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Cytoskeleton of Apicomplexan Parasites

Department of Molecular Microbiology, Washington University School of Medicine, St. Louis, Missouri 63110

SUMMARY

THE APICOMPLEXA

MICROTUBULES IN THE APICOMPLEXA

Organization

Conoid

Role in apicomplexan replication

Tubulin and Microtubule-Associated Proteins

SUBPELLICULAR NETWORK OF THE APICOMPLEXA

Proteins of the Subpellicular Network

Organization of the Inner Membrane Complex and the Subpellicular Network

ACTIN AND MYOSIN IN THE APICOMPLEXA

Properties and Localization of Actin

Motility and Invasion

Actin and Actin Binding Proteins

Myosin

MANIPULATION OF THE HOST CYTOSKELETON BY APICOMPLEXAN PARASITES

Reorganization of the Microvilli of Intestinal Epithelia by Cryptosporidium

Plasmodium Modification and Mimicry of Erythrocyte Cytoskeletal Proteins

Theileria Exploitation of Host Cell Microtubules

CONCLUSIONS

ACKNOWLEDGMENTS

REFERENCES

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THE APICOMPLEXA

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Infection by parasitic protozoa of the phylum Apicomplexa causes incalculable morbidity and mortality to humans and agricultural animals (2, 3, 19, 26, 28, 39, 90). Apicomplexan parasites include Plasmodium spp., the agents of malaria; Toxoplasma gondii, a significant opportunistic pathogen in immunocompromised individuals; Eimeria spp., pathogens of chicken and cattle; Theileria spp., tick-borne parasites of cattle in Africa; and Cryptosporidium, an animal parasite as well as an opportunistic pathogen of humans. The phylum also includes gregarines, parasites of the guts of invertebrates including cockroaches and shrimp. This review is restricted to apicomplexan parasites of medical and agricultural importance since the bulk of the research has been done in this area.

All apicomplexans are obligate intracellular parasites. Most apicomplexan parasites grow and replicate within the parasitophorous vacuole, a nonphagosomal, membrane bound compartment that is segregated from most cellular trafficking pathways (78, 110, 143, 186). Proliferation of these organisms occurs by invasion of a host cell and is followed by parasite growth and cell division until the host cell is lysed by the replicating parasites. Parasites released by host cell lysis do not grow or undergo cell division extracellularly and must rapidly reinvade other host cells in order to survive. Repeated cycles of host cell invasion, parasite replication, host cell lysis, and parasite invasion of new cells account for much of the tissue damage associated with apicomplexan infections. Although apicomplexans are haploid for the bulk of their life cycles, they have complex life cycles, involving differentiation to forms that invade distinct tissues and hosts (Fig. 1). Differentiation can generate gametes that undergo fusion to generate a transient diploid zygote. The zygote immediately undergoes meiosis to reestablish haploid organisms. In some cases, differentiation also permits infection of organisms (such as mosquitoes or ticks) that serve as vectors to transmit parasites from host to host (2, 26, 39, 60, 67, 90, 157).

View larger version (54K): [in this window] [in a new window]   FIG. 1. Life cycle of apicomplexan parasites. For the faint of heart or those not interested in details, the center circle illustrates a generic apicomplexan life cycle. The rapidly proliferating haploid form has the capacity to differentiate into gametes that fuse to produce a diploid zygote. Meiosis reestablishes the haploid state and leads to sporozoites, the form most widely associated with establishment of new infections after inoculation of a naive host by an infected vector. The outer two circles represent the specific life cycles for T. gondii and P. falciparum. The bradyzoite form of Toxoplasma (*) is responsible for reactivation of latent infection and is an obligatory stage between tachyzoites and gametes. Plasmodium spp. do not have an analogous stage, although "hypnozoites" (latent sporozoites) are implicated in reactivation by P. vivax and P. ovale.

View larger version (44K): [in this window] [in a new window]   FIG. 2. The morphology of apicomplexan parasites. Apicomplexans are highly polarized cells containing a collection of organelles that are specific to the phylum. Rhoptries, micronemes, and dense granules are secretory organelles that contain products required for motility, invasion, and establishment of the parasitophorous vacuole. The conoid is a small cone-shaped spiral of unidentified filaments. It is thought to play a mechanical role in invasion and can be protruded from or retracted into the apical polar ring. The apical polar ring serves as an MTOC for the subpellicular microtubules. The spirally arranged subpellicular microtubules closely follow the serpentine body shape of apicomplexans. The parasites are bounded by the pellicle, a composite structure consisting of the plasma membrane and the closely apposed IMC. The endoplasmic reticulum surrounds the nucleus, and the Golgi body is immediately above it. The apicoplast is immediately adjacent to the Golgi body.

View larger version (90K): [in this window] [in a new window]   FIG.3. Cytoskeletal elements in the Apicomplexa. (A) The subpellicular microtubules radiate out of the apical polar ring and run down the cytosolic face of the IMC. The spindle microtubules are nucleated from spindle pole plaques within the nuclear membrane. Centrioles (consisting of nine singlet microtubules surrounding a central singlet microtubule) are adjacent to the spindle pole plaques. (a) Isolated, negatively strained conoid (co), apical polar ring (apr), and subpellicular microtubules from T. gondii (113). (B) A network of intermediate filaments, the subpellicular network, underlies the length of the IMC. The lower right inset illustrates the pattern of IMPs revealed by freeze fracture of the IMC. These particles may represent the transmembrane domains of receptors that link the subpellicular network to the cytoplasmic face of the inner membrane complex. (b) Glycerol- and detergent-extracted, freeze-dried replicas of Toxoplasma tachyzoites illustrate the regular array of the subpellicular network filaments. (Image of the Toxoplasma network kindly provided by J. Heuser.) (C) Actin is localized between the plasma membrane and the IMC. When the plasma membrane is separated from the IMC, actin remains associated with the IMC and myosin is associated with the plasma membrane. (c) Actin filaments protrude beyond the conoid (co) of Toxoplasma tachyzoites treated with jasplakinolide, a drug that induces actin polymerization. (Panel c reprinted from reference (151) with the permission of the publisher.)

MICROTUBULES IN THE APICOMPLEXA

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The arrangement of subpellicular microtubules varies among apicomplexan species, but the number, length, and organization are absolutely stereotyped within the life cycle stage of a species. The number of subpellicular microtubules ranges from a band of 3 or 4 in the tiny Plasmodium falciparum merozoites to 60 in the considerably larger Plasmodium ookinetes (2, 3, 12, 26, 136, 156, 157). In coccidian parasites (Toxoplasma and Eimeria), the subpellicular microtubules are evenly spaced beneath the periphery of the pellicle; however, in Plasmodium species, most of the microtubules occupy two-thirds of the circumference and one microtubule is centered within the latter one-third of the pellicle (3, 157, 180). P. falciparum merozoites are an exception to this spacing generalization; they have a reduced number (three or four) of subpellicular microtubules termed f-MAST (falciparum merozoite-associated assemblage of subellicular microtubules) that extend down one side of the merozoite membrane from the apex toward the posterior (12, 59). Other P. falciparum life cycle stages (sporozoites and ookinetes) have a full complement of subpellicular microtubules, as do merozoites of other Plasmodium species. Theileria sporozoites also deviate from this pattern. Although the tick-borne kinete stage of Theileria contains subpellicular microtubules and an IMC, Theileria sporozoites lack subpellicular microtubules and the IMC altogether and enter host cells in a distinct fashion (see below) (49, 50, 147, 148, 152).

Replicating parasites employ spindle microtubules during mitosis. Nuclear division proceeds without nuclear membrane breakdown (12, 26, 59, 79, 130, 161). Spindle microtubules are nucleated from electron-dense, amorphous plaques associated with nuclear invaginations and embedded in the nuclear membrane (12, 26, 82, 109, 140, 146, 157). This spindle-organizing structure has been variously referred to as the centrocone, the centriolar plaque, the spindle pole body, or the centriolar equivalent. We have chosen to characterize this structure as a "spindle pole plaque" to distinguish it from the adjacent centrioles that are sometimes present in members of the Apicomplexa. Centrioles are apparently not required for spindle assembly, since Plasmodium merozoites and Theileria sporozoites lack these structures and construct spindles by using only the spindle pole plaques (5, 12, 51, 140, 150, 157, 159). However, it is also possible that centrioles exist in these organisms and are obscured by inclusion in the electron-dense spindle pole plaque.

In many apicomplexans, centrioles are located in the cytoplasm close to but separate from the spindle pole plaques (26, 31, 42, 82, 100, 116, 157). Centrioles are highly ordered MTOCs typically consisting of a 9+0 structure of nine triplet microtubule blades organized in a turbine fashion. Apicomplexan centrioles have an unconventional form consisting of a central single microtubule surrounded by nine singlet microtubules, a deviation from the canonical structure (31, 40, 42, 82, 157, 165, 182). It is curious that apicomplexans apparently contain both spindle pole plaque structures and centrioles in addition to the apical polar ring to organize microtubules. It may be that the centrioles are maintained throughout the asexual life cycle in order to serve as a template for construction of basal bodies that nucleate flagellar axonemes in the male gametes. The centriole is also associated with inheritance of the apicoplast during replication in Toxoplasma (169). One intriguing possibility is that the centrioles function as a "super-organizing center" coordinating the apical polar ring MTOC and the spindle pole plaque MTOC.

Although most parasite replication occurs by asexual division, apicomplexans also differentiate to gametes that fuse to form a diploid zygote. The male gamete (microgamete) is flagellated and swims to the female gamete (macrogamete) to carry out fertilization. The anterior end of apicomplexan microgametes is pointed and contains three basal bodies in close proximity to the apical pole (116, 137, 158, 181). These basal bodies nucleate two or three flagella that extend past the nucleus and away from the apical end. Additional microtubules originate in the basal apparatus zone and extend to the posterior end of the microgamete. Two of the flagella are long and are free from association with the gamete body. The third flagellum is shorter and is attached to surface of gamete at its anterior end. In some species this third flagellum is present only in rudimentary form as a band of microtubules that extends along the length of the gamete. In contrast to the atypical centrioles observed in other stages, the basal body of male gametes has a typical triplet microtubule structure with ninefold symmetry and the flagellar axoneme contains a conventional 9+2 arrangement of doublet microtubules surrounding the central pair of microtubules (137, 158-160, 181). In Plasmodium, genesis of the basal bodies involves an intermediate 9+1 singlet form similar to the centrioles in other apicomplexans (157).

View larger version (33K): [in this window] [in a new window]   FIG. 4. Replication by the Apicomplexa. (A) Endodyogeny proceeds without loss of maternal cell shape and apical polarity. Formation of a curved nucleus is coupled to construction of two buds composed of nascent daughter IMC and subpellicular microtubules. The daughter cells develop surrounded by their own subpellicular microtubules and IMC in the fully polarized mother cell. Once the daughter cells are mature, they bud out of the remnants of the mother cell. (B) Schizogeny proceeds with loss of apical polarity. Invasion of a polarized elongated parasite is followed by disassembly of the subpellicular microtubules and IMC and by extensive cell growth and nuclear divisions. To reassemble polarized daughter cells, the multiple nuclei align with individual sets of apical organelles, subpellicular microtubules, and IMC at the periphery of the replicating cell.

The microtubules of extracellular apicomplexans are not dynamic and are therefore impervious to microtubule-disrupting drugs (135). Assembly of microtubules occurs in the course of replication; therefore, intracellular parasites are susceptible to microtubule-depolymerizing drugs (Table 1) (168). In Toxoplasma and Plasmodium, the spindle and subpellicular microtubule populations are differentially stable to disruption by oryzalin or colchicine (14, 115). Lower concentrations (0.5 µM oryzalin or 1.0 mM colchicine) shorten microtubules. Under these conditions, Toxoplasma and Plasmodium continue to undergo nuclear division and budding but lack functional subpellicular microtubules and are incapable of invading new host cells. When removed from 0.5 µM oryzalin, Toxoplasma recovers normal morphology and is invasive (115). In contrast, higher concentrations of drug (2.5 µM oryzalin or 5.0 to 10.0 mM colchicine) disrupt both subpellicular and spindle microtubules (149, 168). Parasites under these conditions are incapable of nuclear division or budding, although cell growth, DNA synthesis, and centriole replication continue unchecked (115, 149, 168). When removed from 2.5 µM oryzalin, Toxoplasma tachyzoites attempt to bud as crescent-shaped parasites. Since the polyploid nuclear mass cannot be correctly segregated, daughter parasites are made that lack nuclei altogether (115).

Microtubule-associated proteins (MAPs) are clearly critical to the highly organized structure of apicomplexan parasites. Bridges connecting the subpellicular microtubules to the inner membrane complex have been observed in thin sections of parasites (3, 131, 195). Isolated frozen-hydrated microtubules of T. gondii have a distinct 32-nm periodicity along their length as revealed by Fourier analysis (113). The periodicity most probably results from a MAP that heavily decorates these microtubules and that may account for their unusual stability after isolation. This MAP may coordinate the close interaction of the subpellicular microtubules with the IMC. The existence of a group of monoclonal antibodies that labels the subpellicular microtubules in Toxoplasma and cross-reacts with Plasmodium suggests that the MAPs may be conserved within the Apicomplexa (112, 114). The Plasmodium and Cryptosporidium genome databases and the Toxoplasma, Eimeria, and Neospora expressed sequence tag (EST) projects have sequences annotated as encoding putative kinesins and dyneins.

SUBPELLICULAR NETWORK OF THE APICOMPLEXA

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ACTIN AND MYOSIN IN THE APICOMPLEXA

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View larger version (19K): [in this window] [in a new window]   FIG. 5. Motility and invasion by the Apicomplexa. (A) Helical gliding is the only behavior associated with motility that leads to long-distance movement across a substrate. During this biphasic behavior, the parasite moves forward a body length and then repositions to repeat this cycle. This sequence illustrates the path taken by a gliding Toxoplasma tachyzoite to move forward one complete cycle. The parasite travels forward by moving a contact zone from its apex to its posterior in a helical path. Because the parasite is crescent shaped, it moves in a "corkscrew" fashion (frames 1 to 5). The crescent shape of the tachyzoite prevents the continuation of this motion because the parasite cannot contact the substrate with its concave face. To rectify this, the parasite rotates 180° without forward motion (frames 6 and 7) before initiating another cycle of ‘corkscrewing’ translational movement (frames 8 and 9). (Modified from reference (66) with the permission of the publisher.) (B) Apicomplexan invasion consists of attachment and apical orientation, induction of a parasitophorous vacuole, and translocation of the parasite into the vacuole. Apically secreted adhesins are capped along the moving junction to the posterior of the parasite. The moving junction is associated with a constriction of parasite shape that moves from the apex to the posterior.

The existence of an actomyosin-based mechanism for invasion was first implied by observations of the effect of cytochalasin B on Plasmodium invasion. Cytochalasin B blocks Plasmodium knowlesii merozoite invasion of red cells (106). Merozoites will attach irreversibly to red cells and form a vestigial parasitophorous vacuole but are inhibited from moving into the cell. Since red cells are unequivocally nonphagocytic, the effects of cytochalasin on Plasmodium invasion are likely to represent drug disruption of parasite (not host cell) microfilaments. However, for other apicomplexans, cytochalasin inhibition of invasion was sometimes attributed to inhibition of induced phagocytosis by the host cell. Active invasion of Toxoplasma tachyzoites can be distinguished from the phagocytic uptake of parasites because invasion of host cells (including macrophages) does not induce host cell membrane ruffling, actin microfilament reorganization, or tyrosine phosphorylation, which are all indicative of phagocytosis (111). Invasion is three to four times faster than phagocytosis (occurring within 25 to 40 s) and is characterized by parasite penetration into a tight-fitting vacuole formed by invagination of the plasma membrane. In contrast, phagocytosis of Toxoplasma involves membrane ruffling and the parasite is captured in a loose-fitting phagosome that forms over 2 to 4 min (78, 111, 121). Phagocytosis involves both reorganization of the host cytoskeleton and tyrosine phosphorylation of host proteins (111).

Experiments with cytochalasin D-resistant T. gondii have definitively established that invasion of Toxoplasma is critically dependent on actin filaments in the parasite but not in the host cell (37). Invasion of cytochalasin D-resistant host cells by wild-type (cytochalasin-sensitive) Toxoplasma tachyzoites is blocked by cytochalasin D. As observed with Plasmodium, attachment and apical orientation of Toxoplasma is normal in the presence of cytochalasin D. Cytochalasin D-resistant Toxoplasma mutants were isolated by chemical mutagenesis and selection for growth in cytochalasin D in resistant host cells. These resistant parasites have a point mutation in the single-copy actin gene ACT1 (A136G) and can invade wild-type host cells in the presence of cytochalasin D. Transformation of the mutant act1 allele into wild-type Toxoplasma confers cytochalasin D-resistant motility and invasion either as an allelic replacement or as a nonhomologous integration, generating a pseudodiploid parasite (37).

Apicomplexan invasion consists of three phases: (i) attachment with apical orientation, (ii) induction of a parasitophorous vacuole, and (iii) translocation of the parasite into the vacuole (Fig. 5B). Parasites attach to host cells and form an intimate connection through apical end contact (37, 53, 106). This results in sequential secretion from the parasite micronemes and rhoptries. Adhesins from the micronemes are translocated along the parasite length and are shed at the site of the moving junction; parasitophorous vacuole components from the rhoptries are secreted into this forming compartment (24). Both tight adhesion to the host cell and secretion into the host cell occur when parasites are immobilized early in invasion by cytochalasin treatment (37, 53, 65, 106).

The "moving junction" which forms during invasion is a circumferential zone of attachment at the orifice of the host cell invagination (7, 105). It is characterized by a markedly thickened host cell membrane with increased electron density and is frequently accompanied by a constriction in the parasite body. The parasite enters the nascent parasitophorous vacuole by capping the moving junction down its body. Ultimately, the parasite becomes enclosed within a cavity delimited by the invaginated host cell membrane. Formation of a moving junction that is capped to the posterior during invasion is likely to be a feature of invasion shared by many apicomplexans (Theileria is an exception [discussed below]). The moving junction is a highly specialized interface of the parasite with the host cell, presumably exploiting cytoskeletal proteins, signaling molecules, and receptors. Very little is known about this interface. P. falciparum MCP-1 (merozoite-capping protein 1) is a 60-kDa merozoite protein that moves from anterior to posterior with the moving junction during merozoite invasion of red cells (86). MCP-1 lacks both a signal sequence and transmembrane domain and is located in the parasite cytosol. The precise role of MCP-1 in invasion remains obscure. The protein has an amino-terminal domain that is conserved in bacterial and eukaryotic oxidoreductases (76). There are also a group of microneme proteins that may play a role in this junction that localize to the moving junction and the (exposed) posterior region during invasion (24, 57, 62).

F-actin affinity chromatography has been used to isolate actin binding proteins from P. knowlesii and P. falciparum merozoites and from Toxoplasma tachyzoites (54, 125, 173). In P. knowlesii, five major proteins with molecular masses of 75, 70, 48, 40, and 32/34 kDa are eluted from F-actin columns (173). The 70-kDa protein has been identified as heat shock protein 70 (HSC70). The 32/34-kDa doublet coelutes with HSC70 from columns or in gel filtration chromatography; however, the identity of these proteins remains unknown. Highly enriched fractions of the Plasmodium HSC70-HSC32-HSC34 complex inhibit rabbit skeletal muscle actin polymerization in vitro. Biochemical experiments have established that this is due to a capping activity that is Ca²⁺ independent and is inhibited by PIP₂.

Homologs of two widely conserved actin-associated proteins, coronin and actin-depolymerizing factor (ADF), have been characterized in the Apicomplexa. Coronin is a WD repeat containing actin binding protein that was first characterized in Dictyostelium discoideum, where it is essential for phagocytosis and motility. WD repeats (a tryptophan-aspartic acid motif) are found in diverse proteins; this motif is thought to mediate protein-protein interactions. Homologs of coronin are found in a large variety of eukaryotes, ranging from humans to C. elegans to yeast. A coronin homolog has been described in P. falciparum and is encoded by a single-copy gene (174). Compared to Dictyostelium coronin, the Plasmodium protein has conserved residues throughout the entire protein. A monoclonal antibody to D. discoideum coronin detects a 42-kDa protein in Triton X-100-insoluble extracts of P. falciparum schizonts. ADF/actophorin/cofilin is a widely conserved low-molecular-weight actin monomer-sequestering protein with filament-severing activity. An ADF homolog has been characterized in T. gondii (9). The single-copy gene encodes a 13.4-kDa protein. Toxoplasma ADF has a high degree of sequence similarity to other ADF homologs, particularly Acanthamoeba actophorin and plant ADFs. Toxoplasma ADF localizes to cytoplasm, especially under the plasma membrane. Recombinant Toxoplasma ADF purified from E. coli binds actin monomers and depolymerizes microfilaments in a pH-independent, concentration-dependent fashion.

One surprising finding is that a homolog of the actin monomer binding protein profilin has not been found in the Apicomplexa. In its place, Toxoplasma has apparently substituted a novel protein, toxofilin (125). Toxofilin is a 27-kDa actin monomer binding protein that was originally isolated from Toxoplasma extracts on G-actin affinity columns. In pyrene actin assays, toxofilin inhibits actin polymerization, acting as an actin-sequestering protein. It also slows microfilament disassembly through a filament end-capping activity. A single-copy gene encodes toxofilin. The protein has a pI of 9.63 and two coiled-coil domains and lacks consensus motifs or any similarity to known proteins. Overexpression of green fluorescent protein (GFP)-tagged toxofilin in vertebrate cells disrupts stress fibers and reduces microfilament levels by half. Toxofilin localizes to the apical cytoplasm in intracellular Toxoplasma but is found at the posterior of invading parasites. In motile parasites, toxofilin is localized throughout the entire cytoplasm.

Apicomplexan myosins are highly atypical and were ultimately cloned in Toxoplasma by using degenerate PCR of conserved regions of the motor domain (70). A similar strategy has been used to identify myosins in Plasmodium, Neospora, Eimeria, Sarcocystis, Babesia, and Cryptosporidium (69). All apicomplexan myosins are extremely similar, suggesting that the diversity of myosins in these parasites is extremely limited (69). Phylogenetic analysis of the myosins places these motors in a novel, highly divergent class (XIV) in the myosin superfamily (69, 70). Apicomplexan myosins range from 91 to 125 kDa and include the smallest myosins characterized thus far (70, 73). There is a high degree of sequence conservation among all apicomplexan myosins throughout their whole length. All have short tails that do not have homology to any other myosin tails. However, these tails do contain a highly basic charge distribution similar to myosin I family members, suggesting that the apicomplexan myosins may interact with membranes. Generally, myosin motors have three domains; the amino terminus contains the motor domain, the central "neck" region binds light chains and acts as a lever arm, and the tail is diverse, carrying out different functions such as targeting to subcellular regions or binding to cargo (18, 102). The apicomplexan myosins do not contain the strictly conserved glycine residue at the fulcrum point of the lever arm and generally lack IQ motifs that bind calmodulin and calmodulin-related proteins (68, 70, 73). Additionally, the Toxoplasma myosins do not follow the TEDS rule, i.e., the presence of an acidic or phosphorylatable residue at a precise site close to the actin binding region (70). In lower eukaryotes, this residue is crucial for stimulation of the ATPase of class I myosins, but other exceptions to this rule have been described. Both the absence of an IQ motif and the nonadherence to the TEDS rule suggest that these motors may be regulated in a novel fashion.

T. gondii expresses five class XIV myosins: TgM-A, TgM-B, TgM-C, TgM-D, and TgM-E (68-70, 73). TgM-A is 93 kDa and lacks a discernible neck domain and IQ motifs (70). Epitope-tagged TgM-A localizes beneath the plasma membrane (73). Mutational analysis has established that a pair of arginine residues is essential to target TgM-A to the periphery (73). Since ectopically expressed TgM-A in HeLa cells does not target to the plasma membrane, peripheral localization in parasites may require a membrane-associated receptor. The P. falciparum homolog of TgM-A (PfM-A/Pf-myo1) is synthesized in mature schizonts and is present in merozoites but vanishes after the parasite enters the red cell (123). PfM-A is associated with the particulate parasite fraction, and immunofluorescence and immunogold analysis shows that PfM-A localizes to the periphery of mature schizonts and merozoites.

TgM-B and TgM-C are the products of differential RNA splicing and are 114 and 125 kDa respectively (70). They are identical throughout their head and neck domains and diverge in their distal tail structures. Both contain a single IQ motif. TgM-B has not been localized, but TgM-C localizes to a juxtanuclear region toward the apical pole of the parasite, consistent with an association with the Golgi apparatus (70, 73). TgM-D is a 91-kDa protein that has a punctate peripheral localization (73). TgM-E is the most recently discovered myosin and is currently being characterized (69, 73). Biochemical studies have established that the myosins bind actin in the absence but not the presence of ATP and that they are tightly associated with membranes (68, 73). The peripheral localization of TgM-A and of the GFP-TgM-A tail fusion is not dependent on an intact F-actin cytoskeleton (73). Truncation of the tail domains of TgM-A or TgM-D abolishes their peripheral localization and tight membrane association; fusion of the TgM-A or TgM-D tail to GFP is sufficient to confer plasma membrane localization (73).

MANIPULATION OF THE HOST CYTOSKELETON BY APICOMPLEXAN PARASITES

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Plasmodium erythrocytic stages also synthesize proteins which are similar to ankyrin and spectrin and which are hypothesized to play a role in reorganization of the cytoskeleton of red cells. Plasmodium chabaudi ROPE (repetitive organellar protein) has a structure similar to that of spectrin (188). This 229-kDa protein is localized to the apical end of merozoites, possibly in the rhoptries. ROPE has characteristics of a cytoskeletal protein. A 364-amino-acid repetitive region based on 32 11-mer repeats suggests that the protein forms an helical coiled-coil triple helix containing a leucine-histidine zipper. Strikingly, this three-dimensional arrangement resembles the structure of spectrin. It has been postulated that ROPE may be involved in invasion, by interacting with the erythrocyte cytoskeleton via molecular mimicry of spectrin. P. falciparum expresses an 88-kDa phosphoprotein that is nearly identical to the amino-terminal region of ankyrin, a region of the protein that binds band 3 (170). This protein may also help the parasite reorganize the membrane skeleton via molecular mimicry.

CONCLUSIONS

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Although researchers studying apicomplexan parasites have amassed many data regarding the cytoskeleton, we still are missing explicit evidence linking cytoskeletal components to cellular properties. The following discussion suggests possible links between the cytoskeletal elements and behavioral traits that are common to the members of the Apicomplexa. One obvious generalization is that in these cells, the subpellicular network and the subpellicular microtubules are critical to cell shape while actin and myosin are essential for motility and invasion. This is not to say that future work will not uncover additional functions for the cytoskeleton.

Although apicomplexan parasites are profoundly deformed during host cell invasion, they retain membrane integrity during host cell entry. This may be ascribed to their robust arrangement of plasma membrane, IMC, and subpellicular network. The newly identified proteins IMC-1 and IMC-2 are implicated in subpellicular network formation (98). In addition to the obvious questions of how these proteins form filaments and how filament assembly is regulated is the issue of how this network associates with the IMC. The lattice of intramembranous particles observed after freeze fracture of the pellicle could reflect the transmembrane domains of receptors for the subpellicular network; however, the identity of these highly organized particles remains undetermined, as does any physical connection between the particles and the lattice proteins (43, 113, 124).

Apicomplexan parasites multiply by endodyogeny or schizogeny. As described above, these processes require independent regulation of spindle and subpellicular microtubules. Perhaps consequently, subpellicular microtubules and spindle microtubules are organized by different MTOCs: the apical polar ring and the spindle pole plaque/centrioles, respectively. In endodyogeny, parasites must discriminate between maternal and daughter apical polar rings and between maternal and daughter subpellicular microtubules. In schizogeny, daughter cell budding is induced after movement of multiple nuclei to the periphery of a maternal cell that lacks subpellicular microtubules. It is likely that the spindle pole plaques then induce or coordinate the formation of the apical polar rings, coupling each nucleus with a set of subpellicular microtubules. Daughter cell budding is distinct from vertebrate cytokinesis. In fact, inhibitor studies suggest that parasite scission may not utilize microfilaments such as are required at the vertebrate cleavage furrow (149). However, a recent study (28a) of the alternatively spliced myosins MyoB and MyoC in Toxoplasma demonstrates that overexpression of MyoB causes defects in cell division, and the parasites make extremely large residual bodies. Tagged MyoB localizes in a punctate cytosolic pattern and tagged MyoC localizes to the apical and posterior polar rings of tachyzoites. These latter observations suggest that MyoB and MyoC may play a role in parasite cell division, implicating an acto-myosin ring in parasite scission. Recent microtubule inhibitor studies show that subpellicular microtubule assembly can be disconnected from nuclear division, creating Toxoplasma tachyzoites that lack nuclei, although budding and scission from the maternal mass is completed (115). Multiple MTOCs permit apicomplexans to control nuclear division independently from cell polarity and cytokinesis. Although this grants greater cell cycle flexibility to these parasites, it abolishes the checks for coregulation of nuclear division and cytokinesis that are found in other eukaryotes.

Rigidity, cell shape, and apical polarity are provided by the subpellicular microtubules, and the apical polar ring organizes these microtubules (120, 134, 168). The apical polar ring represents a MTOC that is unique to the Apicomplexa. We know very little about its genesis and nothing about its component proteins. If the apical polar ring controls daughter cell budding, it must be replicated in a highly regulated fashion. It will also be informative to understand how it nucleates the subpellicular microtubules. The number of subpellicular microtubules and their organization are invariable within a life cycle stage of a particular species of apicomplexan parasite. The apical polar ring is a highly ordered structure and may contain signals that determine the number and placement of subpellicular microtubules.

Like the subpellicular network, the subpellicular microtubules also have intimate connections with the inner membrane complex (3, 113, 131, 195). To understand these interactions, it will be necessary to identify and characterize MAPs. MAPs may dictate subpellicular microtubule length and position under the pellicle. It is unclear why some apicomplexans distribute their microtubules uniformly beneath the pellicle while others center one microtubule beneath one-third of the circumference and evenly space the remainder below the other two-thirds of the pellicle. In studies of motility, it is clear that the convex and concave sides of the parasite are not equivalent. The asymmetry of microtubules in some apicomplexans may simply reflect areas that are more or less closely involved in force generation during motility or other essential functions. Subpellicular microtubules may contribute to motility by providing tracks that direct the acto-myosin-based capping activity. Toxoplasma tachyzoites and Plasmodium merozoites with shortened subpellicular microtubules (due to drug treatment) are noninvasive, supporting this notion (14, 115). However, it is not absolutely clear how microtubules could serve as tracks for the acto-myosin system, since actin and myosin are believed to act between the plasma membrane and the IMC while microtubules localize to the cytoplasmic face of the IMC (35, 120).

Many studies have implicated actin in apicomplexan motility, although apicomplexan microfilaments are apparently quite labile under most circumstances. Polymerized actin is observed only in the presence of jasplakinolide, and in untreated cells nearly all the actin is found as G-actin (36, 151). The apical actin filaments observed after treatment of Toxoplasma with jasplakinolide may reflect the location of actin regulators that nucleate or otherwise facilitate filament polymerization (151). Alternately, the apical localization of an F-actin projection after jasplakinolide treatment may represent the "path of least resistance" since the apical region is the only area of the pellicle not surrounded by three unit membranes and the subpellicular network. The short-lived nature of microfilaments suggests that actin assembly and disassembly are closely regulated. The Plasmodium HSC70 complex caps F-actin, limiting filament growth, and the apicomplexan homologs of ADF/cofilin are likely to sever filaments and sequester monomers, facilitating rapid disassembly of actin filaments (9, 173). Additionally, in Toxoplasma, actin may be kept monomeric by sequestration by toxofilin, a novel monomer binding protein (125). BLAST searches of the Cryptosporidium and Plasmodium genomes do not identify homologs of toxofilin, suggesting that distinct proteins may provide this function in other apicomplexans (unpublished data).

Myosin motors are also implicated in motility and invasion (35, 56, 66, 123). The apicomplexan myosins are quite divergent from myosins in other organisms, constituting a new class of motors in the myosin family (68-70, 73, 123). Apicomplexan myosins are all quite similar but have different subcellular localizations. Myosin-A is most likely to be involved in motility since it is found beneath the plasma membrane, whereas myosin-B is located to the Golgi and myosin-D is found on vesicles, consistent with roles for these latter motors in membrane traffic (68, 73, 123). Ectopic expression of myosin-A has shown that it does not localize to the plasma membrane in nonapicomplexans and therefore must be targeted to this region in parasites by additional proteins (73).

The precise mechanism by which parasites use an acto-myosin motor to generate motility is unclear. Since actin filaments are rare and since the apicomplexan myosins lack typical regulatory domains, it has been suggested that the movement of myosin is limited by filament generation. Consistent with this, jasplakinolide-treated parasites show increased motility, although drug treatment inhibits rather than enhances parasite invasiveness (151). For the myosin motors to have force-generating movement, the microfilaments must be tethered so that they remain in place. Actin filaments may be immobilized by interactions with the IMC, and the rigidity provided by the subpellicular network and the subpellicular microtubules could provide the extra stability required for myosin movement to transport adhesins to the posterior end of the parasite. Myosin movement along the actin filaments would lead to capping of adhesins down the length of the parasite and ultimately to gliding motility or invasion. Gliding motility is a trait shared with gregarines, apicomplexan parasites of invertebrates that are quite distinct from other members of the phylum described here (84, 85, 184).

This review has generalized the behavior of apicomplexans as a group. By doing so, we have undoubtedly glossed over differences, particularly with the more divergent members of this phylum. However, in many cases, atypical attributes are particular to the life cycle stage of the parasite rather than characteristic of the organism as a whole. This is most clearly illustrated with P. falciparum merozoites and Theileria sporozoites. Although most apicomplexans have many subpellicular microtubules and use an acto-myosin mechanism to glide and to invade cells, Plasmodium merozoites have reduced these traits and Theileria sporozoites have eliminated them. P. falciparum merozoites have a drastically scaled-back set of subpellicular microtubules that may reflect the greatly reduced size of merozoites relative to liver and insect stage parasites; both traits may be dictated by the smaller size of the host red cells. Merozoites do not display gliding motility, although they actively invade red cells in an actin-dependent fashion. Other Plasmodium stages are larger, have prominent subpellicular microtubules, and are clearly motile. Similarly, Theileria sporozoites lack subpellicular microtubules and infect lymphocytes after entering in a novel and nonmotile fashion. Tick-stage Theileria kinetes are larger and have subpellicular microtubules. Although very little work has been done on this stage, we assume that they are motile and actively invade host cells. Nonetheless, there are clearly dangers in overgeneralizing the behavior of apicomplexan parasites. Other exceptions to these generalizations will undoubtedly be found within this diverse group of parasites.

At present, the P. falciparum genome project is nearing completion and several genome projects are under way for other Plasmodium species. The Cryptosporidium genome is also nearing completion; genome projects for Toxoplasma, Theileria, and Babesia are under way; and there are ongoing EST projects for Toxoplasma, Neospora, Eimeria, Sarcocystis, and Plasmodium. In the past few years, researchers have developed techniques to transfect Toxoplasma and Plasmodium, to make targeted deletions, and to create gene replacements. With the amassed information about the cytoskeleton and these new resources and tools, we are truly poised to understand the mechanisms underlying cytoskeletal functions in the apicomplexan parasites. Disease caused by these protozoa has tremendous medical and economic impact worldwide. For the cell biologist, the unique biology of the Apicomplexa represents an intriguing departure from standard eukaryotic behaviors; for the clinician, these distinctions may represent unique drug targets.

ACKNOWLEDGMENTS

 
We thank Antonio Barragan, Audra Charron, John Cooper, Susan Dutcher, Olivia Giddings, Dan Goldberg, Wallace Marshall, Alissa Weaver, and Dawn Wetzel for commenting on the manuscript.

N.S.M. is supported by Individual NRSA fellowship F32 GM20484-01A1 and was previously supported by an NIH training grant in Infectious Disease held by the Washington University School of Medicine. (T32; AI-0717221).

FOOTNOTES

 
* Corresponding author. Mailing address: Department of Molecular Microbiology, Washington University School of Medicine, 660 South Euclid Ave., St. Louis, MO 63110. Phone: (314) 362-8874. Fax: (314) 362-3203. E-mail: naomi{at}borcim.wustl.edu.

REFERENCES

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