Ruth E. Fowler

A Brief Illustrated Guide to the Ultrastructure of Plasmodium falciparum Asexual Blood Stages

Interpretation of the new information arising from the Plasmodium falciparum Genome Project requires a good working knowledge of the ultrastructure of the parasite; however many aspects of the morphology of this species remain obscure. Lawrence Bannister, John Hopkins and colleagues here give an illustrated overview of the three-dimensional (3-D) organization of the merozoite, ring, trophozoite and schizont stages of the parasite, based on available data that include 3-D reconstruc-tion from serial electron microscope sections. The review describes the chief organelles present in these stages, emphasizing the continuity of structure in addition to specialized, stage-specific features developed during the asexual erythrocytic cycle.

The plastid in Plasmodium falciparum asexual blood stages: a three-dimensional ultrastructural analysis.

The plastid in Plasmodium falciparum asexual stages is a tubular structure measuring about 0.5 micron x 0.15 micron in the merozoite, and 1.6 x 0.35 microns in trophozoites. Each parasite contains a single plastid until this organelle replicates in late schizonts. The plastid always adheres to the (single) mitochondrion, along its whole length in merozoites and early rings, but only at one end in later stages. Regions of the plastid are also closely related to the pigment vacuole, nuclear membrane and endoplasmic reticulum. In merozoites the plastid is anchored to a band of 2-3 subpellicular microtubules. Reconstructions show the plastid wall is characteristically three membranes thick, with regions of additional, complex membranes. These include inner and outer membrane complexes. The inner complex in the interior lumen is probably a rolled invagination of the plastids inner membrane. The outer complex lies between the outer and middle wall membranes. The interior matrix contains ribosome-like granules and a network of fine branched filaments. Merozoites of P. berghei and P. knowlesi possess plastids similar in structure to those of P. falciparum. A model is proposed for the transfer of membrane lipid from the plastid to other organelles in the parasite.

Ultrastructure of rhoptry development in Plasmodium falciparum erythrocytic schizonts.

Prior to the separation of merozoites from the Plasmodium falciparum schizont, various stage-specific organelles are synthesized and assembled within each merozoite bud. The apical ends of the merozoites are initiated close to the ends of endomitotic spindles. At each of these sites, the nuclear membrane forms coated vesicles, and a single discoidal or cup-like Golgi cisterna appears. Reconstruction from serial sections indicates that this structure receives vesicles from the nuclear envelope and in turn gives off coated vesicles to generate the apical secretory organelles. Rhoptries first form as spheroidal structures and grow by progressive fusion of small vesicles around their margins. As each rhoptry develops, 2 distinctive regions separate within it, an apical reticular zone with electron-lucent areas separated by cords of granular material, and a more homogenously granular basal region. The apical part elongates into the duct, with evidence for further vesicular fusion at the duct apex. The rounded rhoptry base becomes progressively more densely packed to form a spheroidal mass, and compaction also occurs in the duct. Typically, one rhoptry matures before the other. Cryofractured rhoptry membranes show asymmetry in the sizes and numbers of intramembranous particles at the internally- and externally-directed fracture faces.

Motile Systems in Malaria Merozoites: How is the Red Blood Cell Invaded?

The ability of the malaria parasite to invade erythrocytes is central to the disease process, but is not thoroughly understood. In particular, little attention has been paid to the motor systems driving invasion. Here, Jennifer Pinder, Ruth Fowler and colleagues review motility in the merozoite. The components of an actomyosin motor are present, including a novel unconventional class XIV myosin, now called Pfmyo-A, which, because of its time of synthesis and location, is likely to generate the force required for invasion. In addition, there is a subpellicular microtubule assemblage in falciparum merozoites, the f-MAST, the integrity of which is necessary for invasion.

Contractile protein system in the asexual stages of the malaria parasite Plasmodium falciparum.

F-actin was detected in asexual-stage Plasmodium falciparum parasites by fluorescence microscopy of blood films stained with fluorescent phalloidin derivatives. F-actin was present at all stages of development and appeared diffusely distributed in trophic parasites, but merozoites stained strongly at the poles and peripheries. No filament bundles could be discerned. A similar distribution was obtained by immunofluorescence with 2 polyclonal anti-actin antibodies, one of which was directed against a peptide sequence present only in parasite actin (as inferred from the DNA sequence of the gene). A monoclonal anti-actin antibody stained very mature or rupturing schizonts but not immature parasites. Myosin was identified in immunoblots of parasite protein extracts by several monoclonal anti-skeletal muscle myosin antibodies, as well as by a polyclonal antiserum directed against a consensus conserved myosin sequence (IQ motif). The identity of the polypeptides recognised by these antibodies was confirmed by overlaying blots with biotinylated F-actin. The antiserum and one of the monoclonal antibodies were used in immunofluorescence studies and were found to stain all blood-stage parasites, with maximal intensity towards the poles of merozoites. Our results are consistent with the presence of an actomyosin motor system in the blood-stage malaria parasite.

Microtubules in Plasmodium falciparum merozoites and their importance for invasion of erythrocytes

Plasmodium falciparum merozoites have an array of 2-3 subpellicular microtubules, designated f-MAST. We have previously shown that colchicine inhibits merozoite invasion of erythrocytes, indicating a microtubular involvement in this process. Colchicine inhibition of invasion was reduced by the Taxol-stabilization of merozoite microtubules prior to colchicine exposure. Immunofluorescence assays showed that the number and length of f-MASTs were reduced in colchicine-treated merozoites, confirming that microtubules were the target of colchicine inhibition. The dinitroaniline drugs, trifluralin and pendimethalin, were shown by immunofluorescence to depolymerize the f-MAST and both drugs were inhibitory in invasion assays. These results demonstrate that the integrity of the f-MAST is important for successful invasion. Fluorescence imaging demonstrated the alignment of mitochondria to f-MAST, suggesting that mitochondrial transport might be perturbed in merozoites with disorganized f-MAST. Depolymerizing mt in late-stage schizonts did not affect the allocation of mitochondria to merozoites.

Microtubule associated motor proteins of Plasmodium falciparum merozoites

We have studied the occurrence, stage specificity and cellular location of key molecules associated with microtubules in Plasmodium falciparum merozoites. Antibodies to gamma tubulin, conventional kinesin and cytoplasmic dynein were used to determine the polarity of merozoite microtubules (mt), the stage specificity of the motor proteins and their location during merozoite development. We conclude that the minus ends of the mts are located at their apical pole. Kinesin was present throughout the lifecycle, appearing as a distinct crescent at the apex of developing merozoites. The vast majority of cytoplasmic dynein reactivity occurred in late merogony, also appearing at the merozoite apex. Destruction of mt with dinitroanilines did not affect the cellular location of kinesin or dynein. In invasion assays, dynein inhibitors reduced the number of ring stage parasites. Our results show that both conventional kinesin and cytoplasmic dynein are abundant, located at the negative pole of the merozoite mt and, intriguingly, appear there only in very late merogony, prior to merozoite release and invasion.

A role for microtubules in Plasmodium falciparum merozoite invasion

Colchicine, a drug which poisons the polymerization of microtubules, was assayed for effects on the invasion of Plasmodium falciparum merozoites into red cells in order to investigate if merozoite microtubules have a function in invasion. Culture conditions and concentrations of colchicine were established where the maturation and rupture of schizonts was unaffected by the drug. This was judged first by light microscopy, including morphology and counts of nuclear particle numbers, then by ultrastructural studies which excluded deranged organellogenesis as a cause of merozoite failure, and finally by diachronic cultures in which both recruitment and loss of schizonts could be counted. Specific invasion inhibition was seen when 10 microM-1 mM colchicine was present. Red cells pre-incubated in colchicine and then washed showed no reduction in their extent of invasion, and neither red cell lysis, sphering nor blebbing were apparent. We conclude that intact microtubules are necessary for successful merozoite function.

Malaria, Microtubules and Merozoite Invasion: Reply

We agree with the Comment of Hommel and Schrevel (this issue) that the invasion of merozoites into red blood cells is rather poorly understood. We take the view (with which they sympathize) that the physics of invasion precludes a merozoite ‘sliding into’ its vacuole. We expect that its displacement against elastic and hydrostatic forces in the red blood cell requires a motor1, and perhaps longitudinal bracing2. Organellar involvement is complex, and not yet fully understood, but dense granules are released after initial invasion3. The anchorage on the red blood cell, against which motile force is generated, is unknown, but the junctional protein MCP-1 may be involved4. Hudson-Taylor and her colleagues have reported the sequence of MCP-1, and a sub-membranous location where it may interact with merozoite cytoskeletal components5. We have suggested that microtubular