Phospholipid asymmetry in human erythrocyte ghosts

Using phospholipase digestion and the fluorescent probe merocyanine 540 the maintenance of phospholipid asymmetry in the plasma membrane of human erythrocyte ghosts was investigated. Digestion with phospholipase A2 indicated that ghosts prepared in the presence of Mg++ as the only divalent cation retained the normal phospholipid asymmetry characteristic of intact erythrocytes. These ghosts, like normal erythrocytes, also failed to stain with merocyanine 540. However, the presence of as little as 5-10 microM Ca++ during ghost preparation resulted in ghosts in which lipid asymmetry had been abolished, as indicated by phospholipase digestion. Moreover, these ghosts stained with merocyanine 540. In contrast to ghosts, intact erythrocytes treated with ionophore required millimolar levels of Ca++ ions to disrupt membrane lipid asymmetry. To discover the reason for this difference in behavior between ghosts and intact cells, ghosts were prepared from preswollen cells using only small volumes of buffer for lysis. These experiments demonstrated that as the cellular contents of erythrocytes are diluted, the asymmetric arrangement of phospholipids becomes more sensitive to disruption by Ca++.     

Clustering of erythrocytes by fibrinogen is inhibited by carnitine: evidence that sulfhydryl groups on red blood cell membranes are involved in carnitine actions.

Carnitine is bound by intact red blood cells, by red blood cell ghosts, and by glutaraldehyde-fixed human erythrocytes in a non-saturable, temperature-dependent manner. Binding of carnitine by these preparations is blocked by sulfhydryl reagents. Incubation or preincubation of red blood cell preparations with carnitine inhibits the aggregation of erythrocytes otherwise elicited by fibrinogen. Identical effects are obtained with red blood cell ghosts. In contrast, choline, even at high concentrations, is inactive in preventing the aggregation of erythrocytes. We discuss possible mechanisms by which carnitine favors the dispersion of red blood cells, and we present data indicating that sulfhydryl groups on erythrocyte membranes are required to permit these carnitine actions to be manifested.     

Ca-induced K transport in human red blood cell ghosts containing arsenazo III. Transmembrane interactions of Na, K, and Ca and the relationship to the functioning Na-K pump

Increasing free intracellular Ca (Cai) from less than 0.1 microM to 10 microM by means of A23187 activated Ca-stimulated K transport and inhibited the Na-K pump in resealed human red cell ghosts. These ghosts contained 2 mM ATP, which was maintained by a regenerating system, and arsenazo III to measure Cai. Ca-stimulated K transport was activated 50% at 2-3 microM free Cai and the Na-K pump was inhibited 50% by 5-10 microM free Cai. Free Cai from 1 to 8 microM stimulated K efflux before it inhibited the Na-K pump, dissociating the effect of Ca on the two systems. 3 microM trifluoperazine inhibited Ca-stimulated K efflux and 0.5 mM quinidine reduced Na-K pumping by 50%. In other studies, incubating fresh intact cells in solutions containing Ca and 0.5 microM A23187 caused the cells to lose K heterogeneously. Under the same conditions, increasing A23187 to 10 microM initiated a homogeneous loss of K. In ATP-deficient ghosts containing Cai equilibrated with A23187, K transport was activated at the same free Cai as in the ghosts containing 2 mM ATP. Neither Cao nor the presence of an inward Ca gradient altered the effect of free Cai on the permeability to K. In these ghosts, transmembrane interactions of Na and K influenced the rate of Ca-stimulated K efflux independent of Na- and K-induced changes in free Cai or sensitivity to Cai. At constant free Cai, increasing Ko from 0.1 to 3 mM stimulated K efflux, whereas further increasing Ko inhibited it. Increasing Nai at constant Ki and free Cai markedly decreased the rate of efflux at 2 mM Ko, but had no effect when Ko was greater than or equal to 20 mM. These transmembrane interactions indicate that the mechanism underlying Ca-stimulated K transport is mediated. Since these interactions from either side of the membrane are independent of free Cai, activation of the transport mechanism by Cai must be at a site that is independent of those responsible for the interaction of Na and K. In the presence of A23187, this activating site is half-maximally stimulated by approximately 2 microM free Ca and is not influenced by the concentration of ATP. The partial inhibition of Ca-stimulated K efflux by trifluoperazine in ghosts containing ATP suggests that calmodulin could be involved in the activation of K transport by Cai.(ABSTRACT TRUNCATED AT 400 WORDS)     

Characterization of two independent modes of action of ATP on human erythrocyte sugar transport

Intracellular ATP has been reported either to stimulate [Jacquez, J.A. (1983) Biochim. Biophys. Acta 727, 367-378] or to inhibit [Hebert, D. N., & Carruthers, A. (1986) J. Biol. Chem. 261, 10093-10099] human erythrocyte sugar transport. This current study provides a rational explanation for these divergent findings. Protein-mediated 3-O-methyl-alpha-D-glucopyranoside (3OMG) uptake by intact human red blood cells (lacking intracellular sugar) at ice temperature in isotonic KCl containing 2 mM MgCl2, 2 mM EGTA, and 5 mM Tris-HCl, pH 7.4 (KCl medium), is characterized by a Km(app) of 0.4 +/- 0.1 mM and a Vmax of 114 +/- 20 mumol L-1 min-1. Lysis of red cells in 40 volumes of EGTA-containing hypotonic medium and resealing in 10 volumes of KCl medium increase the Km(app) and Vmax for uptake to 7.1 +/- 1.8 mM and 841 +/- 191 mumol L-1 min-1, respectively. Addition of ATP (4 mM) to the resealing medium restores Michaelis and velocity constants for zero-trans 3OMG uptake to 0.42 +/- 0.11 mM and 110 +/- 15 mumol L-1 min-1, respectively. Addition of CaCl2 to extracellular KCl medium (calculated [Ca2+]o = 101 microM) reduces the Vmax for zero-trans 3OMG uptake in intact cells and ATP-containing ghosts by 79 +/- 4% and 61 +/- 9%, respectively. Intracellular Ca2+ (15 microM) reduces the Vmax for 3OMG uptake by ATP-containing ghosts by 38 +/- 12%. In nominally ATP-free ghosts, extracellular (101 microM) and intracellular (11 microM) Ca2+ reduce the Vmax for 3OMG uptake by 96 and 94%, respectively.(ABSTRACT TRUNCATED AT 250 WORDS)     

Fusion of cell ghosts with intact cells in Dictyostelium discoideum: Differential response of opposite mating-type cells

The sexual cycle of Dictyostelium discoideum is initiated by the fusion of cells that are of opposite mating types (e.g. NC4- and HM1-type cells). Cells grown in light on agar plates are not capable of sexual cell fusion, but become capable when cultured in the dark in a liquid medium. Cells in the incapable state are called fusion-incompetent cells, and cells in the latter state, fusion-competent cells. To gain some understanding of the mechanism of cell fusion, cell ghosts prepared by freeze-thawing intact cells were incubated with intact cells. The cell ghosts killed the intact cells by directly fusing with them, the extent of fusion depending on the particular strains employed and the fusion-competency of the intact cells and of the cells from which the cell ghosts had been prepared. A detailed examination revealed that fusion-competent NC4 cells were always more easily killed by cell ghosts than fusion-incompetent NC4 cells. It also became apparent that cell ghosts prepared from fusion-competent NC4 cells killed all cell types far more efficiently than did those prepared from fusion-incompetent NC4 cells. However, fusion-competent and fusion-incompetent HM1 cells were equally sensitive to cell ghosts, and cell ghosts prepared from fusion-competent HM1 cells had the same ability to kill as those prepared from fusion-incompetent HM1 cells. From these findings, it thus appears that opposite mating-type cells have distinct membrane properties related to sexual cell fusion.     

Evidence for the asymmetrical binding of p-chloromercuriphenyl sulphonate to the human erythrocyte nucleoside transporter

Nucleosides cross the human erythrocyte membrane by a facilitated-diffusion process which is selectively inhibited by nanomolar concentrations of nitrobenzylthioinosine (NBMPR). The chemical asymmetry of the transporter was investigated by studying the effects of p-chloromercuriphenyl sulphonate (PCMBS) on uridine transport and high-affinity NBMPR binding in inside-out and right-side-out membrane vesicles, unsealed erythrocyte ghosts and intact cells. PCMBS was an effective inhibitor of the transporter (50% inhibition at 30 microM), but only when the organomercurial had access to the cytoplasmic membrane surface. PCMBS inhibition of NBMPR binding to ghosts was reversed by incubation with dithiothreitol. Both uridine and NBMPR were able to protect the transporter against PCMBS inhibition.     

Effects of metal ions on sphingomyelinase activity of Bacillus cereus

Some divalent metal ions were examined for their effects on sphingomyelinase activity of Bacillus cereus. The enzyme activity toward mixed micelles of sphingomyelin and Triton X-100 proved to be stimulated by Co2+ and Mn2+, as well as by Mg2+. Km's for Co2+ and Mn2+ were 7.4 and 1.7 microM, respectively, being smaller than the Km for Mg2+ (38 microM). Sr2+ proved to be a competitive inhibitor against Mg2+, with a Ki value of 1 mM. Zn2+ completely abolished the enzyme activity at concentrations above 0.5 mM. The concentration of Zn2+ causing 50% inhibition of the enzyme activity was 2.5 microM. Inhibition by Zn2+ was not restored by increasing concentrations of Mg2+ when the concentration of Zn2+ was above 10 microM. Ba2+ was without effect. When sphingomyelinase was incubated with unsealed ghosts of bovine erythrocytes at 37 degrees C, the enzyme was significantly adsorbed onto the membrane in the presence of Mn2+, Co2+, Sr2+ or Ba2+. Incubation with intact or Pronase-treated erythrocytes caused enzyme adsorption only in the presence of Mn2+. In the course of incubation, the enzyme was first adsorbed on the membranes of intact bovine erythrocytes in the presence of Mn2+; then sphingomyelin breakdown proceeded with ensuing desorption of adsorbed enzyme. Hot-cold hemolysis occurred in parallel with sphingomyelin breakdown. In this case, the hydrolysis of membranous sphingomyelin as well as the initial enzyme adsorption took place in the following order: unsealed ghosts greater than Pronase-treated erythrocytes greater than intact erythrocytes.     

Release of liposomal markers by Cerebratulus toxin A-III

Marker release from liposomes induced by the cytolytic protein Cerebratulus lacteus toxin A-III was studied. No phospholipid specificity was apparent, but the sensitivity of liposomes to A-III varied with the membrane fluidity. With dioleylphosphatidylcholine liposomes, complete release occurred at 10-20 micrograms toxin per ml, depending on marker size. Kinetic experiments showed that release was rapid and exhibited no lag phase. The diameter of the A-III produced membrane lesion must exceed 90 A, as tetrameric Concanavalin A is quantitatively released from A-III treated liposomes.     

Photolabeling of the human erythrocyte glucose carrier with androgenic steroids

Androgenic steroids, which are potent inhibitors of facilitated hexose transport in human erythrocytes, were tested as possible natural photolabels of the hexose carrier protein. Androstenedione, which inhibited 3-O-methylglucose uptake half-maximally at 30-50 microM (EC50), was the most potent inhibitor of the photolabile steroids tested. It appeared to interact directly with the carrier, since it (1) inhibited equilibrium [3H]cytochalasin B binding to high affinity D-glucose-sensitive sites in both intact cells (EC50 = 63 microM) and protein-depleted ghosts (EC50 = 61 microM), (2) inhibited cytochalasin B photolabeling of the band 4.5 carrier region in electrophoretic gels of protein-depleted ghosts (EC50 = 50 microM), and (3) underwent photoincorporation into the same gel region in a D-glucose- and cytochalasin B-sensitive fashion. However, Dixon plots for inhibition of both cytochalasin B binding and transport were upward-curving, indicating the binding of more than one molecule of androstenedione to the carrier. The photoincorporation of androstenedione into band 4.5 protein was both time- and concentration-dependent, and not associated with damage to unlabeled carrier. It probably occurred by activation of the alpha, beta-unsaturated ketone on the steroid rather than indirectly by photoactivation of a group on the carrier protein, as occurs with cytochalasin B. Although androstenedione may bind to more than one region of the carrier, as well as to other non-carrier proteins, tryptic digestion of photolabeled ghosts produced a labeled Mr = 18,000-20,000 fragment, the labeling of which was inhibited by cytochalasin B, and which had an electrophoretic mobility similar to the major labeled tryptic fragment in cytochalasin B-labeled ghosts. These data suggest that androstenedione interacts directly with the hexose carrier and that it or other similar naturally photolabile steroids may serve as useful probes for structural dissection of the carrier protein.     

Fusion of cell ghosts with sexually opposite type cells in Dictyostelium discoideum

In the sexual cycle of Dictyostelium discoideum, haploid cells of two opposite mating types, strains HM1 and NC4, acquire fusion-competence under certain conditions, such as suspension culture in the dark, and fuse specifically to form giant zygote cells. Each giant cell engulfs the surrounding cells, gradually increases in size, and finally develops into a macrocyst that is a sexual structure in D. discoideum. Fusion-competent HM1 cells suspended in a solution were frozen and thawed to make cell ghosts. When cell ghosts were introduced into fusion-competent and -incompetent intact NC4 cells, the cell ghosts killed them in a short time, but the fusion-competent cells were killed in preference to the fusion-incompetent cells. This killing occurred through the fusion of the cell ghosts directly to intact cell membranes. Since the fusion was specific, the fusion between ghosts and cells appears to be essentially the same as that between intact cells during the sexual cycle in molecular mechanisms.     

The effects of triethyltin bromide on red cell and brain cyclic AMP-dependent protein kinases

Triethyltin bromide activates the cyclic AMP-dependent protein kinases of human red cell membranes and of bovine brain. Additions of 25-500 microM triethyltin to red cell ghosts resulted in enhanced phosphorylation of ghost proteins. When added to partially purified cyclic AMP-dependent protein kinases from red cell ghosts or bovine brain, stimulation of the phosphorylation of calf thymus histone was observed. The enhancement of kinase activity was due to release of catalytic subunits from the intact protein kinase. Brief exposure of the partially purified enzymes to triethyltin, followed by DE52 chromatography, resulted in elution profiles for regulatory and catalytic subunits that were similar to the profile resulting after cyclic AMP activation. Triethyltin interacts with both regulatory and catalytic subunits. When it was added to the partially purified cyclic AMP-dependent protein kinases from human red cell ghosts or bovine brain, noncompetitive inhibition of cyclic AMP binding to the regulatory subunit of the enzyme was observed. It interacted with the catalytic subunit to produce slow inhibition of catalytic activity. The inhibition was non-competitive with respect to both histone and ATP. When intact red cells were subjected to brief exposure with triethyltin, enhanced phosphorylation of certain membrane proteins occurred, suggesting that the activation of the cyclic AMP protein kinases by triethyltin may be physiologically significant.     

The effects of Ca2+ and guanylnucleotides on isoprenaline-stimulated cyclic AMP formation in rat reticulocyte ghosts

We have studied beta-adrenergic stimulation of cyclic AMP formation in fragmented membranes and in unsealed or resealed ghosts prepared from rat reticulocytes. The maximal rate of isoprenaline-stimulated cyclic AMP formation with saturating MgATP concentrations and in the presence of the phosphodiesterase inhibitor isobutylmethylxanthine was 5-8 nmol/min per ml ghosts and remained constant for at least 15 min. Transition from resealed ghosts to fragmented membranes was associated with a shift of the activation constant (Ka) for (+/-)-isoprenaline from 0.1 to 0.6 microM. THe apparent dissociation constant for propranolol (0.01 microM) remained unchanged. The Ka values for isoprenaline in native reticulocytes and in resealed ghosts were identical. The stimulating effect of NaF on cyclic AMP formation in resealed ghosts reached 15% of maximal beta-adrenergic stimulation. Cyclic AMP formation, both in fragmented membranes and in ghosts, was half-maximally inhibited with Ca2+ concentrations ranging between 0.1 and 1 microM. GTP stimulated isoprenaline-dependent cyclic AMP formation in unsealed ghosts and in fragmented reticulocyte membranes by a factor of 3-5 but did not change the Ka value for isoprenaline. Ka values for the guanylnucleotides in different experiments varied between 0.3 and 2 microM. Ca2+ concentrations up to 4.6 microM reduced the maximal activation by GTP and Gpp(NH)p but did not affect their Ka values. Compared to GTP, maximal activation by Gpp(NH)p was higher in fragmented membranes, but much lower in ghosts. Our results suggest that the native beta-receptor adenylate cyclase system of reticulocytes is more closely approximated in the ghost model than in fragmented membrane preparations. Membrane properties seem to modulate the actions of guanylnucleotides on isoprenaline-dependent cyclic AMP formation in ghosts. Some of these effects are not observed in isolated membranes.     

Association of vanadate-sensitive Mg(2+)-ATPase and shape change in intact red blood cells

Intact human erythrocytes, initially depleted of Mg2+ by EDTA incubation in the presence of A23187, exhibit Mg(2+)-dependent phosphate production of around 1.5 mmol per liter cells.h, half-maximally activated at around 0.4 mM added free Mg2+. This appears to correspond to Mg(2+)-stimulated adenosine triphosphatase (Mg(2+)-ATPase) activity found in isolated membranes, which is known to have a similar activity and affinity for Mg2+. Vanadate (up to 100 microM) inhibited Mg(2+)-dependent phosphate production and ATP breakdown in intact cells. Over a similar concentration range vanadate (3-100 microM) transformed intact cells from normal discocytes to echinocytes within 4-8 h at 37 degrees C, and more rapidly in Mg(2+)-depleted cells. The rate of Ca(2+)-induced echinocytosis was also enhanced in Mg(2+)-depleted cells. These results support previous studies in erythrocyte ghosts suggesting that vanadate-induced shape change is associated with inhibition of Mg(2+)-ATPase activity localized in the plasma membrane of the red blood cell.     

Interaction between phloretin and the red blood cell membrane

Phloretin binding to red blood cell components has been characterized at pH6, where binding and inhibitory potency are maximal. Binding to intact red cells and to purified hemoglobin are nonsaturated processes approximately equal in magnitude, which strongly suggests that most of the red cell binding may be ascribed to hemoglobin. This conclusion is supported by the fact that homoglobin-free red cell ghosts can bind only 10% as much phloretin as an equivalent number of red cells. The permeability of the red cell membrane to phloretin has been determined by a direct measurement at the time-course of the phloretin uptake. At a 2% hematocrit, the half time for phloretin uptake is 8.7s, corresponding to a permeability coefficient of 2 x 10(-4) cm/s. The concentration dependence of the binding to ghosts reveals two saturable components. Phloretin binds with high affinity (K diss = 1.5 muM) to about 2.5 x 10(6) sites per cell; it also binds with lower affinity (Kdiss = 54 muM) to a second (5.5 x 10(7) per cell) set of sites. In sonicated total lipid extracts of red cell ghosts, phloretin binding consists of a single, saturable component. Its affinity and total number of sites are not significantly different from those of the low affinity binding process in ghosts. No high affinity binding of phloretin is exhibited by the red cell lipid extracts. Therefore, the high affinity phloretin binding sites are related to membrane proteins, and the low affinity sites result from phloretin binding to lipid. The identification of these two types of binding sites allows phloretin effects on protein-mediated transport processes to be distinguished from effects on the lipid region of the membrane.     

Effects of N-ethylmaleimide on ouabain-insensitive cation fluxes in human red cell ghosts

In red cells of several species, the sulfhydryl reagent N-ethylmaleimide activates a Cl- -dependent, ouabain-resistant K+ transport pathway. Here we report our attempts to demonstrate ouabain-resistant Cl- -dependent K+ fluxes stimulated by N-ethylmaleimide in resealed human red cell ghosts using Rb+ as a K+ analogue. In contrast to intact cells, the rate constants of the base level Rb+ efflux in ghosts were similar in NaNO3 and NaCl (okRb = 0.535 +/- 0.079 h-1 and 0.534 +/- 0.085 h-1, respectively), while 1 mM N-ethylmaleimide stimulated Rb+ efflux strongly in NaNO3 (okRb = 14.26 +/- 1.32 h-1) and moderately in NaCl (okRb = 2.73 +/- 0.54 h-1). This effect was dependent on the presence of internal ATP. Stimulation of Rb+ efflux was observed in the presence of greater than or equal to 0.2 mM N-ethylmaleimide and increased at pH values approaching 8.0, consistent with titration of SH groups. N-Ethylmaleimide-stimulated Rb+ efflux was approx. 50% inhibited by 100 microM quinine sulfate whereas 1 microM bumetanide had no effect. In NaCl the N-ethylmaleimide-stimulated efflux saturated with initial internal ghost Rb+ concentration, but rates increased linearly in NaNO3. Replacement of external Na+ with glucamine or choline decreased the N-ethylmaleimide-stimulated Rb+ efflux, suggesting a role for external Na+. N-Ethylmaleimide-stimulated Rb+ efflux was greater in buffers with lipophilic anions such as SCN- or NO3- than in solutions with Cl- or acetate. However, the cation selectivity of the pathway studied was low, as Li+ efflux was also stimulated by N-ethylmaleimide. We conclude that the effect of N-ethylmaleimide on ouabain-resistant cation effluxes of human red cell ghosts is very different from the selective action of N-ethylmaleimide on Rb+ influxes in intact red cells.     

T4 phage and T4 ghosts inhibit f2 phage replication by different mechanisms

Both T4 phage and DNA-free ghosts inhibit replication of RNA phage f2. Most but not all of the effects by T4 upon f2 growth can be blocked by the addition of rifampicin prior to T4 superinfection; by contrast, the inhibition of f2 synthesis by T4 ghosts cannot be blocked by rifampicin. This indicates that inhibition by intact T4 requires gene function, while inhibition by ghosts does not. There is a small, multiplicity-dependent inhibition by viable T4 on f2 growth in the presence of rifampicin which may be similar to the gene function-independent inhibition by T4 ghosts. With one viable T4 per cell, there appears to be no effect by viable T4 upon f2 growth which does not require T4 gene action. Moreover, increasing multiplicities of viable T4 appear to inhibit T4 replication as well.In the absence of rifampicin, pre-existing f2 single and double-stranded RNA are degraded after superinfection by viable T4, but remain stable after superinfection by ghosts. However, no new f2 RNA is synthesized after superinfection with either. In the presence of rifampicin, f2-specific protein synthesis is largely unaffected by viable T4, but is completely inhibited by ghosts. Both Escherichia coli, as well as f2-speciflc polysomes disappear in the presence of ghosts.We conclude that, at low multiplicities, T4 phage and T4 ghosts inhibit replication of f2 phage, and presumably host syntheses, by different mechanisms.     

Derivatization of the human erythrocyte glucose transporter using a novel forskolin photoaffinity label

An iodinated photoaffinity label for the glucose transporter, 3-iodo-4-azidophenethylamido-7-O-succinyldeacetyl-forskolin (IAPS-forskolin), has been synthesized, purified, and characterized. The I50 for inhibition of 3-O-methylglucose transport in red blood cells by IAPS-forskolin was found to be 0.05 microM. The carrier free radioiodinated label is a highly specific photoaffinity label for the human erythrocyte glucose transporter. Photolysis of erythrocyte membranes (ghosts) and purified glucose transporter preparations with 1-2 nM [125I]IAPS-forskolin and analysis by sodium dodecyl sulfate-polyacrylamide gel electrophoresis showed specific derivatization of a broad band with an apparent molecular mass of 40-70 kDa. Photoincorporation into erythrocyte membranes using 2 nM [125I]IAPS-forskolin was protected with D-glucose (I50 400 mM), cytochalasin B (I50 0.5 microM), and forskolin (I50 10 microM). No protection was observed with L-glucose (600 mM). Endo-beta-galactosidase digestion of [125I] IAPS-forskolin-labeled ghosts and purified transporter resulted in a dramatic sharpening of the specifically radiolabeled transporter to 40 kDa. Trypsinization of [125I]IAPS-forskolin-labeled ghosts and purified transporter reduced the specifically radiolabeled transporter to a sharp peak at 18 kDa. [125I]IAPS-forskolin will be a useful tool to study the structural aspects of the glucose transporter.     

Microstructure of cell wall-associated melanin in the human pathogenic fungus Cryptococcus neoformans

Melanin is a virulence factor for many pathogenic fungal species, including Cryptococcus neoformans. Melanin is deposited in the cell wall, and melanin isolated from this fungus retains the shape of the cells, resulting in hollow spheres called "ghosts". In this study, atomic force, scanning electron, and transmission electron microscopy revealed that melanin ghosts are covered with roughly spherical granular particles approximately 40-130 nm in diameter, and that the melanin is arranged in multiple concentric layers. Nuclear magnetic resonance cryoporometry indicated melanin ghosts contain pores with diameters between 1 and 4 nm, in addition to a small number of pores with diameters near 30 nm. Binding of the antibodies to melanin reduced the apparent measured volume of these pores, suggesting a mechanism for their antifungal effect. We propose a model of cryptococcal melanin structure whereby the melanin granules are held together in layers. This structural model has implications for cell division, cell wall remodeling, and antifungal drug discovery.