also present in the immunopurified cytosolic particles. Osmiophilic particles were discernible in the cytosol of carnation petal cells by transmission electron microscopy, and the association of H⁺-ATPase catabolites with a subpopulation of these particles was confirmed by immunogold labelling with H⁺-ATPase antiserum. Cross-reaction of the H⁺-ATPase antiserum with elements of the cytosol was also evident by immunofluorescent light microscopy. These observations collectively indicate that lipid-protein particles of plasma membrane origin are present in the cytosol of carnation petal cells and that their formation may serve as a means of removing lipid and protein metabolites from the plasma membrane which would otherwise destabilize its structure.     

Flotation of lipid-protein particles containing triacylglycerol and phospholipid from the cytosol of carnation petals

Lipid-protein particles ranging from 20 to 250 nm in diameter have been isolated from the cytosol of carnation petals by flotation centrifugation and also by ultrafiltration. The cytosolic lipid-protein particles resemble oil bodies, lipid-protein particles found in oil-bearing seeds, in that they contain triacylglycerol, are circumscribed by phospholipid that is not organized in a bilayer, appear to be derived from membranes and can be isolated by flotation. However, the cytosolic particles are distinguishable from oil bodies in that triacylglycerol is not the dominant lipid. Indeed, they contain a spectrum of lipids in addition to phospholipids and triacylglycerol including free fatty acids, sterol and wax esters, phosphatidic acid and diacylglycerol. These same lipids are present in corresponding microsomal membranes as well, but in much smaller proportions relative to phospholipid. The lipid-protein particles from carnation petals contain a 17-kDa protein that is of similar size to oil body oleosin, but does not cross-react with anti-oleosin antibodies. The data indicate that these cytosolic particles are structurally and chemically similar to oil bodies and are consistent with the notion that their genesis may be a means of removing destabilizing lipids from membrane bilayers.     

Regulation of fatty acid unsaturation in encystingAcanthamoeba cells

The degree of fatty acid unsaturation and average chain length are closely similar for microsomal membranes from exponential-phase trophozoites and cysts ofAcanthamoeba castellanii despite significant differences in fatty acid composition. The same trend was apparent for total fatty acids extracted from whole cells. The observations suggest that the organism regulates these lipid parameters during differentiation in order to maintain optimum membrane lipid viscosity, and are consistent with previous electron spin resonance measurements indicating that the fluidity of microsomal membranes does not change during encystment. About 75% of the microsomal fatty acids are unsaturated for both cysts and amoebae. Wide-angle X-ray diffraction of phospholipid liposomes prepared from lipid extracts of the membranes has indicted that this high level of unsaturation renders the phospholipid exclusively liquid-crystalline at temperatures as low as 9°C for rough microsomes and-1.5°C for smooth microsomes. Thus, by retaining a high proportion of unsaturated fatty acids throughout its differentiation cycle, the organism gains some protection in its natural soil habitat against lateral phase separation of membrane lipids.     

COMPOSITION OF CELLULAR MEMBRANES IN THE PANCREAS OF THE GUINEA PIG : II. Lipids

The lipid composition of rough and smooth microsomal membranes, zymogen granule membranes, and a plasmalemmal fraction from the guinea pig pancreatic exocrine cell has been determined. As a group, membranes of the smooth variety (i.e., smooth microsomes, zymogen granule membranes, and the plasmalemma) were similar in their content of phospholipids, cholesterol and neutral lipids, and in the ratio of total lipids to membrane proteins. In contrast, rough microsomal membranes contained much less sphingomyelin and cholesterol and possessed a smaller lipid/protein ratio. All membrane fractions were unusually high in their content of lysolecithin (up to ~20% of the total phospholipids) and of neutral lipids, especially fatty acids. The lysolecithin content was shown to be due to the hydrolysis of membrane lecithin by pancreatic lipase; the fatty acids, liberated by the action of lipase on endogenous triglyceride stores, are apparently scavenged by the membranes from the suspending media. Similar artifactually high levels of lysolecithin and fatty acids were noted in hepatic microsomes incubated with pancreatic postmicrosomal supernatant. E 600, an inhibitor of lipase, largely prevented the appearance of lysolecithin and fatty acids in pancreatic microsomes and in liver microsomes treated with pancreatic supernatant.     

Effects of in vitro treatment with ozone on the physical and chemical properties of membranes

Treatment of microsomal membranes from cotyledons of Phaseolus vulgaris with ozone raises the liquid-crystalline to gel lipid phase transition temperature and results in the formation of distinct domains of gel phase lipid in the membranes. Liposomes prepared from the total lipid extracts of ozone-treated membranes undergo phase separations just a few degrees below the transition temperature for intact membranes, indicating that the formation of gel phase lipids is largely attributable to ozone-induced alterations in the membrane lipids. Levels of unsaturated fatty acids as well as the sterol to phospholipid ratio are markedly reduced in the ozone-treated membranes, and the neutral lipid fraction from treated membranes shows, an increased propensity to induce the formation of gel phase phospholipid when incorporated into liposomes of egg phosphatidylcholine. Since gel phase phospholipid also forms in naturally senescing plant membranes and appears to be attributable to changes in the neutral lipid fraction, the effects of natural senescence and ozone on membranes have been compared.     

Isolation and characterization of deteriosomes from rat liver

Deteriosomes, a new class of microvesicles, have been isolated from rat liver tissue. These microvesicles are similar to those isolated previously from plant tissue [Yao et al., Proc Natl Acad Sci USA 88:2269–2273, 1991] in that they are nonsedimentable and enriched in membrane catabolites, particularly products of phospholipid degradation. Liver deteriosomes range in size from 0.05 μm to 0.11 μm in radius. They are also much more permeable than microsomal membrane vesicles indicating that the deteriosome bilayer is perturbed. The data are consistent with the proposal that deteriosomes are formed from membranes by microvesiculation and that they represent an intermediate stage of membrane deterioration. Furthermore, liver deteriosomes were found to contain phospholipase A₂ activity. This suggests that they not only serve as a means of moving destabilizing macromolecular catabolites out of membranes into the cytosol but also possess enzymatic activity. The fact that the specific activity of phospholipase A₂ is higher in deteriosomes than in deteriosome-free cytosol suggests that some of the enzymatic activity traditionally assumed to be cytosolic may in fact be associated with deteriosomes.     

Incorporation of 2-14C-acetate into isolated nuclei and cytosolic lipids of rat thymus cells

It is generally recognized nowadays that active lipid metabolism takes place in the nucleus of a mammalian cell. Experimental data testify to the biosynthesis of polyphosphoinositides and phosphatidylcholine and reveal corresponding enzymes within nuclei of mammalian cells. These findings suggest that lipidmediated signaling pathways in nuclei operate independently of lipid-mediated regulatory mechanisms functioning in membranes and cytosol. To explore the pathways of intranuclear lipid biosynthesis, we studied incorporation of 2-¹⁴C-acetate into lipids of cytosol and isolated nuclei of rat thymus cells after separate and combined incubation with the labeled precursor. The most efficient incorporation of 2-¹⁴C-acetate into lipids (cholesterol, free fatty acids, and phospholipids) was observed in a reaction mixture containing cytosol. When the reaction mixture contained only nuclei, incorporation of the radioactive precursor into lipids also took place, but specific radioactivity of the lipids was essentially lower than in the cytosol. In both cases, 2-¹⁴C-acetate incorporated into phosphatidylethanolamine, sphingomyelin, phosphatidylserine, phosphatidylinositol, and cardiolipin. Phosphatidylcholine, the most abundant membrane phospholipid, demonstrated the lowest radioactivity, which was significantly lower than that of phosphatidylethanolamine. Incorporation of newly synthesized free fatty acids in nuclear phospholipids was inhibited, if nuclei were incubated with cytosol. As a result, radioactive free fatty acids were accumulated in nuclei, while in cytosol they were efficiently incorporated into phospholipids. The levels of phospholipids and cholesterol remained constant regardless of incubation protocol, while the overall yield of free fatty acids decreased after combined incubation of nuclear and cytosolic fractions or after incubation of cytosol without nuclei. Putative mechanisms underlying the appearance of radioactive lipids in isolated nuclei of thymus cells are discussed.     

In vitro simulation of cytoplasmic membrane senescence in cotyledons

The loss of microsomal NADH-cytochrome c reductase activity (EC 1.6.99.3) in cotyledons, known to accompany germination of Phaseolus vulgaris and thought to reflect the progress of cytoplasmic membrane senescence, can be simulated in an in vitro system in which isolated microsomes from 2-day-old tissue are treated with cytosol fractions (microsomal supernatants). Inactivation of the enzyme is comparatively low when the microsomes are treated for 4 hours with cytosol fractions from 1- and 2-day-old tissue, but increases to about 68% upon treatment with a corresponding fraction from 3-day-old cotyledons. This temporal pattern is consistent with the pronounced in situ decline in NADH-cytochrome c reductase detectable between the 2nd and 4th days of germination. Extensive in vitro inactivation was also effected by cytosol fractions prepared from older tissue, including that harvested after 9 days of germination by which time the cotyledons were beginning to abscise.     

Lipid composition and molecular organization in plasma membrane-enriched fractions from senescing cotyledons

Plasma membrane fractions isolated from cotyledons of Phaseolus vulgaris L. cv. Kinghorn at various stages of senescence showed no significant change in fatty acid saturation with advancing senescence. However, the steroliphospholipid ratio increased by about 400% as senescence intensified. The lipid phase transition temperature of the membranes, which was measured by wide-angle x-ray diffraction, also rose from a point well below the growing temperature for young tissue to about 50°C for membrane from extensively senescent 9-day-old tissue. This means that by day 4 of germination there was a mixture of liquid-crystalline and gel phase phospholipid in the membrane matrices. Crystallinity attributable to sterol-sterol interaction was also apparent in the diffraction patterns for senescent membranes. The co-existence of gel and liquid-crystalline phase phospholipid in the aging membranes as well as the crystalline sterol aggregates presumably render the storage cells of cotyledons leaky and may thus facilitate the translocation of hydrolyzed food reserves into the vascular network.     

Retinal fatty acid binding protein reduce lipid peroxidation stimulated by long-chain fatty acid hydroperoxides on rod outer segments

In the present study we have investigated the effect of partially purified retinal fatty acid binding protein (FABP) against nonenzymatic lipid peroxidation stimulated by hydroperoxides derived from fatty acids on rod outer segment (ROS) membranes. Linoleic acid hydroperoxide (LHP), arachidonic acid hydroperoxide (AHP) and docosahexaenoic acid hydroperoxide (DHP) were prepared from linoleic acid, arachidonic acid and docosahexaenoic acid, respectively, by means of lipoxidase. ROS membranes were peroxidized using an ascorbate-Fe(+2) experimental system. The effect on the peroxidation of ROS containing different amounts of lipid hydroperoxides (LOOH) was studied; ROS deprived of exogenously added LOOH was utilized as control. The degradative process was measured simultaneously by determining chemiluminescence and fatty acid composition of total lipids isolated from ROS. The addition of hydroperoxides to ROS produced a marked increase in light emission. This increase was hydroperoxide concentration-dependent. The highest value of activation was produced by DHP. The decrease percentage of the more polyunsaturated fatty acids (PUFAs) (20:4 n6 and 22:6 n3) was used to evaluate the fatty acid alterations observed during the process. We have compared the fatty acid composition of total lipids isolated from native ROS and peroxidized ROS that were incubated with and without hydroperoxides. The major difference in the fatty acid composition was found in the docosahexaenoic acid content, which decreased by 45.51+/-1.07% in the peroxidized group compared to native ROS; the decrease was even higher, 81.38+/-1.11%, when the lipid peroxidation was stimulated by DHP. Retinal FABP was partially purified from retinal cytosol. Afterwards, we measured its effect on the reaction of lipid peroxidation induced by LOOH. As a result, we observed a decrease of chemiluminescence (inhibition of lipid peroxidation) when adding increasing amounts (0.2 to 0.6 mg) of retinal FABP to ROS. The inhibitory effect reaches its highest value in the presence of DHP (41.81+/-10.18%). Under these conditions, bovine serum albumin (BSA) produces a smaller inhibitory effect (20.2+/-7.06%) than FABP.     

Changes in Lipids of Bean Seeds (Phaseolus vulgaris) and Corn Caryopses (Zea mays) Aged in Contrasting Environments

Seeds of French bean (Phaseolus vulgaris L. cv. The Prince)and caryopses of sweet corn (maize; Zea mays L.F1 hybrid Firstof All) were stored in environments of 79% relative humidityand 25 °C, 80% r.h. and 40 °C or 100% r.h. and 45 °C,giving ageing (loss of gcrminability and vigour) over periodsof months, weeks or days, respectively. The relationship betweenchanges in lipids and changes in germinability or vigour wasunaffected in general by the speed of ageing. In the corn caryopsesthere was no evidence of hydrolysis of phospholipids or peroxidationof fatty acids during ageing. In the bean seeds phosphatidicacid increased during the ageing period and phosphatyl cholinedeclined. The percentage of fatty acids as hnolenic acid initiallyfell during bean ageing, but in the slower ageing conditionsit rose again as germination reached zero. In bean, the presenceof phosphatidic acid could be a sensitive indictator of lossof vigour, but relative proportions of the different fatty acidswould be a misleading indicator of quality. Rapid artificialageing may be an adequate model, in some species, of ageingat moderate speeds and of ageing under some ambient conditions. French bean (Phaseolus vulgaris L. cv. The Prince), sweet corn (maize, Zea mays L., Fl hybrid First of All), seed ageing, phospholipids, fatty acids     

Fluorescence emitted from microsomal membranes by lipid peroxidation

The fluorescence emitted from rat liver microsomal membranes which had undergone enzymatic and nonenzymatic lipid peroxidation was detected directly. This fluorescence produced in peroxidized membranes increased progressively with peroxidation reaction time, and the fluorescent substances produced were retained in the membranes without being released into the aqueous phase. Extracts of the peroxidized membranes with organic solvents (chloroform/methanol) emitted fluorescence which was also dependent on the peroxidation reaction time. The generation profiles of fluorescence emitted from both the peroxidized membranes and their extracted membrane lipids differed essentially from that of thiobarbituric acid-reactive substances which reached a plateau at a relatively early stage of peroxidation reaction. These results indicate that lipid peroxidation induces stepwise chemical and physical changes in membranes and that the fluorescence from peroxidized membranes will be useful in studying such changes occurring in biological membranes.     

Lipid crystallization in senescent membranes from cotyledons

Lipid transition temperatures for rough and smooth microsomal membranes isolated from bean (Phaseolus vulgaris) cotyledon tissue at various stages of germination were determined by wide angle x-ray diffraction. The transition temperatures were established by recording diffraction patterns through a temperature series until a sharp x-ray reflection centered at a Bragg spacing of 4.15 Å and denoting the presence of crystalline lipid was discernible. For rough and smooth microsomes from 2-day-old tissue, the transitions occurred at 0 C and 3 C, respectively, indicating that at this early stage in the germination sequence the membrane lipid is entirely liquid-crystalline at physiological temperature. By the 4th day of germination, the transition temperatures had increased to 32 C for smooth microsomes and 35 C for rough microsomes, indicating that at 29 C, which was the growth temperature, portions of the membrane lipid were crystalline. During the later stages of germination, the transition temperature for smooth microsomes continued to rise through 44 C at day 7 to 56 C at day 9, by which time the cotyledons were extensively senescent and beginning to abscise. There was also a dramatic increase in the proportion of membrane lipid in the crystalline phase at 29 C. By contrast, the rough microsomes showed little change in transition temperature and only a slight increase in the proportion of crystalline lipid during this late period in germination. The data indicate that substantial amounts of the lipid is senescing membranes are crystalline even at physiological temperature. Moreover, there is a temporal correlation between the appearance of this crystallinity and loss of membrane function, suggesting that the two may be causally related.