TgICMAP1 Is a Novel Microtubule Binding Protein in Toxoplasma gondii

The microtubule cytoskeleton provides essential structural support for all eukaryotic cells and can be assembled into various higher order structures that perform drastically different functions. Understanding how microtubule-containing assemblies are built in a spatially and temporally controlled manner is therefore fundamental to understanding cell physiology. Toxoplasma gondii, a protozoan parasite, contains at least five distinct tubulin-containing structures, the spindle pole, centrioles, cortical microtubules, the conoid, and the intra-conoid microtubules. How these five structurally and functionally distinct sets of tubulin containing structures are constructed and maintained in the same cell is an intriguing problem. Previously, we performed a proteomic analysis of the T. gondii apical complex, a cytoskeletal complex located at the apical end of the parasite that is composed of the conoid, three ring-like structures, and the two short intra-conoid microtubules. Here we report the characterization of one of the proteins identified in that analysis, TgICMAP1. We show that TgICMAP1 is a novel microtubule binding protein that can directly bind to microtubules in vitro and stabilizes microtubules when ectopically expressed in mammalian cells. Interestingly, in T. gondii, TgICMAP1 preferentially binds to the intra-conoid microtubules, providing us the first molecular tool to investigate the intra-conoid microtubule assembly process during daughter construction

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

F-actin and myosin F control apicoplast elongation dynamics which drive apicoplast-centrosome association in Toxoplasma gondii

ABSTRACT Toxoplasma gondii contains an essential plastid organelle called the apicoplast that is necessary for fatty acid, isoprenoid, and heme synthesis. Perturbations affecting apicoplast function or inheritance lead to parasite death. The apicoplast is a single copy organelle and, therefore, must be divided so that each daughter parasite inherits an apicoplast during cell division. In this study, we identify new roles for F-actin and an unconventional myosin motor, TgMyoF, in this process. First, loss of TgMyoF and actin lead to an accumulation of apicoplast vesicles in the cytosol indicating a role for this actomyosin system in apicoplast protein trafficking or morphological integrity of the organelle. Second, live cell imaging reveals that during division the apicoplast is highly dynamic, exhibiting branched, U-shaped and linear morphologies that are dependent on TgMyoF and actin. In parasites where movement was inhibited by the depletion of TgMyoF, the apicoplast fails to associate with the parasite centrosomes. Thus, this study provides crucial new insight into mechanisms controlling apicoplast-centrosome association, a vital step in the apicoplast division cycle, which ensures that each daughter inherits a single apicoplast. IMPORTANCE Toxoplasma gondii and most other parasites in the phylum Apicomplexa contain an apicoplast, a non-photosynthetic plastid organelle required for fatty acid, isoprenoid, iron-sulfur cluster, and heme synthesis. Perturbation of apicoplast function results in parasite death. Thus, parasite survival critically depends on two cellular processes: apicoplast division to ensure every daughter parasite inherits a single apicoplast, and trafficking of nuclear encoded proteins to the apicoplast. Despite the importance of these processes, there are significant knowledge gaps in regards to the molecular mechanisms which control these processes; this is particularly true for trafficking of nuclear-encoded apicoplast proteins. This study provides crucial new insight into the timing of apicoplast protein synthesis and trafficking to the apicoplast. In addition, this study demonstrates how apicoplast-centrosome association, a key step in the apicoplast division cycle, is controlled by the actomyosin cytoskeleton

The motility of a human parasite, Toxoplasma gondii, is regulated by a novel lysine methyltransferase.

Protozoa in the phylum Apicomplexa are a large group of obligate intracellular parasites. Toxoplasma gondii and other apicomplexan parasites, such as Plasmodium falciparum, cause diseases by reiterating their lytic cycle, comprising host cell invasion, parasite replication, and parasite egress. The successful completion of the lytic cycle requires that the parasite senses changes in its environment and switches between the non-motile (for intracellular replication) and motile (for invasion and egress) states appropriately. Although the signaling pathway that regulates the motile state switch is critical to the pathogenesis of the diseases caused by these parasites, it is not well understood. Here we report a previously unknown mechanism of regulating the motility activation in Toxoplasma, mediated by a protein lysine methyltransferase, AKMT (for Apical complex lysine (K) methyltransferase). AKMT depletion greatly inhibits activation of motility, compromises parasite invasion and egress, and thus severely impairs the lytic cycle. Interestingly, AKMT redistributes from the apical complex to the parasite body rapidly in the presence of egress-stimulating signals that increase [Ca²⁺] in the parasite cytoplasm, suggesting that AKMT regulation of parasite motility might be accomplished by the precise temporal control of its localization in response to environmental changes

TgMORN1 is a key organizer for the basal complex of Toxoplasma gondii.

Toxoplasma gondii is a leading cause of congenital birth defects, as well as a cause for ocular and neurological diseases in humans. Its cytoskeleton is essential for parasite replication and invasion and contains many unique structures that are potential drug targets. Therefore, the biogenesis of the cytoskeletal structure of T. gondii is not only important for its pathogenesis, but also of interest to cell biology in general. Previously, we and others identified a new T. gondii cytoskeletal protein, TgMORN1, which is recruited to the basal complex at the very beginning of daughter formation. However, its function remained largely unknown. In this study, we generated a knock-out mutant of TgMORN1 (DeltaTgMORN1) using a Cre-LoxP based approach. We found that the structure of the basal complex was grossly affected in DeltaTgMORN1 parasites, which also displayed defects in cytokinesis. Moreover, DeltaTgMORN1 parasites showed significant growth impairment in vitro, and this translated into greatly attenuated virulence in mice. Therefore, our results demonstrate that TgMORN1 is required for maintaining the structural integrity of the parasite posterior end, and provide direct evidence that cytoskeleton integrity is essential for parasite virulence and pathogenesis

Actin and an unconventional myosin motor, TgMyoF, control the organization and dynamics of the endomembrane network in Toxoplasma gondii.

Toxoplasma gondii is an obligate intracellular parasite that relies on three distinct secretory organelles, the micronemes, rhoptries, and dense granules, for parasite survival and disease pathogenesis. Secretory proteins destined for these organelles are synthesized in the endoplasmic reticulum (ER) and sequentially trafficked through a highly polarized endomembrane network that consists of the Golgi and multiple post-Golgi compartments. Currently, little is known about how the parasite cytoskeleton controls the positioning of the organelles in this pathway, or how vesicular cargo is trafficked between organelles. Here we show that F-actin and an unconventional myosin motor, TgMyoF, control the dynamics and organization of the organelles in the secretory pathway, specifically ER tubule movement, apical positioning of the Golgi and post-Golgi compartments, apical positioning of the rhoptries, and finally, the directed transport of Rab6-positive and Rop1-positive vesicles. Thus, this study identifies TgMyoF and actin as the key cytoskeletal components that organize the endomembrane system in T. gondii

Toxoplasma gondii actin filaments are tuned for rapid disassembly and turnover

Abstract The cytoskeletal protein actin plays a critical role in the pathogenicity of the intracellular parasite, Toxoplasma gondii, mediating invasion and egress, cargo transport, and organelle inheritance. Advances in live cell imaging have revealed extensive filamentous actin networks in the Apicomplexan parasite, but there are conflicting data regarding the biochemical and biophysical properties of Toxoplasma actin. Here, we imaged the in vitro assembly of individual Toxoplasma actin filaments in real time, showing that native, unstabilized filaments grow tens of microns in length. Unlike skeletal muscle actin, Toxoplasma filaments intrinsically undergo rapid treadmilling due to a high critical concentration, fast monomer dissociation, and rapid nucleotide exchange. Cryo-EM structures of jasplakinolide-stabilized and native (i.e. unstabilized) filaments show an architecture like skeletal actin, with differences in assembly contacts in the D-loop that explain the dynamic nature of the filament, likely a conserved feature of Apicomplexan actin. This work demonstrates that evolutionary changes at assembly interfaces can tune the dynamic properties of actin filaments without disrupting their conserved structure

Cytoskeletal Dependence of Insulin Granule Movement Dynamics in INS-1 Beta-Cells in Response to Glucose

<div><p>For pancreatic β-cells to secrete insulin in response to elevated blood glucose, insulin granules retained within the subplasmalemmal space must be transported to sites of secretion on the plasma membrane. Using a combination of super-resolution STORM imaging and live cell TIRF microscopy we investigate how the organization and dynamics of the actin and microtubule cytoskeletons in INS-1 β-cells contribute to this process. GFP-labeled insulin granules display 3 different modes of motion (stationary, diffusive-like, and directed). Diffusive-like motion dominates in basal, low glucose conditions. Upon glucose stimulation no gross rearrangement of the actin cytoskeleton is observed but there are increases in the 1) rate of microtubule polymerization; 2) rate of diffusive-like motion; and 3) proportion of granules undergoing microtubule-based directed motion. By pharmacologically perturbing the actin and microtubule cytoskeletons, we determine that microtubule-dependent granule transport occurs within the subplasmalemmal space and that the actin cytoskeleton limits this transport in basal conditions, when insulin secretion needs to be inhibited.</p></div

Effect of microtubule perturbation on insulin granule dynamics.

<p>(A and B: Upper Image Panels) Outline of a representative INS-1 cells with insulin granule trajectories overlaid in low and high glucose conditions after treatment with taxol (A) and nocodazole (B). Left inset: image of INS-1 cells with eGFP-labeled insulin granules. Right inset: 4× magnification of trajectories in the black box. Trajectories are color-coded to depict mode of granule motion. Red indicates stationary granule, blue indicates diffusive-like motion, and green indicates directed motion. (A and B: Lower Panel) Bar charts depict the percentage of granules exhibiting stationary, diffusive-like, and directed motions in low and high glucose conditions. Black arrows/dashed lines indicate the percentage of granules in each population in control cells from <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0109082#pone-0109082-g002" target="_blank">Figure 2A</a>. Taxol: 17 cells/282 granules in low glucose and 15 cells/289 granules in high glucose conditions were analyzed. Nocodazole: 18 cells/321 granules in low glucose and 23 cells/428 granules in high glucose conditions were analyzed.</p