Osmotic Vesicle Collapse of Sealed Inside-Out Membrane Vesicles From Red Blood Cells.

The preparation of plasma membrane vesicles from a large variety of cells has contributed a wealth of information on the identity and vectorial properties of membrane transporters and enzymes. Vesicles from red blood cell (RBC) membranes are generated in media of extremely low tonicity. For functional studies, it is required to suspend the vesicles in higher tonicity media in order to bring the concentrations of the substrates of transporters and enzymes under investigation within the physiological ranges. We investigated the effects of hypertonic transitions on the vesicle morphology using transmission electron microscopy. The results show that hypertonic transitions cause an irreversible osmotic collapse of sealed membrane vesicles. Awareness of the collapsed condition of vesicles during functional studies is critical for the proper interpretation of experimental results.

Evidence against a Role of Elevated Intracellular Ca2+ during Plasmodium falciparum Preinvasion.

Severe malaria is primarily caused by Plasmodium falciparum parasites during their asexual reproduction cycle within red blood cells. One of the least understood stages in this cycle is the brief preinvasion period during which merozoite-red cell contacts lead to apical alignment of the merozoite in readiness for penetration, a stage of major relevance in the control of invasion efficiency. Red blood cell deformations associated with this process were suggested to be active plasma membrane responses mediated by transients of elevated intracellular calcium. Few studies have addressed this hypothesis because of technical challenges, and the results remained inconclusive. Here, Fluo-4 was used as a fluorescent calcium indicator with optimized protocols to investigate the distribution of the dye in red blood cell populations used as P. falciparum invasion targets in egress-invasion assays. Preinvasion dynamics was observed simultaneously under bright-field and fluorescence microscopy by recording egress-invasion events. All the egress-invasion sequences showed red blood cell deformations of varied intensities during the preinvasion period and the echinocytic changes that follow during invasion. Intraerythrocytic calcium signals were absent throughout this interval in over half the records and totally absent during the preinvasion period, regardless of deformation strength. When present, calcium signals were of a punctate modality, initiated within merozoites already poised for invasion. These results argue against a role of elevated intracellular calcium during the preinvasion stage. We suggest an alternative mechanism of merozoite-induced preinvasion deformations based on passive red cell responses to transient agonist-receptor interactions associated with the formation of adhesive coat filaments.

On the Mechanism of Human Red Blood Cell Longevity: Roles of Calcium, the Sodium Pump, PIEZO1, and Gardos Channels.

In a healthy adult, the transport of O2 and CO2 between lungs and tissues is performed by about 2 · 1013 red blood cells, of which around 1.7 · 1011 are renewed every day, a turnover resulting from an average circulatory lifespan of about 120 days. Cellular lifespan is the result of an evolutionary balance between the energy costs of maintaining cells in a fit functional state versus cell renewal. In this Review we examine how the set of passive and active membrane transporters of the mature red blood cells interact to maximize their circulatory longevity thus minimizing costs on expensive cell turnover. Red blood cell deformability is critical for optimal rheology and gas exchange functionality during capillary flow, best fulfilled when the volume of each human red blood cell is kept at a fraction of about 0.55-0.60 of the maximal spherical volume allowed by its membrane area, the optimal-volume-ratio range. The extent to which red blood cell volumes can be preserved within or near these narrow optimal-volume-ratio margins determines the potential for circulatory longevity. We show that the low cation permeability of red blood cells allows volume stability to be achieved with extraordinary cost-efficiency, favouring cell longevity over cell turnover. We suggest a mechanism by which the interplay of a declining sodium pump and two passive membrane transporters, the mechanosensitive PIEZO1 channel, a candidate mediator of Psickle in sickle cells, and the Ca2+-sensitive, K+-selective Gardos channel, can implement red blood cell volume stability around the optimal-volume-ratio range, as required for extended circulatory longevity.

Quantitation of malaria parasite-erythrocyte cell-cell interactions using optical tweezers.

Erythrocyte invasion by Plasmodium falciparum merozoites is an essential step for parasite survival and hence the pathogenesis of malaria. Invasion has been studied intensively, but our cellular understanding has been limited by the fact that it occurs very rapidly: invasion is generally complete within 1 min, and shortly thereafter the merozoites, at least in in vitro culture, lose their invasive capacity. The rapid nature of the process, and hence the narrow time window in which measurements can be taken, have limited the tools available to quantitate invasion. Here we employ optical tweezers to study individual invasion events for what we believe is the first time, showing that newly released P. falciparum merozoites, delivered via optical tweezers to a target erythrocyte, retain their ability to invade. Even spent merozoites, which had lost the ability to invade, retain the ability to adhere to erythrocytes, and furthermore can still induce transient local membrane deformations in the erythrocyte membrane. We use this technology to measure the strength of the adhesive force between merozoites and erythrocytes, and to probe the cellular mode of action of known invasion inhibitory treatments. These data add to our understanding of the erythrocyte-merozoite interactions that occur during invasion, and demonstrate the power of optical tweezers technologies in unraveling the blood-stage biology of malaria.

Dynamic morphology and cytoskeletal protein changes during spontaneous inside-out vesiculation of red blood cell membranes.

Vesicle preparations from cell plasma membranes, red blood cells in particular, are extensively used in transport and enzymic studies and in the fields of drug delivery and drug-transport interactions. Here we investigated the role of spectrin-actin, the main components of the red cell cortical cytoskeleton, in a particular mechanism of vesicle generation found to be relevant to the egress process of Plasmodium falciparum merozoites from infected red blood cells. Plasma membranes from red blood cells lysed in ice-cold media of low ionic strength and free of divalent cations spontaneously and rapidly vesiculate upon incubation at 37 °C rendering high yields of inside-out vesicles. We tested the working hypothesis that the dynamic shape transformations resulted from changes in spectrin-actin configuration within a disintegrating cytoskeletal mesh. We showed that cytoskeletal-free membranes behave like a two-dimensional fluid lacking shape control, that spectrin-actin remain attached to vesiculating membranes for as long as spontaneous movement persists, that most of the spectrin-actin detachment occurs terminally at the time of vesicle sealing and that naked membrane patches increasingly appear during vesiculation. These results support the proposed role of spectrin-actin in spontaneous vesiculation. The implications of these results to membrane dynamics and to the mechanism of merozoite egress are discussed.

An automated live imaging platform for studying merozoite egress-invasion in malaria cultures.

Most cases of severe and fatal malaria are caused by the intraerythrocytic asexual reproduction cycle of Plasmodium falciparum. One of the most intriguing and least understood stages in this cycle is the brief preinvasion period during which dynamic merozoite-red-cell interactions align the merozoite apex in preparation for penetration. Studies of the molecular mechanisms involved in this process face formidable technical challenges, requiring multiple observations of merozoite egress-invasion sequences in live cultures under controlled experimental conditions, using high-resolution microscopy and a variety of fluorescent imaging tools. Here we describe a first successful step in the development of a fully automated, robotic imaging platform to enable such studies. Schizont-enriched live cultures of P. falciparum were set up on an inverted stage microscope with software-controlled motorized functions. By applying a variety of imaging filters and selection criteria, we identified infected red cells that were likely to rupture imminently, and recorded their coordinates. We developed a video-image analysis to detect and automatically record merozoite egress events in 100% of the 40 egress-invasion sequences recorded in this study. We observed a substantial polymorphism of the dynamic condition of pre-egress infected cells, probably reflecting asynchronies in the diversity of confluent processes leading to merozoite release.

Elevated intracellular Ca2+ reveals a functional membrane nucleotide pool in intact human red blood cells.

Elevated intracellular calcium generates rapid, profound, and irreversible changes in the nucleotide metabolism of human red blood cells (RBCs), triggered by the adenosine triphosphatase (ATPase) activity of the powerful plasma membrane calcium pump (PMCA). In the absence of glycolytic substrates, Ca(2+)-induced nucleotide changes are thought to be determined by the interaction between PMCA ATPase, adenylate kinase, and AMP-deaminase enzymes, but the extent to which this three-enzyme system can account for the Ca(2+)-induced effects has not been investigated in detail before. Such a study requires the formulation of a model incorporating the known kinetics of the three-enzyme system and a direct comparison between its predictions and precise measurements of the Ca(2+)-induced nucleotide changes, a precision not available from earlier studies. Using state-of-the-art high-performance liquid chromatography, we measured the changes in the RBC contents of ATP, ADP, AMP, and IMP during the first 35 min after ionophore-induced pump-saturating Ca(2+) loads in the absence of glycolytic substrates. Comparison between measured and model-predicted changes revealed that for good fits it was necessary to assume mean ATPase V(max) values much higher than those ever measured by PMCA-mediated Ca(2+) extrusion. These results suggest that the local nucleotide concentrations generated by ATPase activity at the inner membrane surface differed substantially from those measured in bulk cell extracts, supporting previous evidence for the existence of a submembrane microdomain with a distinct nucleotide metabolism.

X-ray microanalysis investigation of the changes in Na, K, and hemoglobin concentration in plasmodium falciparum-infected red blood cells.

Plasmodium falciparum is responsible for severe malaria. During the ∼48 h duration of its asexual reproduction cycle in human red blood cells, the parasite causes profound alterations in the homeostasis of the host red cell, with reversal of the normal Na and K gradients across the host cell membrane, and a drastic fall in hemoglobin content. A question critical to our understanding of how the host cell retains its integrity for the duration of the cycle had been previously addressed by modeling the homeostasis of infected cells. The model predicted a critical contribution of excess hemoglobin consumption to cell integrity (the colloidosmotic hypothesis). Here we tested this prediction with the use of electron-probe x-ray microanalysis to measure the stage-related changes in Na, K, and Fe contents in single infected red cells and in uninfected controls. The results document a decrease in Fe signal with increased Na/K ratio. Interpreted in terms of concentrations, the results point to a sustained fall in host cell hemoglobin concentration with parasite maturation, supporting a colloidosmotic role of excess hemoglobin digestion. The results also provide, for the first time to our knowledge, comprehensive maps of the elemental distributions of Na, K, and Fe in falciparum-infected red blood cells.

Hydrogen ion dynamics in human red blood cells.

Our understanding of pH regulation within red blood cells (RBCs) has been inferred mainly from indirect experiments rather than from in situ measurements of intracellular pH (pH(i)). The present work shows that carboxy-SNARF-1, a pH fluorophore, when used with confocal imaging or flow cytometry, reliably reports pH(i) in individual, human RBCs, provided intracellular fluorescence is calibrated using a 'null-point' procedure. Mean pH(i) was 7.25 in CO(2)/HCO(3)(-)-buffered medium and 7.15 in Hepes-buffered medium, and varied linearly with extracellular pH (slope of 0.77). Intrinsic (non-CO(2)/HCO(3)(-)-dependent) buffering power, estimated in the intact cell (85 mmol (l cell)(-1) (pH unit)(-1) at resting pH(i)), was somewhat higher than previous estimates from cell lysates (50-70 mmol (l cell)(-1) (pH unit)(-1)). Acute displacement of pH(i) (superfusion of weak acids/bases) triggered rapid pH(i) recovery. This was mediated via membrane Cl(-)/HCO(3)(-) exchange (the AE1 gene product), irrespective of whether recovery was from an intracellular acid or base load, and with no evident contribution from other transporters such as Na(+)/H(+) exchange. H(+)-equivalent flux through AE1 was a linear function of [H(+)](i) and reversed at resting pH(i), indicating that its activity is not allosterically regulated by pH(i), in contrast to other AE isoforms. By simultaneously monitoring pH(i) and markers of cell volume, a functional link between membrane ion transport, volume and pH(i) was demonstrated. RBC pH(i) is therefore tightly regulated via AE1 activity, but modulated during changes of cell volume. A comparable volume-pH(i) link may also be important in other cell types expressing anion exchangers. Direct measurement of pH(i) should be useful in future investigations of RBC physiology and pathology.

Quantitative imaging of human red blood cells infected with Plasmodium falciparum.

During its 48 h asexual reproduction cycle, the malaria parasite Plasmodium falciparum ingests and digests hemoglobin in excess of its metabolic requirements and causes major changes in the homeostasis of the host red blood cell (RBC). A numerical model suggested that this puzzling excess consumption of hemoglobin is necessary for the parasite to reduce the colloidosmotic pressure within the host RBC, thus preventing lysis before completion of its reproduction cycle. However, the validity of the colloidosmotic hypothesis appeared to be compromised by initial conflicts between model volume predictions and experimental observations. Here, we investigated volume and membrane area changes in infected RBCs (IRBCs) using fluorescence confocal microscopy on calcein-loaded RBCs. Substantial effort was devoted to developing and testing a new threshold-independent algorithm for the precise estimation of cell volumes and surface areas to overcome the shortfalls of traditional methods. We confirm that the volume of IRBCs remains almost constant during parasite maturation, suggesting that the reported increase in IRBCs' osmotic fragility results from a reduction in surface area and increased lytic propensity on volume expansion. These results support the general validity of the colloidosmotic hypothesis, settle the IRBC volume debate, and help to constrain the range of parameter values in the numerical model.

Biophotonic techniques for the study of malaria-infected red blood cells.

Investigation of the homeostasis of red blood cells upon infection by Plasmodium falciparum poses complex experimental challenges. Changes in red cell shape, volume, protein, and ion balance are difficult to quantify. In this article, we review a wide range of optical techniques for quantitative measurements of critical homeostatic parameters in malaria-infected red blood cells. Fluorescence lifetime imaging and tomographic phase microscopy, quantitative deconvolution microscopy, and X-ray microanalysis, are used to measure haemoglobin concentration, cell volume, and ion contents. Atomic force microscopy is briefly reviewed in the context of these optical methodologies. We also describe how optical tweezers and optical stretchers can be usefully applied to empower basic malaria research to yield diagnostic information on cell compliance changes upon malaria infection. The combined application of these techniques sheds new light on the detailed mechanisms of malaria infection providing potential for new diagnostic or therapeutic approaches.

Detection of Plasmodium falciparum-infected red blood cells by optical stretching.

We present the application of a microfluidic optical cell stretcher to measure the elasticity of malaria-infected red blood cells. The measurements confirm an increase in host cell rigidity during the maturation of the parasite Plasmodium falciparum. The device combines the selectivity and sensitivity of single-cell elasticity measurements with a throughput that is higher than conventional single-cell techniques. The method has potential to detect early stages of infection with excellent sensitivity and high speed.

Effects of elevated intracellular calcium on the osmotic fragility of human red blood cells.

High throughput methodologies that measure the distribution of osmotic fragilities in red blood cell populations have enabled the investigation of dynamic changes in red cell homeostasis and membrane permeability in health and disease. The common assumption in the interpretation of dynamic changes in osmotic fragility curves is that left or right shifts reflect a decreased or increased hydration state of the cells, respectively, allowing direct inferences on membrane transport from osmotic fragility measurements. However, the assumed correlation between shifts in osmotic fragility and hydration state has never been directly explored, and may prove invalid in certain conditions. We investigated here whether this correlation holds for red cells exposed to elevated intracellular calcium. The results showed that elevated cell calcium causes a progressive increase in osmotic fragility with minimal contribution from cell hydration (<8%). Loss of membrane area by the release of 160+/-40nm diameter (mean+/-SD) vesicles is shown to be a major contributor, but may not account for the full non-hydration component. The rest must reflect a specific calcium-induced lytic vulnerability of the membrane causing rupture before the cells attain their maximal spherical volumes. The implications of these findings are discussed.

The homeostasis of Plasmodium falciparum-infected red blood cells.

The asexual reproduction cycle of Plasmodium falciparum, the parasite responsible for severe malaria, occurs within red blood cells. A merozoite invades a red cell in the circulation, develops and multiplies, and after about 48 hours ruptures the host cell, releasing 15-32 merozoites ready to invade new red blood cells. During this cycle, the parasite increases the host cell permeability so much that when similar permeabilization was simulated on uninfected red cells, lysis occurred before approximately 48 h. So how could infected cells, with a growing parasite inside, prevent lysis before the parasite has completed its developmental cycle? A mathematical model of the homeostasis of infected red cells suggested that it is the wasteful consumption of host cell hemoglobin that prevents early lysis by the progressive reduction in the colloid-osmotic pressure within the host (the colloid-osmotic hypothesis). However, two critical model predictions, that infected cells would swell to near prelytic sphericity and that the hemoglobin concentration would become progressively reduced, remained controversial. In this paper, we are able for the first time to correlate model predictions with recent experimental data in the literature and explore the fine details of the homeostasis of infected red blood cells during five model-defined periods of parasite development. The conclusions suggest that infected red cells do reach proximity to lytic rupture regardless of their actual volume, thus requiring a progressive reduction in their hemoglobin concentration to prevent premature lysis.

Preserved function of the plasma membrane calcium pump of red blood cells from diabetic subjects with high levels of glycated haemoglobin.

The activity of the plasma membrane Ca(2+)-pump decreases steeply throughout the 120 days lifespan of normal human red blood cells. Experiments with isolated membrane preparations showed that glycation of a lysine residue near the catalytic site of the pump ATPase had a powerful inhibitory effect. This prompted the question of whether glycation is the mechanism of age-related decline in pump activity in vivo. It is important to investigate this mechanism because the Ca(2+) pump is a major regulator of Ca(2+) homeostasis in all cells. Its impaired activity in diabetic patients, continuously exposed to high glycation rates, may thus contribute to varied tissue pathology in this disease. We measured Ca(2+)-pump activity as a function of red cell age in red cells from diabetics continuously exposed to high glucose concentrations, as documented by their high mean levels of glycated haemoglobin. The distribution of Ca(2+)-pump activities was indistinguishable from that in non-diabetics, and the pattern of activity decline with cell age in the diabetics' red cells was identical to that observed in red cells from non-diabetics. These results indicate that in intact cells the Ca(2+) pump is protected from glycation-induced inactivation.

FRET imaging of hemoglobin concentration in Plasmodium falciparum-infected red cells.

BACKGROUND: During its intraerythrocytic asexual reproduction cycle Plasmodium falciparum consumes up to 80% of the host cell hemoglobin, in large excess over its metabolic needs. A model of the homeostasis of falciparum-infected red blood cells suggested an explanation based on the need to reduce the colloid-osmotic pressure within the host cell to prevent its premature lysis. Critical for this hypothesis was that the hemoglobin concentration within the host cell be progressively reduced from the trophozoite stage onwards. METHODOLOGY/PRINCIPAL FINDINGS: The experiments reported here were designed to test this hypothesis by direct measurements of the hemoglobin concentration in live, infected red cells. We developed a novel, non-invasive method to quantify the hemoglobin concentration in single cells, based on Förster resonance energy transfer between hemoglobin molecules and the fluorophore calcein. Fluorescence lifetime imaging allowed the quantitative mapping of the hemoglobin concentration within the cells. The average fluorescence lifetimes of uninfected cohorts was 270+/-30 ps (mean+/-SD; N = 45). In the cytoplasm of infected cells the fluorescence lifetime of calcein ranged from 290+/-20 ps for cells with ring stage parasites to 590+/-13 ps and 1050+/-60 ps for cells with young trophozoites and late stage trophozoite/early schizonts, respectively. This was equivalent to reductions in hemoglobin concentration spanning the range from 7.3 to 2.3 mM, in line with the model predictions. An unexpected ancillary finding was the existence of a microdomain under the host cell membrane with reduced calcein quenching by hemoglobin in cells with mature trophozoite stage parasites. CONCLUSIONS/SIGNIFICANCE: The results support the predictions of the colloid-osmotic hypothesis and provide a better understanding of the homeostasis of malaria-infected red cells. In addition, they revealed the existence of a distinct peripheral microdomain in the host cell with limited access to hemoglobin molecules indicating the concentration of substantial amounts of parasite-exported material.

Is invasion efficiency in malaria controlled by pre-invasion events?

The invasion efficiency of Plasmodium falciparum merozoites was found to decrease with increasing red blood cell density, a finding relevant to protection strategies against falciparum malaria. The mechanism of this 'density effect' remained unexplained. Searching for possible explanations, we studied selected video recordings of the dynamic events during merozoite invasion and identified a pre-invasion stage as a crucial mediator of invasion efficiency. We suggest that the role of the pre-invasion stage is to induce the apical alignment of the merozoite, and propose a working hypothesis on its mechanism, with a crucial role for elevated intracellular Ca2+.

Age decline in the activity of the Ca2+-sensitive K+ channel of human red blood cells.

The Ca(2+)-sensitive K(+) channel of human red blood cells (RBCs) (Gardos channel, hIK1, hSK4) was implicated in the progressive densification of RBCs during normal senescence and in the mechanism of sickle cell dehydration. Saturating RBC Ca(2+) loads were shown before to induce rapid and homogeneous dehydration, suggesting that Gardos channel capacity was uniform among the RBCs, regardless of age. Using glycated hemoglobin as a reliable RBC age marker, we investigated the age-activity relation of Gardos channels by measuring the mean age of RBC subpopulations exceeding a set high density boundary during dehydration. When K(+) permeabilization was induced with valinomycin, the oldest and densest cells, which started nearest to the set density boundary, crossed it first, reflecting conservation of the normal age-density distribution pattern during dehydration. However, when Ca(2+) loads were used to induce maximal K(+) fluxes via Gardos channels in all RBCs (F(max)), the youngest RBCs passed the boundary first, ahead of the older RBCs, indicating that Gardos channel F(max) was highest in those young RBCs, and that the previously observed appearance of uniform dehydration concealed a substantial degree of age scrambling during the dehydration process. Further analysis of the Gardos channel age-activity relation revealed a monotonic decline in F(max) with cell age, with a broad quasi-Gaussian F(max) distribution among the RBCs.

Effects of age-dependent membrane transport changes on the homeostasis of senescent human red blood cells.

Little is known about age-related changes in red blood cell (RBC) membrane transport and homeostasis. We investigated first whether the known large variation in plasma membrane Ca(2+) (PMCA) pump activity was correlated with RBC age. Glycated hemoglobin, Hb A1c, was used as a reliable age marker for normal RBCs. We found an inverse correlation between PMCA strength and Hb A1c content, indicating that PMCA activity declines monotonically with RBC age. The previously described subpopulation of high-Na(+), low-density RBCs had the highest Hb A1c levels, suggesting it represents a late homeostatic condition of senescent RBCs. Thus, the normal densification process of RBCs with age must undergo late reversal, requiring a membrane permeability increase with net NaCl gain exceeding KCl loss. Activation of a nonselective cation channel, Pcat, was considered the key link in this density reversal. Investigation of Pcat properties showed that its most powerful activator was increased intracellular Ca(2+). Pcat was comparably selective to Na(+), K(+), choline, and N-methyl-D-glucamine, indicating a fairly large, poorly selective cation permeability pathway. Based on these observations, a working hypothesis is proposed to explain the mechanism of progressive RBC densification with age and of the late reversal to a low-density condition with altered ionic gradients.