Ca<sup>2+</sup>‐Dependence of Conoid Extrusion in Toxoplasma gondii Tachyzoites

Mondragón, Ricardo; Frixione, Eugenio

doi:10.1111/j.1550-7408.1996.tb04491.x

Cited by 144 publications

(107 citation statements)

References 34 publications

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“…Secretion is increased in response to agents that elevate intracellular calcium and decreased by treatment with cell-permeable calcium chelators or xestospongin (13,14,19). With the exception of Inhibitor 6 (see below), none of the secretion inhibitors affect conoid extension, another calcium-dependent process (12), suggesting that they are unlikely to inhibit secretion via a direct effect on parasite intracellular calcium. However, direct measurement of parasite intracellular calcium levels in response to each of the secretion inhibitors will be an important area for future investigation.…”

Section: Discussionmentioning

confidence: 99%

“…The conoid extends and retracts repeatedly as the parasite moves along the surface of a cell. Conoid extension can be experimentally induced by treating extracellular parasites with calcium ionophores, such as ionomycin (12). The effect of each invasion inhibitor on conoid extension was examined by adding buffer containing either ionomycin or DMSO to inhibitorpretreated parasites, then visualizing extended conoids by phase microscopy.…”

Section: Resultsmentioning

confidence: 99%

“…Actin sedimentation was assayed as described (11). Parasite motility, conoid extension, microneme secretion, and intracellular calcium levels were assayed as described (6,(12)(13)(14) with the modifications described in Supporting Methods.…”

Section: Methodsmentioning

confidence: 99%

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A small-molecule approach to studying invasive mechanisms of Toxoplasma gondii

Carey

Westwood

Mitchison

et al. 2004

Proc. Natl. Acad. Sci. U.S.A.

128

163

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Toxoplasma gondii is the most common protozoan parasite of humans. Infection with T. gondii can lead to life-threatening disease as a result of repeated cycles of host cell invasion, parasite replication, and host cell lysis. Relatively little is known about the invasive mechanisms of T. gondii and related parasites within the Phylum Apicomplexa (including Plasmodium spp., the causative agents of malaria), due to difficulties associated with studying genes essential to invasion in haploid obligate intracellular organisms. To circumvent this problem, we have developed a highthroughput microscope-based assay, which we have used to screen a collection of 12,160 structurally diverse small molecules for inhibitors of T. gondii invasion. A total of 24 noncytotoxic invasion inhibitors were identified. Secondary assays demonstrated that different inhibitors perturb different aspects of invasion, including gliding motility, secretion of host cell adhesins from apical organelles (the micronemes), and extension of a unique tubulinbased structure at the anterior of the parasite (the conoid). Unexpectedly, the screen also identified six small molecules that dramatically enhance invasion, gliding motility, and microneme secretion. The small molecules identified here reveal a previously unrecognized complexity in the control of parasite motility and microneme secretion, and they constitute a set of useful probes for dissecting the invasive mechanisms of T. gondii and related parasites. Small-molecule-based approaches provide a powerful means to address experimentally challenging problems in host-pathogen interaction, while simultaneously identifying new potential targets for drug development. N early one-third of all deaths in the world today are caused by infectious disease. The development of new preventative and therapeutic strategies relies on an improved understanding of the interaction between pathogens and their hosts. In many pathogenic systems, this presents a formidable experimental challenge, because standard genetic tools are either rudimentary or unavailable. Biochemical, genomic, and other approaches exist, but these lack the assumption-free power of a genetic screen.An alternative nongenetic approach to studying mechanisms of host-pathogen interaction involves screening large structurally diverse collections of small molecules for those that disrupt the interaction. Once identified, the small molecules (or their derivatives) are used to determine the cellular components that function in the process (reviewed in refs. 1-3). The approach relies on the demonstrated ability of many small molecules to interact specifically with their targets (e.g., ref. 4). As with classical forward genetic screens, the approach is assumptionfree: by sampling large unbiased collections of structurally diverse small molecules, the screen ''selects'' structures that perturb the process under study. Such phenotype-based highthroughput screening has recently gained momentum in the academic setting due to technological advances and the av...

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Section: Discussionmentioning

confidence: 99%

Section: Resultsmentioning

confidence: 99%

See 1 more Smart Citation

A small-molecule approach to studying invasive mechanisms of Toxoplasma gondii

Carey

Westwood

Mitchison

et al. 2004

Proc. Natl. Acad. Sci. U.S.A.

128

163

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“…Careful EM analysis will be necessary to determine how the ultrastructural arrangement of this tubulin is related to the lattice arrangement of typical microtubules or other tubulin polymers (33,34). Because the conoid protrudes during parasite invasion (14,17), it will be interesting to determine whether a microtubule-based motor protein of the dynein or kinesin family is present. Our measurements also suggest that the amount of tubulin in the conoid decreases as daughters mature into adults.…”

Section: Discussionmentioning

confidence: 99%

“…Based on morphological studies, the subpellicular microtubules are thought to help maintain the integrity of the cell surface as well as play a role in active shape changes during invasion (2,7,8,14). The apical region of most apicomplexan parasites contains a specialized cytoskeletal structure, the conoid (15,16), that is actively protruded and retracted during the invasion process (14,17). In electron microscopy (EM) studies, the conoid appears as a tightly coiled spiral of 10-12 fibrous elements of unknown composition (6)(7)(8).…”

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confidence: 99%

Measuring tubulin content in Toxoplasma gondii : A comparison of laser-scanning confocal and wide-field fluorescence microscopy

Swedlow

Andrews

et al. 2002

Proc. Natl. Acad. Sci. U.S.A.

133

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Toxoplasma gondii is an intracellular parasite that proliferates within most nucleated cells, an important human pathogen, and a model for the study of human and veterinary parasitic infections. We used a stable yellow fluorescent protein-␣-tubulin transgenic line to determine the structure of the microtubule cytoskeleton in T. gondii. Imaging of living yellow fluorescent protein-␣-tubulin parasites by laser-scanning confocal microscopy (LSCM) failed to resolve the 22 subpellicular microtubules characteristic of the parasite cytoskeleton. To understand this result, we analyzed sources of noise in the LSCM and identified illumination fluctuations on time scales from microseconds to hours that introduce significant amounts of noise. We confirmed that weakly fluorescent structures could not be imaged in LSCM by using fluorescent bead standards. By contrast, wide-field microscopy (WFM) did visualize weak fluorescent standards and the individual microtubules of the parasite cytoskeleton. We therefore measured the fluorescence per unit length of microtubule by using WFM and used this information to estimate the tubulin content of the conoid (a structure important for T. gondii infection) and in the mitotic spindle pole. The conoid contains sufficient tubulin for Ϸ10 microtubule segments of 0.5-m length, indicating that tubulin forms the structural core of the organelle. We also show that the T. gondii mitotic spindle contains Ϸ1 microtubule per chromosome. This analysis expands the understanding of structures used for invasion and intracellular proliferation by an important human pathogen and shows the advantage of WFM combined with image deconvolution over LSCM for quantitative studies of weakly fluorescent structures in moderately thin living cells.

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