A Small-Molecule Inhibitor of T. gondii Motility Induces the Posttranslational Modification of Myosin Light Chain-1 and Inhibits Myosin Motor Activity

Toxoplasma gondii is an obligate intracellular parasite that enters cells by a process of active penetration. Host cell penetration and parasite motility are driven by a myosin motor complex consisting of four known proteins: TgMyoA, an unconventional Class XIV myosin; TgMLC1, a myosin light chain; and two membrane-associated proteins, TgGAP45 and TgGAP50. Little is known about how the activity of the myosin motor complex is regulated. Here, we show that treatment of parasites with a recently identified small-molecule inhibitor of invasion and motility results in a rapid and irreversible change in the electrophoretic mobility of TgMLC1. While the precise nature of the TgMLC1 modification has not yet been established, it was mapped to the peptide Val46-Arg59. To determine if the TgMLC1 modification is responsible for the motility defect observed in parasites after compound treatment, the activity of myosin motor complexes from control and compound-treated parasites was compared in an in vitro motility assay. TgMyoA motor complexes containing the modified TgMLC1 showed significantly decreased motor activity compared to control complexes. This change in motor activity likely accounts for the motility defects seen in the parasites after compound treatment and provides the first evidence, in any species, that the mechanical activity of Class XIV myosins can be modulated by posttranslational modifications to their associated light chains.     

Expression of a nonpolymerizable actin mutant in Sf9 cells

We have succeeded in expressing actin in the baculovirus/Sf9 cell system in high yield. The wild-type (WT) actin is functionally indistinguishable from tissue-purified actin in its ability to activate ATPase activity and to support movement in an in vitro motility assay. Having achieved this feat, we used a mutational strategy to express a monomeric actin that is incapable of polymerization. Native actin requires actin binding proteins or chemical modification to maintain it in a monomeric state. The mutant actin sediments in the analytical ultracentrifuge as a homogeneous monomeric species of 3.2 S in 100 mM KCl and 2 mM MgCl(2), conditions that cause WT actin to polymerize. The two point mutations that render actin nonpolymerizable are in subdomain 4 (A204E/P243K; "AP-actin"), distant from the myosin binding site. AP-actin binds to skeletal myosin subfragment 1 (S1) and forms a homogeneous complex as demonstrated by analytical ultracentrifugation. The ATPase activity of a cross-linked AP-actin.S1 complex is higher than that of S1 alone, although less than that supported by filamentous actin (F-actin). AP-Actin is an excellent candidate for structural studies of complexes of actin with motor proteins and other actin-binding proteins.     

Gliding Associated Proteins Play Essential Roles during the Formation of the Inner Membrane Complex of Toxoplasma gondii

The inner membrane complex (IMC) of apicomplexan parasites is a specialised structure localised beneath the parasite’s plasma membrane, and is important for parasite stability and intracellular replication. Furthermore, it serves as an anchor for the myosin A motor complex, termed the glideosome. While the role of this protein complex in parasite motility and host cell invasion has been well described, additional roles during the asexual life cycle are unknown. Here, we demonstrate that core elements of the glideosome, the gliding associated proteins GAP40 and GAP50 as well as members of the GAPM family, have critical roles in the biogenesis of the IMC during intracellular replication. Deletion or disruption of these genes resulted in the rapid collapse of developing parasites after initiation of the cell cycle and led to redistribution of other glideosome components.     

Intracellular transport based on actin polymerization

In addition to the intracellular transport of particles (cargo) along microtubules, there are in the cell two actin-based transport systems. In the actomyosin system the transport is driven by myosin, which moves the cargo along actin microfilaments. This transport requires the hydrolysis of ATP in the myosin molecule motor domain that induces conformational changes in the molecule resulting in the myosin movement along the actin filament. The other actin-based transport system of the cell does not involve myosin or other motor proteins. This system is based on a unidirectional actin polymerization, which depends on ATP hydrolysis in actin polymers and is initiated by proteins bound to the surface of transported particles. Obligatory components of the actin-based transport are proteins of the WASP/Scar family and a complex of Arp2/3 proteins. Moreover, the actin-based systems often contain dynamin and cortactin. It is known that a system of actin filaments formed on the surface of particles, the so-called “comet-like tail”, is responsible for intracellular movements of pathogenic bacteria, micropinocytotic vesicles, clathrin-coated vesicles, and phagosomes. This movement is reproduced in a cell-free system containing extract of Xenopus oocytes. The formation of a comet-like structure capable of transporting vesicles from the plasma membrane into the cell depth has been studied in detail by high performance electron microscopy combined with electron tomography. A similar mechanism provides the movement of vesicles containing membrane rafts enriched with sphingolipids and cholesterol, changes in position of the nuclear spindle at meiosis, and other processes. This review will consider current ideas about actin polymerization and its regulation by actin-binding proteins and show how these mechanisms are realized in the intracellular actin-based vesicular transport system.     

Immobilization of the type XIV myosin complex in Toxoplasma gondii

The substrate-dependent movement of apicomplexan parasites such as Toxoplasma gondii and Plasmodium sp. is driven by the interaction of a type XIV myosin with F-actin. A complex containing the myosin-A heavy chain, a myosin light chain, and the accessory protein GAP45 is attached to the membranes of the inner membrane complex (IMC) through its tight interaction with the integral membrane glycoprotein GAP50. For the interaction of this complex with F-actin to result in net parasite movement, it is necessary that the myosin be immobilized with respect to the parasite and the actin with respect to the substrate the parasite is moving on. We report here that the myosin motor complex of Toxoplasma is firmly immobilized in the plane of the IMC. This does not seem to be accomplished by direct interactions with cytoskeletal elements. Immobilization of the motor complex, however, does seem to require cholesterol. Both the motor complex and the cholesterol are found in detergent-resistant membrane domains that encompass a large fraction of the inner membrane complex surface. The observation that the myosin XIV motor complex of Toxoplasma is immobilized within this cholesterol-rich membrane likely extends to closely related pathogens such as Plasmodium and possibly to other eukaryotes.     

Molecular dissection of host cell invasion by the apicomplexans: the glideosome

Gliding motility is an essential and fascinating apicomplexan-typical adaptation to an intracellular lifestyle. Apicomplexan parasites rely on gliding motility for their migration across biological barriers and for host cell invasion and egress. This unusual substratedependent mode of locomotion involves the concerted action of secretory adhesins, a myosin motor, factors regulating actin dynamics and proteases. During invasion, complexes of soluble and transmembrane micronemes proteins (MICs) and rhoptry neck proteins (RONs) are discharged to the apical pole of the parasite, some protein acts as adhesins and bind to host cell receptors whereas others are involved in the moving junction formation. These complexes redistribute towards the posterior pole of the parasite via a physical connection to the parasite actomyosin system and are eventually released from the parasite surface by the action of parasite proteases.     

Use of the myosin motor domain as large-affinity tag for the expression and purification of proteins in Dictyostelium discoideum

The cellular slime mold Dictyostelium discoideum is increasingly be used for the overexpression of proteins. Dictyostelium is amenable to classical and molecular genetic approaches and can easily be grown in large quantities. It contains a variety of chaperones and folding enzymes, and is able to perform all kinds of post-translational protein modifications. Here, new expression vectors are presented that have been designed for the production of proteins in large quantities for biochemical and structural studies. The expression cassettes of the most successful vectors are based on a tandem affinity purification tag consisting of an octahistidine tag followed by the myosin motor domain tag. The myosin motor domain not only strongly enhances the production of fused proteins but is also used for a fast affinity purification step through its ATP-dependent binding to actin. The applicability of the new system has been demonstrated for the expression and purification of subunits of the dynein-dynactin motor protein complex from different species.     

Microneme proteins: structural and functional requirements to promote adhesion and invasion by the apicomplexan parasite Toxoplasma gondii

Host-cell invasion by apicomplexan parasites is extremely rapid and relies on a sequence of events that are tightly controlled in time and space. In most Apicomplexa, the gliding motility and host-cell invasion are tightly coupled to the release of microneme proteins at the apical tip of the parasites and their redistribution toward the posterior pole. This movement is dependent on an intact parasite actomyosin system. Micronemes are involved in the trafficking and storage of ligands (MICs) for host-cell receptors that are not only structurally related but also functionally conserved among the Apicomplexa. In Toxoplasma gondii, the repertoire of membrane-spanning microneme proteins includes adhesins such as TgMIC2 and escorters such as TgMIC6. The latter forms a complex with the soluble adhesins, TgMIC1 and TgMIC4 and assures their proper sorting to the mironemes. Escorters are also anticipated to bridge host-cell receptors to the parasite membrane during invasion. Most TgMICs are proteolytically cleaved either during their transport along the secretory pathway and/or after exocytosis. The biological significance of these processing events is largely unknown. One of these processing events targets a conserved motif close to the membrane-spanning domain causing the release of the processed form of the micronemes from the parasite surface. The cleavages occurring after release might contribute to the disassembly of the complexes and thus to fission between the parasitophorous vacuole and the host plasma membrane at the end of the invasion process. Gliding motility and host-cell penetration involve the redistribution of the micronemes toward the posterior pole of the parasites. This capping process involves actin polymerisation, myosin adenosine triphosphatase activation and the establishment of a connection between the MICs-receptor complexes and the actomyosin system of the parasite. The most carboxy-terminal end of the MICs cytoplasmic tails is implicated in this process, but the precise nature of the connection with the actomyosin system remains to be elucidated.     

Toxoplasma gondii myosin A and its light chain: a fast, single-headed, plus-end-directed motor

Successful host cell invasion is a prerequisite for survival of the obligate intracellular apicomplexan parasites and establishment of infection. Toxoplasma gondii penetrates host cells by an active process involving its own actomyosin system and which is distinct from induced phagocytosis. Toxoplasma gondii myosin A (TgMyoA) is presumed to achieve power gliding motion and host cell penetration by the capping of apically released adhesins towards the rear of the parasite. We report here an extensive biochemical characterization of the functional TgMyoA motor complex. TgMyoA is anchored at the plasma membrane and binds a novel type of myosin light chain (TgMLC1). Despite some unusual features, the kinetic and mechanical properties of TgMyoA are unexpectedly similar to those of fast skeletal muscle myosins. Microneedle-laser trap and sliding velocity assays established that TgMyoA moves in unitary steps of 5.3 nm with a velocity of 5.2 microm/s towards the plus end of actin filaments. TgMyoA is the first fast, single-headed myosin and fulfils all the requirements for power parasite gliding.     

Identification and characterization of Toxoplasma SIP,a conserved apicomplexan cytoskeleton protein involved in maintaining the shape,motility and virulence of the parasite

Apicomplexa possess a complex pellicle that is composed of a plasma membrane and a closely apposed inner membrane complex (IMC) that serves as a support for the actin‐myosin motor required for motility and host cell invasion. The IMC consists of longitudinal plates of flattened vesicles, fused together and lined on the cytoplasmic side by a subpellicular network of intermediate filament‐like proteins. The spatial organization of the IMC has been well described by electron microscopy, but its composition and molecular organization is largely unknown. Here, we identify a novel protein of the IMC cytoskeletal network in Toxoplasma gondii, called TgSIP, and conserved among apicomplexan parasites. To finely pinpoint the localization of TgSIP, we used structured illumination super‐resolution microscopy and revealed that it likely decorates the transverse sutures of the plates and the basal end of the IMC. This suggests that TgSIP might contribute to the organization or physical connection among the different components of the IMC. We generated a T.gondii SIP deletion mutant and showed that parasites lacking TgSIP are significantly shorter than wild‐type parasites and show defects in gliding motility, invasion and reduced infectivity in mice.     

The melanosome as a model to study organelle motility in mammals

Melanosomes are lysosome-related organelles within which melanin pigment is synthesized. The molecular motors that allow these organelles to move within melanocytes have been the subject of intense study in several organisms. In mammals, melanosomes travel bi-directionally along microtubule tracks. The anterograde movement, i.e., towards microtubule plus-ends at the periphery, is accomplished by proteins of the kinesin superfamily, whereas the retrograde movement, i.e., towards microtubule minus-ends at the cell center, is achieved by dynein and dynein-associated proteins. At the periphery, melanosomes interact with the actin cytoskeleton via a tripartite complex formed by the small GTPase Rab27a, melanophilin and myosin Va, an actin-based motor. This interaction is essential for the maintenance of a dispersed state of the melanosomes, as shown by the perinuclear clustering of organelles in mutants in any of the referred proteins. In the retinal pigment epithelium, a similar complex formed by Rab27a, a melanophilin homolog called MyRIP and myosin VIIa is probably responsible for the tethering of melanosomes to the actin cytoskeleton. The coordination of motor activities is still poorly characterized, although some models have emerged in recent years and are discussed here. Unraveling regulatory mechanisms responsible for melanosome motility in pigmented cells will provide general insights into organelles dynamics within eukaryotic cells.     

A dibasic motif in the tail of a class XIV apicomplexan myosin is an essential determinant of plasma membrane localization

Obligate intracellular parasites of the phylum Apicomplexa exhibit gliding motility, a unique form of substrate-dependent locomotion essential for host cell invasion and shown to involve the parasite actin cytoskeleton and myosin motor(s). Toxoplasma gondii has been shown to express three class XIV myosins, TgM-A, -B, and -C. We identified an additional such myosin, TgM-D, and completed the sequences of a related Plasmodium falciparum myosin, PfM-A. Despite divergent structural features, TgM-A purified from parasites bound actin in an ATP-dependent manner. Isoform-specific antibodies revealed that TgM-A and recombinant mycTgM-A were localized right beneath the plasma membrane, and subcellular fractionation indicated a tight membrane association. Recombinant TgM-D also had a peripheral although not as sharply defined localization. Truncation of their respective tail domains abolished peripheral localization and tight membrane association. Conversely, fusion of the tails to green fluorescent protein (GFP) was sufficient to confer plasma membrane localization and sedimentability. The peripheral localization of TgM-A and of the GFP-tail fusion did not depend on an intact F-actin cytoskeleton, and the GFP chimera did not localize to the plasma membrane of HeLa cells. Finally, we showed that the specific localization determinants were in the very C terminus of the TgM-A tail, and site-directed mutagenesis revealed two essential arginine residues. We discuss the evidence for a proteinaceous plasma membrane receptor and the implications for the invasion process.     

Calcium-dependent phosphorylation alters class XIVa myosin function in the protozoan parasite Toxoplasma gondii

Class XIVa myosins comprise a unique group of myosin motor proteins found in apicomplexan parasites, including those that cause malaria and toxoplasmosis. The founding member of the class XIVa family, Toxoplasma gondii myosin A (TgMyoA), is a monomeric unconventional myosin that functions at the parasite periphery to control gliding motility, host cell invasion, and host cell egress. How the motor activity of TgMyoA is regulated during these critical steps in the parasite''s lytic cycle is unknown. We show here that a small-molecule enhancer of T. gondii motility and invasion (compound 130038) causes an increase in parasite intracellular calcium levels, leading to a calcium-dependent increase in TgMyoA phosphorylation. Mutation of the major sites of phosphorylation altered parasite motile behavior upon compound 130038 treatment, and parasites expressing a nonphosphorylatable mutant myosin egressed from host cells more slowly in response to treatment with calcium ionophore. These data demonstrate that TgMyoA undergoes calcium-dependent phosphorylation, which modulates myosin-driven processes in this important human pathogen.     

The regulation of myosin II in Dictyostelium

Dictyostelium conventional myosin (myosin II) is an abundant protein that plays a role in various cellular processes such as cytokinesis, cell protrusion and development. This review will focus on the signal transduction pathways that regulate myosin II during cell movement. Myosin II appears to have two modes of action in Dictyostelium: local stabilization of the cytoskeleton by myosin filament association to the actin meshwork (structural mode) and force generation by contraction of actin filaments (motor mode). Some processes, such as cell movement under restrictive environment, require only the structural mode of myosin. However, cytokinesis in suspension and uropod retraction depend on motor activity as well. Myosin II can self-assemble into bipolar filaments. The formation of these filaments is negatively regulated by heavy chain phosphorylation through the action of a set of novel alpha kinases and is relatively well understood. However, only recently it has become clear that the formation of bipolar filaments and their translocation to the cortex are separate events. Translocation depends on filamentous actin, and is regulated by a cGMP pathway and possibly also by the cAMP phosphodiesterase RegA and the p21-activated kinase PAKa. Myosin motor activity is regulated by phosphorylation of the regulatory light chain through myosin light chain kinase A. Unlike conventional light chain kinases, this enzyme is not regulated by calcium but is activated by cGMP-induced phosphorylation via an upstream kinase and subsequent autophosphorylation.     

Roles of myosin phosphatase during Drosophila development

Myosins are a superfamily of actin-dependent molecular motor proteins, among which the bipolar filament forming myosins II have been the most studied. The activity of smooth muscle/non-muscle myosin II is regulated by phosphorylation of the regulatory light chains, that in turn is modulated by the antagonistic activity of myosin light chain kinase and myosin light chain phosphatase. The phosphatase activity is mainly regulated through phosphorylation of its myosin binding subunit MYPT. To identify the function of these phosphorylation events, we have molecularly characterized the Drosophila homologue of MYPT, and analyzed its mutant phenotypes. We find that Drosophila MYPT is required for cell sheet movement during dorsal closure, morphogenesis of the eye, and ring canal growth during oogenesis. Our results indicate that the regulation of the phosphorylation of myosin regulatory light chains, or dynamic activation and inactivation of myosin II, is essential for its various functions during many developmental processes.     

Structural basis for the interaction of the myosin light chain Mlc1p with the myosin V Myo2p IQ motifs

Calmodulin, regulatory, and essential myosin light chain are evolutionary conserved proteins that, by binding to IQ motifs of target proteins, regulate essential intracellular processes among which are efficiency of secretory vesicles release at synapsis, intracellular signaling, and regulation of cell division. The yeast Saccharomyces cerevisiae calmodulin Cmd1 and the essential myosin light chain Mlc1p share the ability to interact with the class V myosin Myo2p and Myo4 and the class II myosin Myo1p. These myosins are required for vesicle, organelle, and mRNA transport, spindle orientation, and cytokinesis. We have used the budding yeast model system to study how calmodulin and essential myosin light chain selectively regulate class V myosin function. NMR structural analysis of uncomplexed Mlc1p and interaction studies with the first three IQ motifs of Myo2p show that the structural similarities between Mlc1p and the other members of the EF-hand superfamily of calmodulin-like proteins are mainly restricted to the C-lobe of these proteins. The N-lobe of Mlc1p presents a significantly compact and stable structure that is maintained both in the free and complexed states. The Mlc1p N-lobe interacts with the IQ motif in a manner that is regulated both by the IQ motifs sequence as well as by light chain structural features. These characteristic allows a distinctive interaction of Mlc1p with the first IQ motif of Myo2p when compared with calmodulin. This finding gives us a novel view of how calmodulin and essential light chain, through a differential binding to IQ1 of class V myosin motor, regulate this activity during vegetative growth and cytokinesis.