J. Cell Set. 81, 1-16 (1986)Printed in Great Britain © The Company of Biologists Limited 1986

DYNAMIC REARRANGEMENTS OF ERYTHROCYTE

MEMBRANE INTERNAL ARCHITECTURE INDUCED

BY INFECTION WITH PLASMODIUM FALCIPARUM

DAVID R. ALLRED*, JEAN E. GRUENBERGj AND IRWIN W.SHERMAN

Department of Biology, University of California, Riverside, CA 92521, U.SA.

SUMMARY

Cultured human erythrocytes infected with Plasmodium falciparum were studied by freeze-fracture electron microscopy. Special emphasis was placed upon the formation of the membranesurface excrescences ('knobs') found on red cells containing mature parasites. Knobs werevisualized as conoid projections of the protoplasmic fracture face (PF) and depressions of theexoplasmic fracture face (EF). Knob formation was correlated with parasite growth and, on thebasis of the organization of intramembranous particles (IMP) in the PF leaflet, a series of changesassociated with parasite maturation was discerned:' (1) a focal IMP cluster with minimalerythrocyte membrane elevation; (2) an elevated central IMP cluster surrounded by an IMP-freezone and concentric IMP ring; (3) maximal erythrocyte membrane deformation, concomitant witha loss of obvious IMP organization. Subtle changes in PF IMP organization were seen with knobformation and parasite maturation, including an apparent lateral partitioning of endogenous redcell membrane proteins between knobby or knob-free membrane areas in trophozoite-infectedcells. IMP size distributions of the PF were shifted toward smaller particles in schizont-infectedcells. Parasite development did not affect IMP densities in the PF; however, a decrease from464 ±106 fim~2 to 374 ± 94 /tfn~2 was seen in the EF of schizont-infected cells. IMP densities weresimilar over knobs and knob-free areas of either membrane leaflet, and there was no apparent EFIMP reorganization associated with the presence of knobs. These findings indicate that dynamicmembrane changes are associated with knob formation and parasite maturation.

INTRODUCTION

Plasmodium falciparum undergoes a 48-h cycle of asexual reproduction withinhuman red blood cells. The first 18-20 h are spent as a 'ring' form, whichsubsequently grows and differentiates into the trophozoite stage. The last 24 hinvolve further growth, maturation and differentiation of the trophozoite to give themultinucleate schizont stage. The schizont undergoes cytokinesis to yield numerousmerozoites — the stage responsible for reinvasion of other red cells.

• Author for correspondence at: Department of Molecular, Cellular and Developmental Biology,University of Colorado, Boulder, CO 80309, U.S.A.

tPresent address: European Molecular Biology Laboratories, Postfach 102209, 6900Heidelberg, West Germany.

Key words: Plasmodium falciparum, freeze-fracture, erythrocyte membrane, membranealterations, malaria.

2 D. R. Mired, J. E. Gruenberg and I. W. Sherman

The surface of human red blood cells infected with P. falciparum undergoesultrastructural, antigenic and biochemical modifications (Langreth, Jensen, Reese &Trager, 1978; Sherman, 1979; Howard, 1982). Electron-dense surface excrescences(knobs) are formed on P. falciparum -infected cells containing trophozoite orschizont-stage parasites. Knobs are thought to mediate binding of infected cells tothe capillary endothelium in vivo (Luse & Miller, 1971), and to cultured endothelial(Udeinya et al. 1981) and amelanotic melanoma cells (Schmidt et al. 1982) in vitro.The mechanism of this binding is unknown.

The origin of knobs remains a point of controversy. One opinion is that knobs aremade of proteins of parasitic origin; several lines of evidence indirectly support thishypothesis. Thus, schizont-synthesized glycoproteins were detected on the infectedred cell surface (Perkins, 1982), 'neoglycoproteins' occur on red cells obtained fromhumans infected with P. falciparum (Howard et al. 1981), and areas of red cellmembrane immediately overlying knobs appear antigenically distinct from theremainder of the erythrocyte surface (Kilejian, Abati & Trager, 1977; Langreth &Reese, 1979). A correlation has also been made between knob formation and thepresence of an 88xlO3Mr protein, which may be associated with the erythrocytecytoskeleton (Kilejian, 1979; Leech et al. 1984). Despite such presumptive evi-dence, it has been demonstrated that parasite-synthesized proteins are neitherstructural components of the knob itself (Gruenberg & Sherman, 1983), nor do theyappear to be inserted through the erythrocyte membrane (Allred, 1982; Allred &Sherman, 1983; Allred, Gruenberg & Sherman, 1983).

Freeze-fracture electron-microscopic studies of malaria-infected erythrocytes havebeen performed using P. knowlesi-infected Rhesus monkey cells. In one such studyerythrocyte invasion by P. knowlesi merozoites was associated with a movingjunction of rhomboidally arrayed red cell membrane intramembranous particles(IMPs) (Aikawa, Miller, Johnson & Rabbege, 1978), although the origin of thejunction IMPs was not established. Despite the fact that P. knowlesi does not induceknob formation, IMP rearrangements and a decrease in IMP density upon infectionwere revealed (McLaren, Bannister, Trigg & Butcher, 1977, 1979); these results,however, are controversial (Wunderlich, Stubig & Konigk, 1982). More recently,P. falciparum-inf ected human and Aotus sp. monkey red cells were similarly studied.No gross alterations associated with knob formation were found (Aikawa, Rabbege,Udeinya & Miller, 1983), although evidence of subtle reorganizations of membraneprotein complexes was reported (Allred, Gruenberg & Sherman, 1983).

These reports, in conflict with one another in several respects, neither prove nordisprove the involvement of parasite-derived proteins in the membrane alterationsfound in malaria-infected cells. To determine if knob formation is reflected internallyin the erythrocyte membrane, and to gain insight into the mechanisms of knobformation, we have analysed P. falciparum-iniected human erythrocytes by freeze-fracture electron microscopy. Our results suggest that several levels of membranemodification do occur, but provide no evidence for the structural involvement ofparasite-derived proteins in the process.

Red cell membrane alterations in malaria 3

MATERIALS AND METHODS

ParasitesA cloned strain olP.falciparum FCR-3, which produces knobs and adheres to endothelial cells

in vitro (Udeinya et air 1981), was generously provided by Dr L. H. Miller of the NationalInstitutes of Health. Parasites were cultured by the method of Trager & Jensen (1976) in RPMI1640 tissue-culture medium (Grand Island Biological Co., Grand Island, NY) supplemented with0-2% (w/v) sodium bicarbonate, 10% (v/v) normal human serum and 2SmM-HEPES buffer(./V-2-hydroxyethylpiperazine-./V'-2-ethanesulphonic acid; Calbiochem, La Jolla, CA). Parasitedevelopment was synchronized by double sorbitol lysis at 36-h intervals (Lambros & Vanderberg,1980). Parasitized red cells containing schizonts (at 36 h of development) were concentrated from5-10% parasitaemia to 70-90% using a modification of the technique of Pasvol, Wilson, Smalley& Brown (1978). Briefly, a 25 % (v/v) cell suspension in complete culture medium was added to anequal volume of Physiogel (Hausmann Laboratories, St Gallen, Switzerland), mixed, incubated30 min at 37°C, and the supernatant was collected and washed twice with complete culture mediumby centrifugation (1500grim, 4°C).

Transmission electron microscopyCells were fixed for 1 h at 37°C by addition of 10 vol. of fixative (2% (w/v) glutaraldehyde in

0-1 M-sodium phosphate, pH 7-40, containing 4% (w/v) sucrose and 2x 10~s M-CaCl2, 37°C), thenwashed in 0-1 M-phosphate buffer, pH7-4, dehydrated in ethanol, cleared in propylene oxide, andembedded in Epon 812. Sections were stained with 2% (w/v) ethanolic uranyl acetate and leadcitrate (Reynolds, 1963).

Freeze-fracture electron microscopyParasitized cells (5-10% parasitaemia) were collected at appropriate times, concentrated when

possible (see above), washed in culture medium, then made up to a 20% (v/v) suspension in thesame medium. The cell suspension (37°C) was placed in 10 vol. of 37°C fixative for 30 min, washedwith isotonic phosphate-buffered saline (PBS; 0-01 M-sodium phosphate, 0-15M-NaCl, pH7-40),and cryoprotected by incubation in 20% (v/v) glycerol for 30—60 min at room temperature. Afterlight pelleting, cells were loaded onto gold planchets and frozen from 37 °C in liquid propane cooledwith liquid nitrogen. Fracturing was done in a Balzers freeze-etch apparatus (model BAF301) at-120°C under a vacuum of s=2xlO~6Torr. Replicas were cleaned overnight on 5-25% (w/v)sodium hypochlorite.

Statistical analysisElectron micrographs were prepared at a standard magnification of 75 000X. IMPs were

measured to 0-1 mm, using a magnifying ocular, and both size and location of the IMPs wererecorded. IMPs were measured on four cells per sample type, with an average of >300 measuredper uninfected cell, and >400 measured per infected cell. For the determination of IMP sizedistributions and densities, data from all cells within a population were pooled; all data wereaccounted separately for subsequent statistical analyses. The experiment was repeated at least fivetimes for both synchronized populations of parasitized cells. Micrographs for analysis were chosenfrom all experiments based on replica and image quality, and for purposes of IMP measurementswere encoded to prevent prior identification of trie cell population. IMP size distributions wereanalysed statistically according to the method of deLaat, Tertoolen & Bluemink (1981). Todescribe the general nature of IMP distributions, the 'Z value' (approximate normal deviate) wasdetermined for each area of the infected cell membrane. Absolute Z values greater than 1-65 arestatistically significant, with positive values indicating IMP aggregation, negative values IMPdispersion, and values near zero random IMP organization. Cumulative frequency distributions ofdifferent IMP populations were compared using the Kolmogorov-Smirnov test, as described bydeLaat et al. (1981). Mean values of IMP density and Z values were compared by a two-tailedStudent's t test modified for small sample populations (Bailey, 1981).

D. R. Mired, J. E. Gruenberg and I. W. Sherman

1Fig. 1. Thin-section transmission electron micrograph of a P. falciparum trophozoite-infected red cell, k, knobs;/), parasite; e, erythrocyte. X60000. Bar, 0-5/Jxn.

RESULTS

Knob formation

Knob structure, as seen by thin-section transmission electron microscopy, isshown in Fig. 1. In cross-section, knobs appear to be conoid in shape and directlyapposed to the cytoplasmic surface of the red cell membrane. When sectionedtangentially to the red cell surface, a doughnut-like structure with a rim of veryelectron-dense material is seen. This appearance of knobs is in agreement with thatpreviously reported (Langreth et al. 1978). From a correlation of parasite maturityand organization of the protoplasmic fracture face (PF), knob formation could bedenned morphologically by three stages (Fig. 2). Because our observations weremade on synchronous populations and correlated directly with parasite development,we believe these three stages indicate the progress of knob maturation: (1) IMPs overforming knobs were clustered at discrete foci, but membrane elevation was notapparent and there was no overt IMP organization; (2) membrane elevation was inevidence and the central IMP cluster was surrounded by an IMP-free zone and one tothree concentric IMP rings (see inset, Fig. 2A); (3) membrane deformation wasmaximal, the central IMP cluster of the knob was dispersed, and the ordered IMPring and IMP-free zone were lost. Knobs in all three stages of formation were found

Fig. 2. Protoplasmic fracture face (PF) of infected and uninfected red cells; X60000.A. Trophozoite-infected cell; inset (X200000) shows the substructure of a stage (2) knob.B. Schizont-infected cell. C. Uninfected erythrocyte. k, knobs; arrows, IMP-free zone;arrowheads, organized IMP ring. Platinum-carbon shadowing direction is from thebottom in all cases. Bar, 0-5/an.

Red cell membrane alterations in malaria

6 D. R. Mired, jf. E. Gruenberg and I. W. Sherman

on trophozoite-infected cells; however, with maturation to the schizont stage, stage(3) knobs were the predominant form found, and no further knob maturation wasseen (Fig. 2B), although knob elevation sometimes decreased again. The obvious PFIMP rearrangements associated with knob formation were not reflected in theexoplasmic fracture face (EF) (Fig. 3), indicating a probable lack of physicalcoupling between the integral membrane proteins residing in each leaflet.

PF IMP densities

PF IMP densities were unchanged by infection of the red cell or by parasitematuration. Uninfected cells had a density of 4808 ± 667 /xm~2, compared with4771 ± 437 fim~2 for trophozoite-infected and 4795 ± 570jUm~2 for schizont-infectedcells. This result is consistent with data from P. knowlesi-'mfected Rhesus monkeycells (Wunderlich et al. 1982). IMP densities also were not significantly differentbetween knobby and knob-free areas of infected cell membranes at either stage ofparasite development (summarized in Table 1).

PF IMP distribution

In contrast to the PF IMP densities, striking differences in mean PF IMPdiameters (Table 1) and frequency distributions of IMP size classes were observedbetween knobby and knob-free membrane areas, and between each area and themembrane as a whole (Figs 4, 5). Trophozoite-infected cell membranes were not,however, significantly different from uninfected cells when total PF IMP popu-lations were compared. The PF IMP size frequency distributions for uninfected, andtrophozoite- and schizont-infected cells are compared in Fig. 4 (cumulative relativefrequency data are not shown).

At the trophozoite stage, IMPs from the knob-free areas of the red cell membraneswere right-shifted in size (i.e. toward larger particle sizes) when compared withuninfected cells (P — 5X 10~4) (Fig. 4G), and were especially so when compared withknobby areas (P = 2x 10~s). Knob PF IMPs were left-shifted (i.e. a tendency towardsmaller particles) relative to uninfected cells (Fig. 4H), although not significantly(P = 0-20). The shifts are clearly shown in Fig. 5, which indicates the magnitude ofthe change and the size classes primarily involved. Knobs in all three stages offormation (as defined earlier) were considered together for this analysis since thestages represent points on a continuum. In addition, a separate record was kept ofobvious organized IMP rings of stage (2) knobs, on the premise that these IMPsmight represent a population of special origin, e.g. parasite proteins inserted into thered cell membrane. The size distribution of stage (2) knob IMP rings was some-what right-shifted (i.e. toward larger particles; Fig. 4F) relative to uninfected cells(P = 0-06), and thus resembled the knob-free rather than the knobby areas of theinfected cell membrane (Fig. 5c). In contrast, schizont-infected cell membraneparticles were significantly left-shifted in comparison with uninfected cells (Figs 4B,5D; P = 2X 10~4 for the knob-free areas of the membrane, P = 5 X 10~8 for knobs, and/> = 8xl0~8 for the membrane as a whole), although there were no differencesbetween knobby and knob-free areas.

Red cell membrane alterations in malaria 7

PF IMP general organization

PF IMPs of uninfected red cells (Fig. 2c) had a mildly dispersed organizationunder the present preparative conditions, with a mean Z value of —1-8 (P = 0-05).The PF of infected red cells displayed an even more pronounced dispersion of thetotal IMP population, with mean Z values of —3-8 and — 3-2 for trophozoites andschizonts, respectively (P = 0-01—0-001). These data are summarized in Table 2.

The overall nature of IMP organization over knobs was not significantly differentfrom that of knob-free membrane areas, and was similarly unaffected by parasitematuration. Mean Z values for trophozoite-infected red cells were —2-8 and —2-6 forknobby and knob-free areas, respectively, whereas those for schizont-infected cellswere —1-9 for knobby area IMPs and —2-5 for knob-free areas.

It is possible to determine the spatial organization of particular IMP populationsby grouping discrete IMP size classes together and performing the same type ofanalysis. The results of such an analysis could provide clues as to whether the totalIMP population, or different IMP sub-populations are affected. Fig. 6 graphicallypresents the Z value data for the IMP size groups 4-0—6-0nm (A), 6-0—8-7 nm (B),8-7-12-7nm (c), and larger than 12-7 nm (D) in diameter. PF IMPs were analysedfor both knobby (Fig. 6, closed circles) and knob-free (open circles) membrane areasas a function of parasite maturation. From this analysis, it is clear that the varioussize groups are affected differently.

EF IMP density and organization

As mentioned earlier, the EF leaflet of infected cells (Fig. 3) did not reflect thesame overt IMP reorganization found in the PF leaflet. The density of EF IMPs,however, was found to be reduced from 464 ± 106 [im~2 in uninfected cells to354 ± 40/im~2 in trophozoite-infected and 374 ± 94/im~2 in schizont-infected cells;these differences were not statistically significant (P=>0-10). Also, a tendencytoward IMP aggregation appeared, with Z values changing from +0-6 for uninfectedcells to +0-9 in trophozoite-infected and +1-9 in schizont-infected cells. Although atrend appears to exist, there was considerable variation between individual cells, andthe differences were again not statistically significant. In contrast to the PF IMPdata, relative frequency size distributions for infected cell EF IMPs were notsignificantly different from those of uninfected cells (Figs 5,7).

DISCUSSION

Knob formation is a continuous process that does not occur synchronously overthe surface of an infected cell (Gruenberg, Allred & Sherman, 1983; this study).Ultrastructurally, three major stages could be defined by the degree of PF IMPaggregation and organization, as well as the extent of membrane elevation (seeFig. 2). Knobs in all three stages of formation were typically found simultaneouslyon individual trophozoite-infected cells. Since schizont-infected cells possessedknobs predominantly of the stage (3) form, and further maturational changes were

D. R. Mired, J. E. Gruenberg and I. W. Sherman

Red cell membrane alterations in malaria

Table 1. Intramembranoiis particle densities

Sample

Normal red bloodPFEF

Mean density (/an 2)

cells4808

464

Trophozoite-infected cells; total membrane*PF 4771EF 354

Trophozoite-infected cells; knobby areasPF 4870

Trophozoite-infected cells; knob-free areasPF 4682

Schizont-infectedPFEF

Schizont-infectedPF

Schizont-infectedPF

cells; total membrane*4795

374

cells; knobby areas4694

cells; knob-free areas4896

• Total membrane refers to data combined from

±ls .D.

667106

43740

274

583

57094

481

693

both knobby and

Mean diam. (nm)

9-1510-52

9-3110-18

8-99

9-53

8-6510-53

8-53

8-76

knob-free areas.

not found, it appears that knob-induced membrane modifications (as detected byalterations in PF) terminate with stage (3).

Membranes of trophozoite-infected cells showed no change in PF IMP densities orin the size frequency distribution of the total PF IMP population. Interestingly,areas over forming knobs showed a preponderance of smaller IMPs when comparedwith apparently unaffected regions of the membrane or with the concentric IMP ringsurrounding stage (2) knobs. Therefore: (1) it is unlikely that any significant changein the population of proteins making up the PF IMP population occurred; (2) aselective lateral movement of endogenous red cell proteins could account for, and isconsistent with, the IMP size separation in the two areas; and (3) the characteristicsof the IMP rings surrounding stage (2) knobs are consistent with reorganization ofendogenous host cell membrane proteins. If specific parasite-synthesized proteinswere inserted into the red cell membrane, significant local changes in IMP sizefrequency distribution and, or, density might be expected, since a priori parasiteproteins cannot be expected to have the same size frequency distribution asendogenous red cell proteins. Such changes were not found.

How could the described IMP reorganizations occur? It is possible thatassociations form between endogenous red cell integral membrane proteins andaccumulating knob materials, owing to a loss of the normal diffusional constraintsimposed upon such proteins by the red cell cytoskeleton. This situation would be

Fig. 3. Exoplasmic fracture face (EF) of infected and uninfected red cells; X60000.A. Trophozoite-infected cell. B. Schizont-infected cell. C. Uninfected erythrocyte.k, knobs. Platinum-carbon shadowing direction is from the bottom in all cases. Bar,0-5/im.

10 D. R. Mired, J. E. Gruenberg and I. W. Sherman

IMP diameter (nm)

Fig. 4. PF IMP size frequency distributions. A. Uninfected red cells, B - D . Schizont-infected cells: B, total membrane; C, knob-free areas; D, knobs. E - H . Trophozoite-infected cells: E, total membrane; F, stage (2) knob organized IMP rings; G, knob-freeareas; H, knobs. Bars indicate frequency value ranges.

consistent with our data, since band 3 - the major protein giving rise to PF IMPs(Edwards, Mueller & Morrison, 1978; Gratzer, 1981; Morrison, Mueller &Edwards, 1981) — is normally associated with haemoglobin (Sayare & Fikiet, 1981)and spectrin (Bennett & Stenbuck, 1979) in vivo, both of which are degraded inmalaria-infected red cells (see Sherman, 1979, for a review).

The organization of stage (2) knobs is of special interest because it may reflect themechanism of knob formation. The IMP population making up the organized ringsis characterized by: (1) a somewhat larger size than that of uninfected cells; (2) verysimilar size distribution to the IMPs in knob-free areas of trophozoite-infected cells;(3) a wide distribution of IMP sizes; and (4) a high degree of organization. Thenature of the IMP population and the degree of order of these IMP rings are suchthat their formation is likely to result from extrinsically imposed ordering effectsacting upon endogenous red cell proteins recruited from knob-free areas of themembrane, e.g. by a modified cytoskeleton or by association with accumulatingunderlying knob materials. Such a contention is compatible with the increase in knobspatial density and concomitant loss of obvious patterns of knob arrangement (rows,clusters, etc.) during parasite maturation (Gruenberg et al. 1983).

Parasite maturation from trophozoite to schizont was accompanied by a shift in thecumulative size frequency distribution toward smaller particles (P= 1X1O~6), andincluded selective increases and decreases in particular PF IMP class frequencies,but no change in overall PF IMP density. The cause of this overall shift towardsmaller PF IMP sizes is unclear. Importantly, knobby and knob-free areas ofschizont-infected cell membranes were no longer different from one another in

Red cell membrane alterations in malaria 11

size frequency distribution. There are several possible explanations for theseobservations: (1) removal or degradation of integral membrane proteins or wholemembrane fragments by parasite activities, followed by subsequent replacement

0-06

0-04

0-03

0-02

0-01

0

0-01

0-02

0-03

0-04

0-05

0-06

0-07

0-06

0-05

0-04

0-03

0'02

0-01

0

0-01

0-02

0-03

0-04

0-05

n.rn

•

-

•

•

.

•

1 1

i-

•

•

•

•

-

-

-

-

•

Fig. 5. PF IMP size relative frequency difference histograms. A. (Trophozoite-infectedcell knob-free areas) minus (uninfected erythrocytes). B. (Trophozoite-infected cellknobs) minus (uninfected erythrocytes). C. (Trophozoite-infected cell knobs) minus(organized stage (2) knob IMP rings). D. (Schizont-infected cell total membrane area)minus (uninfected erythrocytes).

12 D.R. Alfred, J. E. Gruenberg and I. W. Sherman

with parasite-derived proteins or membrane fragments; (2) alteration of specificprotein complexes within the membrane bilayer; (3) covalent modification ofendogenous red cell membrane proteins; or (4) modification of red cell membranelipids during parasite growth. The first possibility (based upon the IMP size relativefrequency difference histogram; Fig. 5D) would involve about 10% of the integralproteins giving rise to IMPs, an amount clearly inconsistent with data obtained frommetabolically labelled P. faldparum-iniected cells, in which evidence for parasiteproteins in the red cell membrane was not found (Allred, 1982; Allred & Sherman,1983; Gruenberg & Sherman, 1983; Allred et al. 1983). The second possibility isquite plausible, provided the assumption is made that only 1 part of a disruptedcomplex is still resolved as an IMP, leaving IMP densities unchanged but IMPdiameters reduced. Evidence has accumulated in other malaria/host systemsregarding the last two possibilities (for reviews, see Sherman, 1979; Howard, 1982).Covalent modification of integral erythrocyte membrane proteins seems unlikely toaffect IMP diameters, except for the effect of such modification on protein complexstability, as in point (2) above. Significant proteolytic modification, althoughpossible, is unlikely due to the relatively large population of proteins involved and thefailure to detect this in other studies. Membrane lipid alterations, however, areextensive (Sherman, 1979). Altered membrane fluidity has been shown to affect theapparent vertical partitioning of some integral membrane proteins (Shinitzky &Rivnay, 1977; Borochov, Abbott, Schachter & Shinitzky, 1979), as well as theantigenic reactivity of red cell Rho(D) antigen (Shinitzky & Souroujon, 1979; Basuet al. 1980). Membrane fluidity of infected red blood cells is increased (Howard &

Table 2. Intramembranous particle organization: Z valuesSample Mean Z value Z value range

Normal red blood cellsPF -1-79 -0-63 to-2-48EF +0-55 -0-04 to+0-86

Trophozoite-infected cells; total membrane*PF -3-82 -3-48 to-4-00EF +0-89 -0-60 to+2-56

Trophozoite-infected cells; knobby areasPF -2-80 -1-46 to-3-52

Trophozoite-infected cells; knob-free areasPF -2-56 -1-71 to-3-66

Schizont-infected cells; total membrane*PF -3-16 -2-66 to-3-75EF +1-87 -0-26 to+4-42

Schizont-infected cells; knobby areasPF -1-85 - l -22 to -2 -34

Schizont-infected cells; knob-free areasPF -2-53 -1-97 to -2-89

• Total membrane refers to data compiled from both of the two separately considered areas of themembrane (i.e. knobby and knob-free areas).

Red cell membrane alterations in malaria 13

4

3

2

1

0

-1

-2

- 3

-4

4

3

2

1

0

- 1

- 2

- 3

-4

• A

B

• c

D o

N 24 36 N 24 36

Fig. 6. Z value changes for grouped PF IMP sizes during parasite development.A. 4-0-6-7nm; B, 6-7-8-7nm; c, 8-7-12-7nm; D, 12-7nm and larger. ( • ) Values forknobs; (O) knob-free areas; (A) data from uninfected red cells. N, uninfected red cells;24, trophozoite-infected cells at 24h of parasite development; 36, schizont-infected cellsat 36 h of development.

Sawyer, 1980; Allred, Sterling & Morse, 1983; Butler, Deslauriers & Smith, 1984),and could conceivably affect protein conformations, complex stability, or 'verticalpartitioning', and therefore apparent IMP diameters. A possible non-biologicalexplanation is that these data merely reflect variations in the quality of shadowingand, or, water vapour deposition in different experiments — potential artifacts in thistype of study (see, e.g., Schotton, 1982). However, this is quite unlikely, since allthree sample types were included in each experiment, and the micrographs used foranalysis were chosen from different experiments solely on the basis of overall replicaquality and uniform shadow angle.

The dynamic changes in integral protein spatial organization found in infectedcells are illustrated in Fig. 6. Of interest is the different effect seen on IMPorganization in knobby and knob-free areas of the membrane. For this situation tooccur, there must exist a heterogeneity in the forces affecting protein behaviour ineach area.

Our results have several implications: (1) knob formation in P. falciparum-infected cells is a dynamic process, and is probably not associated with the suddenappearance of pre-packaged knob materials; (2) association of knob materials with

14 D. R. Mired, J. E. Gruenberg and I. W. Sherman

3

0-40

0-30

0-20

0-10

0

0-30

0-20

0-30

0-20

0-10

0

rJr/h—rh

o00

o r- o

IMP diameter (nm)

Fig. 7. EF IMP size relative frequency distributions. A. Uninfected red cells; B,trophozoite-infected cells; c, schizont-infected cells. Bars indicate frequency valueranges.

the cytoplasmic surface of the membrane is not obviously reflected in theorganization of integral membrane protein complexes of the EF leaflet and mayimply, but does not prove, that any transmembrane effect does not directly involvesuch proteins; and (3) there appears to be no stable association of a specificmembrane protein (i.e. particle size class) with knobs, either during or after theirformation.

The authors thank Drs Edward Platzer and William Thomson for their critical review of themanuscript. This manuscript represents work performed by D.R.A. in partial fulfilment of therequirements for the Doctor of Philosophy degree. This work was supported by a research grantfrom the National Institutes of Health (AI 20456). J.G. received funding through the UNDP/WHO Special Programme for Research and Training in Tropical Diseases.

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parasites: a moving junction between erythrocyte and parasite. J. Cell Biol. 77, 72—82.AJKAWA, M., RABBEGE, J. R., UDEINYA, I. & MILLER, L. H. (1983). Electron microscopy of knobs

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Red cell membrane alterations in malaria 15

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(Received 24 July 1985 -Accepted 29 August 1985)