REPORTS

Acknowledgments: We thank V. Toxavidis and J. Tigges (Beth

Israel Deaconess Medical Center flow cytometry facility) for
support with flow cytometry applications; X. Yang and
S. Breitkopf for technical assistance with mass spectrometry;
Ross Dickins and Scott Lowe (Cold Spring Harbor Laboratory)
for the gift of tamoxifen-inducible Cre-expressing retroviral
plasmid; and C. Benes, N. Wu, A. Shaywitz, B. Emerling,
A. Saci, G. DeNicola, K. Courtney, A. Couvillon, S. Soltoff,
A. Carracedo, A. Grassian, J. Brugge, and members of the
Cantley lab for helpful discussions. L.C.C. is a cofounder of
Agios Pharmaceuticals, a company that seeks to develop novel

therapeutics that target cancer metabolism. G.P. is a Pfizer

Fellow of the Life Sciences Research Foundation. A.T.S. is a
Genentech Fellow and supported by the Japanese Society for
the Promotion of Science and the Kanae Foundation for
Research Abroad. M.G.V.H. is supported by the NIH
(R03MH085679 and 1P30CA147882), the Burroughs
Wellcome Fund, the Damon Runyon Cancer Research
Foundation, and the Smith family. This work was supported
by grants from the NIH (R01-GM056203-13, P01-CA089021,
and P01-CA117969-04 to L.C.C), the Starr Cancer
Consortium (L.C.C. and M.G.V.H.), the Molecular Libraries
Initiative of the National Institutes of Health Roadmap for
Medical Research, and the Intramural Research Program of
the National Human Genome Research Institute, NIH

Hemoglobins S and C Interfere with

Actin Remodeling in Plasmodium
falciparumInfected Erythrocytes
Marek Cyrklaff,1* Cecilia P. Sanchez,1 Nicole Kilian,1 Cyrille Bisseye,2 Jacques Simpore,2
Friedrich Frischknecht,1 Michael Lanzer1*
The hemoglobins S and C protect carriers from severe Plasmodium falciparum malaria. Here, we found
that these hemoglobinopathies affected the trafficking system that directs parasite-encoded proteins
to the surface of infected erythrocytes. Cryoelectron tomography revealed that the parasite generated a
host-derived actin cytoskeleton within the cytoplasm of wild-type red blood cells that connected the
Maurers clefts with the host cell membrane and to which transport vesicles were attached. The actin
cytoskeleton and the Maurers clefts were aberrant in erythrocytes containing hemoglobin S or
C. Hemoglobin oxidation products, enriched in hemoglobin S and C erythrocytes, inhibited actin
polymerization in vitro and may account for the protective role in malaria.
he malaria parasite Plasmodium falciparum
has exerted a selective pressure on the human population that has led to the emergence of several polymorphisms within the human
genome that protect carriers from severe malariarelated disease and death (1). The best-known
examples are the structural hemoglobinopathies
S (sickle cell trait; HbS) and C (HbC), in which
glutamate at the sixth position within the b-globin
chain is replaced by valine and lysine, respectively
(2, 3). Protection against severe malaria correlates
with a distorted display of parasite-encoded adhesins on the surface of infected erythrocytes
(4, 5). By reducing the cytoadhesive capacity of
parasitized erythrocytes, HbS and HbC seem to
mitigate the life-threatening complications resulting from the sequestration of infected erythrocytes in postcapillary microvessels of the brain
and other organs. How HbS and HbC bring about
this effect is unclear. We tested the hypothesis
that HbS and HbC interfere with the machinery
that directs parasite-encoded proteins to the erythrocyte surface.

1
Department of Infectious Diseases, Parasitology, Heidelberg
University, Im Neuenheimer Feld 324, 69120 Heidelberg,
Germany. 2Biomolecular Research Center Pietro Annigoni, 01
BP 364 Ouagadougou, Burkina Faso.

*To whom correspondence should be addressed. E-mail:

michael_lanzer@med.uni-heidelberg.de (M.L.); marek.
cyrklaff@med.uni-heidelberg.de (M.C.)

Human erythrocytes lack a secretory system

and are rapidly cleared from circulation by the
spleen when damaged or infected. To develop
within human erythrocytes and to avoid passage
through the spleen, P. falciparum extensively
modifies its host cell (6), for example, by placing the disease-mediating immunovariant adhesin
PfEMP1 in knob-like protrusions in the erythrocyte plasma membrane (7). To direct PfEMP1
and other determinants of virulence and pathology to the erythrocytes plasma membrane, the
parasite establishes a trafficking system within the
cytoplasm of its host cell, of which a prominent
feature are Maurers clefts, unilamellar membrane
profiles that serve as intermediary compartments
for proteins en route to the erythrocyte surface
(810). To better define the elements of this machinery and how they are altered when P. falciparum develops in HbS and HbC erythrocytes, we
applied electron tomography to parasitized erythrocytes preserved by rapid freezing (fig. S1).
We initially investigated P. falciparum
infected erythrocytes (at the trophozoite stage,
20 to 26 hours after invasion) containing the
wild-type hemoglobin HbA (homozygous). The
tomograms revealed the erythrocyte plasma
membrane, the knobs, and the Maurers clefts
(Fig. 1A). In addition, we observed an extended
network of long, sometimes branched filaments
that connected the Maurers clefts with the knobs.
Of the 20 knobs identified in 12 tomograms, all

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(M.B.B., J. J., M.S., D.S.A., and C.J.T.). We apologize to

colleagues whose work we could not cite because of space
limitations.

Supporting Online Material

www.sciencemag.org/cgi/content/full/science.1211485/DC1
Materials and Methods
Figs. S1 to S10
References
20 July 2011; accepted 24 October 2011
Published online 3 November 2011;
10.1126/science.1211485

were connected to Maurers clefts by filaments.

The filaments were between 40 and 950 nm long
(Fig. 1B) and 6.8 T 0.5 nm in diameter (Fig. 1C).
Some of the filaments branched at main angles of
70 T 5 and 110 T 5 (Fig. 1D).
The tomograms further revealed vesicles of
various sizes, ranging from 20 nm to more than
200 nm in diameter (fig. S2). About 70% of the
observed vesicles (47 out of 68) were attached to
filaments (Fig. 1E), independent of their size. The
remaining vesicles were associated with Maurers
clefts or appeared free in the erythrocyte cytosol.
Some vesicles carried PfEMP1 (Fig. 1F) (9, 11).
Tomograms taken in areas more than 5 mm
distant from Maurers clefts also revealed vesicles and a filamentous network (Fig. 2A). However, these filaments were shorter than those
observed in the vicinity of Maurers clefts (Fig.
1B). Moreover, the two main branching angles of
the filaments were equally distributed, whereas
close to Maurers clefts the filaments preferentially branched at an angle of 70 T 5 in the
direction of the Maurers clefts (Fig. 1D).
The filaments had features reminiscent of
actin filaments, including diameter and branching
pattern (Fig. 1, C and D) (12). Indeed, treating
P. falciparuminfected erythrocytes (trophozoites) for 10 min with the actin depolymerizing
agent cytochalasin D (1 mM) destroyed the filaments and altered the morphology of the
Maurers clefts (Fig. 2B). Vesicles were also not
observed under these conditions. Immunolabeling of high-pressure frozen electron microscopy
(EM) sections, using a gold-coupled monoclonal
antibody specific for b-actin (13), provided further evidence that the long filaments contained actin
(Fig. 2C and fig. S3). We noted a higher density
of actin labeling in the area between the Maurers
clefts and the erythrocyte plasma membrane compared with areas elsewhere in the erythrocyte cytoplasm (fig. S3), supporting the conclusion that actin
filaments connect the Maurers clefts with the erythrocyte plasma membrane (13). Ankyrin may anchor
the actin filaments to the Maurers clefts (14).
The erythrocyte owes its shape and physical
properties to a membrane skeleton that is primarily composed of spectrin tetramers joined by
a junctional complex that is mainly composed
of actin protofilaments (15). The length of the actin protofilaments is tightly regulated and is restricted to 14 to 16 monomers (15). Tomograms

2 DECEMBER 2011

Downloaded from www.sciencemag.org on May 4, 2015

28. M. B. Boxer et al., J. Med. Chem. 53, 1048 (2010).

29. C. Le Goffe et al., Biochem. J. 364, 349 (2002).

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Fig. 1. Structural features in the cytoplasm of P. falciparuminfected erythrocytes. (A) Section through a cryoelectron tomogram (left) and corresponding surface-rendered view (right) of a trophozoite-infected HbAA erythrocyte.
Labeling and color code in all figures: PM, erythrocyte plasma membrane
(dark blue); K, knobs (red); V, vesicles (cyan); MC, Maurers clefts (cyan);
filaments (yellow) indicated by arrowheads. (B) Frequency histogram of
filament lengths in infected (filled circles) and uninfected (open circles)
erythrocytes containing HbAA, HbSC, and HbCC. T, trophozoite; R, ring.
MC, close to (+), or far from (), Maurers clefts. n > 150 filaments in each
case. **P < 0.001 compared with uninfected HbAA erythrocytes (Students
t test). (C) Mean densitogram across 30 filaments. Dotted lines indicate
width of filament. (D) Distribution of branching angles in filaments in
P. falciparuminfected erythrocytes containing HbAA (blue, close to; brown,
far from Maurers clefts), HbSC (gray), HbCC (green). (E) Filament-vesicle
contacts, indicated by arrowheads. (F) Different planes of a tomogram of a
Tokuyasu section and surface rendered view, showing immunolabeling of
vesicles with PfEMP1 antibodies (red, 10-nm gold). Scale bars: (A) and (F),
100 nm; (E), 50 nm.

Fig. 2. Organization of host actin in infected and uninfected HbAA erythrocytes.

Tomograms of trophozoite-infected erythrocytes: (A) distant from Maurers clefts;
(B) after treatment with cytochalasin D (1 mM). (C) Immuno-EM of infected

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erythrocytes, using a gold-coupled monoclonal antibody to b-actin (see also fig.

S3A). Tomograms of uninfected (D) and ring-stageinfected (E) HbAA erythrocytes. Cyan, vesicles and early-stage Maurers clefts. Scale bars, 100 nm.
SCIENCE

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REPORTS

Fig. 3. Aberrant actin cytoskeleton and Maurers clefts in trophozoite-infected

HbSC and HbCC erythrocytes. Tomograms of infected HbSC (A) and HbCC (B)
erythrocytes. (C) Immunolabeling of infected HbCC erythrocyte, using an
antiserum against the Maurers clefts marker PfSBP1. (D) Tomogram of an
uninfected HbCC erythrocyte. Scale bars, 100 nm.

Fig. 4. Inhibition of actin polymerization in vitro. (A) Actin filament length in the presence of total protein
extracts from erythrocytes containing HbAA, HbSC, and HbCC. (B) Effect of 5 mM of hemoglobin (Hb),
methemoglobin (met-Hb), and ferryl hemoglobin (ferryl-Hb) on actin filament length. (C) Concentrationdependent decrease of actin filament length by ferryl hemoglobin. The percentage of ferryl hemoglobin of
total hemoglobin (5 mM) is indicated. The means T SEM of at least four independent experiments are
shown. **P < 0.01 compared with HbAA extracts and hemoglobin.

of plunge-frozen uninfected human erythrocytes

revealed short filaments of 30 to 40 nm underneath the erythrocyte plasma membrane (Figs.
2D and 1B)which in terms of length, thickness
(6.8 T 0.5 nm), and location are consistent with
actin protofilaments. The spectrin filaments are
significantly thinner than actin filaments (16)
and were not detected in our tomograms but were
visible by classical EM in negatively stained
preparations of the membrane skeleton (fig. S4).
The hexagonal arrangements characterizing the

membrane skeleton of uninfected red blood cells

(17) were not observed in membrane skeletal preparations from parasitized erythrocytes (fig. S4).
These data suggest that the parasite remodels
the membrane skeleton of its host cell during
intra-erythrocytic development to build an actin
network of its own design. Consistent with this
hypothesis, the parasite-generated actin cytoskeleton in ring-stage-infected erythrocytes (10
to 14 hours after invasion) was less pronounced
in areas close to Maurers clefts (Fig. 2E), with

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short filaments of 20 to 60 nm dominating (Fig.

1B) (P < 0.001), suggesting that actin remodeling
occurs as the parasite develops.
We next analyzed tomograms of P. falciparum
infected erythrocytes (at the trophozoite stage)
containing homozygous HbCC and heterozygous
HbSC. The tomograms revealed the erythrocyte
plasma membrane with knobs, vesicles of different sizes (ranging from 20 to 100 nm in diameter)
(fig. S2), and large amorphous membrane conglomerates, but no membrane profiles characteristic of Maurers clefts (Fig. 3, A and B). However,
an antiserum against the Maurers clefts marker
PfSBP1 identified the amorphous membrane conglomerates as aberrant Maurers clefts (Fig. 3C).
Classical EM of chemically fixed or high-pressure
frozen cells confirmed that in HbSC- and HbCCcontaining erythrocytes, Maurers clefts did not
form the elaborated, multilayered membrane stacks
seen in HbAA erythrocytes (fig. S5). Despite the
altered morphology of Maurers clefts, parasites
appeared to develop normally in HbSC and HbCC
erythrocytes (figs. S6 and S7).
The tomograms further revealed actin filaments underneath the erythrocyte plasma membrane in both HbSC and HbCC erythrocytes.
However, the filaments were significantly shorter
than those seen in infected HbAA erythrocytes
(P < 0.001) (Fig. 1B), and the two main branching
angles were equally distributed (Fig. 1D). Only a
few actin filaments (8 of 150 in nine tomograms)

2 DECEMBER 2011

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REPORTS
were attached to the degenerated Maurers clefts,
and they did not interconnect them with the
knobs. Only 15% (6 out of 40) of the vesicles
observed were attached to filaments; 85% were
free in the cytoplasm.
Erythrocytes containing HbS and HbC have a
dysfunctional cytoskeleton among other abnormalities (18), which we confirmed in uninfected
HbCC erythrocytes by cryoelectron tomography
(compare Figs. 2D and 3D). The actin filaments
appeared less confined to the volume underneath
the erythrocyte plasma membrane (Fig. 3D) and
were significantly longer (P < 0.001) than those
in uninfected HbAA erythrocytes (Fig. 1B).
These changes in actin organization result
from endogenous factors and may involve oxidized forms of hemoglobin (19). Both HbS and
HbC are unstable and readily oxidize to methemoglobin and finally to hemichromes, which
accumulate more than 10-fold over normal levels
(18, 20). Hemoglobin and methemoglobin have a
stabilizing effect on the membrane skeleton by
promoting the self-association of spectrin dimers
to tetramers (19, 21), whereas hemichromes can
destabilize the erythrocyte membrane skeleton by
decreasing the spectrin-protein 4.1-actin interaction (19). Thus, oxidized hemoglobins might interfere with the actin reorganization brought about
by the parasite in HbS and HbC erythrocytes.
Consistent with this hypothesis, shorter actin filaments were formed in in vitro actin polymerization assays in the presence of total protein extracts
(5 mM) from HbSC and HbCC erythrocytes compared with extracts from HbAA erythrocytes (P <
0.001) (Fig. 4A). This inhibitory effect could be
reproduced by ferryl hemoglobin, but not by hemoglobin or methemoglobin, and was dependent
on the percentage of ferryl hemoglobin of total
hemoglobin (Fig. 4, B and C, and fig. S8). Ferryl
hemoglobin is readily formed in human erythrocytes under conditions of oxidative stress (22, 23),
as occurs in HbS and HbC erythrocytes (18). Ferryl
hemoglobin is also known to oxidize actin and to
alter actin filament dynamics (24).
Here, we have shown that P. falciparum establishes an actin cytoskeleton of its own design
within the host cell cytoplasm. The parasites own
actin seems not to play a role in this process because Plasmodium actin is not known to be exported into the erythrocyte cytoplasm (25).
Instead, the parasite seems to establish this actin
cytoskeleton by mining the actin and, possibly,
other components from the erythrocyte membrane skeleton. The parasite-organized actin cytoskeleton connects the Maurers clefts with the
knobs, defines the morphology of the Maurers
clefts, and may facilitate the movement of cargo
vesicles from the Maurers clefts to the erythrocyte plasma membrane.
The predicted secretome of P. falciparum
(26, 27) is expected to contain actin regulatory
factors that help to generate and maintain the
parasite-generated actin cytoskeleton. Other proteins of parasite origin may fix the actin-deprived
membrane skeleton of the host cell, thereby re-

1286

defining the mechanical and morphological properties of the infected erythrocyte (28, 29). Oxidized
forms of hemoglobins, in particular ferryl hemoglobin, might interfere with actin remodeling,
thereby preventing the parasite from creating its
own actin cytoskeleton within the host cell cytoplasm. As a result, Maurers clefts do not properly
form, vesicular transport is impaired, and the export of parasite-encoded disease-mediating adhesins to the erythrocyte surface is distorted. It seems
to be through this mechanism that HbS and HbC
confer their protective role against malaria.
References and Notes
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18. R. P. Hebbel, Blood 77, 214 (1991).

19. P. Jarolim, M. Lahav, S. C. Liu, J. Palek, Blood 76,
2125 (1990).
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Hematology 13, 187 (2008).
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Acknowledgments: We thank S. Prior, L. Lemgruber, and
D. Ouermi for technical assistance and help. We thank the
Max Planck Institutes (Martinsried and Frankfurt), the
European Molecular Biology Laboratory, Centre for Organismal
Studies, and Bioquant (Heidelberg) for access to EM facilities.
Supported by the Deutsche Forschungsgemeinschaft (research
focus Host-Parasite Coevolution); the Bundesministerium fr
Bildung, Wissenschaft, Forschung und Technologie, and the
Chica and Heinz Schaller Foundation. M.L. and F.F. are
members of the Heidelberg Excellence Cluster CellNetworks
and the European Network of Excellence EVIMalaR.

Supporting Online Material

www.sciencemag.org/cgi/content/full/science.1213775/DC1
Materials and Methods
Figs. S1 to S8
References (3044)
9 September 2011; accepted 19 October 2011
Published online 10 November 2011;
10.1126/science.1213775

Robust Crossover Assurance and

Regulated Interhomolog Access
Maintain Meiotic Crossover Number
Simona Rosu,1 Diana E. Libuda,2 Anne M. Villeneuve1,2*
Most organisms rely on interhomolog crossovers (COs) to ensure proper meiotic chromosome segregation
but make few COs per chromosome pair. By monitoring repair events at a defined double-strand break
(DSB) site during Caenorhabditis elegans meiosis, we reveal mechanisms that ensure formation of the
obligate CO while limiting CO number. We find that CO is the preferred DSB repair outcome in the
absence of inhibitory effects of other (nascent) recombination events. Thus, a single DSB per chromosome
pair is largely sufficient to ensure CO formation. Further, we show that access to the homolog as a repair
template is regulated, shutting down simultaneously for both CO and noncrossover (NCO) pathways. We
propose that regulation of interhomolog access limits CO number and contributes to CO interference.
rossover (CO) recombination during meiosis is initiated by induction of doublestrand breaks (DSBs) (1). Interhomolog
(IH) COs provide connections (chiasmata) that
enable homologous chromosomes to orient and

1
Department of Genetics, Stanford University School of Medicine, Stanford, CA 94305, USA. 2Department of Developmental
Biology, Stanford University School of Medicine, Stanford, CA
94305, USA.

*To whom correspondence should be addressed. E-mail:

annev@stanford.edu

2 DECEMBER 2011

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segregate to opposite poles at the meiosis I

division (2). To ensure formation of IH COs,
DSB repair is modulated to (i) promote use of
the homologous chromosome as a repair template, as opposed to the sister chromatid (the
preferred template in other contexts), and (ii)
promote maturation of IH recombination intermediates via a pathway that yields CO rather
than noncrossover (NCO) products (3, 4). However, more DSBs are formed than IH COs, indicating that excess DSBs must be repaired in

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Hemoglobins S and C Interfere with Actin Remodeling in Plasmodium

falciparum Infected Erythrocytes
Marek Cyrklaff et al.
Science 334, 1283 (2011);
DOI: 10.1126/science.1213775

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