First published online 5 August 2003
doi: 10.1242/jcs.00665
Plasmodium falciparum apical membrane antigen 1 (PfAMA-1) is translocated within micronemes along subpellicular microtubules during merozoite development
Lawrence H. Bannister1,*,
John M. Hopkins1,2,
Anton R. Dluzewski1,2,
Gabriele Margos1,2,
Ian T. Williams2,
Michael J. Blackman3,
Clemens H. Kocken4,
Alan W. Thomas4 and
Graham H. Mitchell2
1 Department of Anatomy, Cell and Human Biology, Guy's, King's and St Thomas'
School of Biomedical Science, Guy's Hospital, London SE1 1UL, UK
2 Department of Immunobiology, Guy's, King's and St Thomas' School of Medicine,
Guy's Hospital, London SE1 9RT, UK
3 Division of Parasitology, National Institute for Medical Research, Mill Hill,
London NW7 1AA, UK
4 Department of Parasitology, Biomedical Primate Research Centre, Lange Kleiweg,
2280GH, Rijswijk, The Netherlands
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Fig. 1. Electron micrographs showing developing merozoites in late stages schizonts
of Plasmodium falciparum. (A) Transmission electron micrograph (TEM)
of a red blood cell containing a late-stage schizont that is in the process of
budding off merozoites. The arrow indicates the residual body containing
haemozoin crystals. Scale bar, 1 µm. (B) Scanning electron micrograph of a
schizont from which the surrounding red blood cell and parasitophorous vacuole
membranes have been lost to expose a series of merozoites budding off from the
larger residual body (arrow). Scale bar, 1 µm. (C) TEM of a late-stage
merozoite bud within a red blood cell, showing a rhoptry (rh) at the apical
end, with closely grouped micronemes (white arrow). Two dense granules (dg)
are also visible close to the nucleus (nuc). Scale bar, 200 nm. (D) TEM of the
apical region of a merozoite, showing the positions of a rhoptry (rh),
micronemes (mn) and three polar rings in section (black arrowheads). Scale
bar, 100 nm. (E) TEM of a longitudinally sectioned merozoite within a red
blood cell, showing an earlier stage of development than in (C) and (D).
Micronemes (small black arrows) have not yet reached their apical position but
are spread out between the Golgi cisterna (go) and the apical prominence
(left). Notice the two dense granules (dg) near the Golgi cisterna, part of
the nucleus (nuc) and a cluster of coated vesicles (cv) between the nucleus
and the Golgi cisterna. Scale bar, 200 nm.
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Fig. 2. A reconstruction of a late stage merozoite bud, still attached
(constriction ring) to the parasite's residual body. At this stage of
development, the micronemes are migrating apically from the Golgi cisterna
beneath the f-MAST (in this example, composed of three microtubules) but the
formation of dense granules has not yet commenced. This reconstruction was
made from a series of 19 consecutive longitudinal sections.
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Fig. 3. Details of microneme and related apical structures in the developing
merozoite apex. (A) A longitudinally sectioned microneme, showing the basal
bulb (bu) and narrow neck (white arrows). A transversely sectioned neck is
also visible (arrowhead). Scale bar, 100 nm. (B) A group of micronemes
clustered close to the surface of a rhoptry (rh). Notice the radiating
filaments attached perpendicularly to the microneme surfaces. Scale bar, 100
nm. (C) Another group of micronemes transversely sectioned and image-enhanced
to clarify their radial surface filaments. Scale bar, 100 nm. (D) Part of a
transversely sectioned merozoite apex, enhanced to show filamentous
connections (arrowhead) between two rhoptries (rh) and a polar ring (pr).
Scale bar, 200 nm. (E) A freeze-fracture preparation showing two micronemes.
Notice their bottle-like appearance and the presence of small numbers of
intramembranous particles (e.g. arrow). Scale bar, 50 nm.
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Fig. 4. Sections through developing merozoites illustrating the route taken by
micronemes from the Golgi body to the apex in relation to the subpellicular
microtubules of the f-MAST. (A) Part of a merozoite transversely sectioned
through the region of the vesicle budding zone at the periphery of the Golgi
cisterna, an edge of which is visible in oblique section (go); three
subpellicular microtubules are visible on the left (white arrows). Scale bar,
200 nm. (B) The microtubules of the f-MAST in transverse section (short
arrows), close to micronemes (asterisk). The three membranes of the merozoite
pellicle are also visible (long arrows); to the left of the merozoite surface
is the membrane of the parasitophorous vacuole. (C) Structures similar to
those in (B) with a more clearly defined microneme (asterisk). Scale bar for B
and C, 100 nm. (D,E) Stereoscopic pair showing the apical region of a
developing merozoite with migrating micronemes (black arrows) associated with
a pair of subpellicular microtubules (white arrows) attached on the left to
the polar rings (pr). Angle of tilt, 12°. Scale bar, 100 nm. (F,G) Another
stereoscopic pair showing micronemes migrating in relation to a subpellicular
microtubule (mt) attached to the apical polar rings (pr). The electron
micrographs have been enhanced to show filamentous connections between the
micronemes and microtubule (indicated by small arrows). Part of a rhoptry (rh)
is also visible. Angle of tilt, 12°. Scale bar, 100 nm.
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Fig. 5. Immunogold labelling of maturing schizonts with antibody against PfAMA-1
whole ectodomain. (A) An obliquely sectioned merozoite apex with a cluster of
micronemes strongly labelled by Pab 1, surrounding the tips of two unlabelled
rhoptries (rh). Scale bar, 100 nm. (B) Two micronemes (black arrows) labelled
by Pab 3 and a longitudinally sectioned rhoptry devoid of labelling, including
its narrow apical end (white arrow). Scale bar, 100 nm. (C,D) PfAMA-1
labelling by Pab 3 around the peripheries of micronemes. The microneme in (D)
was situated near the Golgi cisterna and represents an early stage in this
organelle's migration to the apex. Scale bars, 50 nm.
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Fig. 6. Western blots of the polyclonal antibodies (Pab 1, Pab 2 and Pab 3, and
control Pab 4) used to study PfAMA-1 expression in extracts of late-stage ITO4
schizont-parasitized red blood cells (p) and uninfected red blood cell (r)
samples for each. Lanes: (A) p and r incubated with (control) antibody against
Pichia expression contaminants; (B-D) incubated with anti-PfAMA-1 Pab
1 (B), Pab 2 (C) and Pab 3 (D). All anti-PfAMA-1 antibodies stain both the
unprocessed (PfAMA-183) and processed (PfAMA-166) forms
of PfAMA-1. Lower molecular weight bands represent further cleavage products,
variably detected by the three anti-PfAMA-1 antibodies.
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Fig. 7. The trafficking routes of the three major classes of secretory organelles
in the Plasmodium falciparum merozoite, numbered 1-3 to reflect their
times of origin from the Golgi cisterna. Routes 1 (rhoptry-directed vesicles)
and 3 (dense granules) are independent of microtubules, whereas route 2
represents the microtubule-related targeting of micronemes to the merozoite
apex. Other details of merozoite structure, including the pellicular system
and merozoite coat are not depicted.
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© The Company of Biologists Ltd 2003
