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Time-lapse video microscopy of gliding motility in Toxoplasma gondii reveals a novel, biphasic mechanism of cell locomotion.

ABSTRACT: Toxoplasma gondii is a member of the phylum Apicomplexa, a diverse group of intracellular parasites that share a unique form of gliding motility. Gliding is substrate dependent and occurs without apparent changes in cell shape and in the absence of traditional locomotory organelles. Here, we demonstrate that gliding is characterized by three distinct forms of motility: circular gliding, upright twirling, and helical rotation. Circular gliding commences while the crescent-shaped parasite lies on its right side, from where it moves in a counterclockwise manner at a rate of approximately 1.5 microm/s. Twirling occurs when the parasite rights itself vertically, remaining attached to the substrate by its posterior end and spinning clockwise. Helical gliding is similar to twirling except that it occurs while the parasite is positioned horizontally, resulting in forward movement that follows the path of a corkscrew. The parasite begins lying on its left side (where the convex side is defined as dorsal) and initiates a clockwise revolution along the long axis of the crescent-shaped body. Time-lapse video analyses indicated that helical gliding is a biphasic process. During the first 180(o) of the turn, the parasite moves forward one body length at a rate of approximately 1-3 microm/s. In the second phase, the parasite flips onto its left side, in the process undergoing little net forward motion. All three forms of motility were disrupted by inhibitors of actin filaments (cytochalasin D) and myosin ATPase (butanedione monoxime), indicating that they rely on an actinomyosin motor in the parasite. Gliding motility likely provides the force for active penetration of the host cell and may participate in dissemination within the host and thus is of both fundamental and practical interest.

SUBMITTER: Hakansson S

PROVIDER: S-EPMC25631 | biostudies-literature | 1999 Nov

REPOSITORIES: biostudies-literature

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Time-lapse video microscopy of gliding motility in Toxoplasma gondii reveals a novel, biphasic mechanism of cell locomotion.

Håkansson S S Morisaki H H Heuser J J Sibley L D LD

Molecular biology of the cell 19991101 11

Toxoplasma gondii is a member of the phylum Apicomplexa, a diverse group of intracellular parasites that share a unique form of gliding motility. Gliding is substrate dependent and occurs without apparent changes in cell shape and in the absence of traditional locomotory organelles. Here, we demonstrate that gliding is characterized by three distinct forms of motility: circular gliding, upright twirling, and helical rotation. Circular gliding commences while the crescent-shaped parasite lies on ...[more]

PMID: 10564254

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Project description:Toxoplasma gondii is a highly successful parasite that infects approximately one-third of the human population and can cause fatal disease in immunocompromised individuals. Systemic parasite dissemination to organs such as the brain and eye is critical to pathogenesis. T. gondii can disseminate via the circulation, and both intracellular and extracellular modes of transport have been proposed. However, the processes by which extracellular tachyzoites adhere to and migrate across vascular endothelium under the conditions of rapidly flowing blood remain unknown. We used microfluidics and time-lapse fluorescence microscopy to examine the interactions between extracellular T. gondii and primary human endothelial cells under conditions of physiologic shear stress. Remarkably, tachyzoites adhered to and glided on human vascular endothelium under shear stress conditions. Compared to static conditions, shear stress enhanced T. gondii helical gliding, resulting in a significantly greater displacement, and increased the percentage of tachyzoites that invaded or migrated across the endothelium. The intensity of the shear forces (from 0.5 to 10 dynes/cm(2)) influenced both initial and sustained adhesion to endothelium. By examining tachyzoites deficient in the T. gondii adhesion protein MIC2, we found that MIC2 contributed to initial adhesion but was not required for adhesion strengthening. These data suggest that under fluidic conditions, T. gondii adhesion to endothelium may be mediated by a multistep cascade of interactions that is governed by unique combinations of adhesion molecules. This work provides novel information about tachyzoite interactions with vascular endothelium and contributes to our understanding of T. gondii dissemination in the infected host. IMPORTANCE Toxoplasma gondii is a global parasite pathogen that can cause fatal disease in immunocompromised individuals. An unresolved question is how the parasites circulate in the body to tissues to cause disease. T. gondii parasites are found in the bloodstream of infected animals and patients, and they have been shown to adhere to and cross the endothelial cells that line blood vessel walls. To investigate these interactions, we devised a microfluidic system to visualize parasites interacting with vascular endothelium under conditions similar to those found in the bloodstream. Interestingly, parasite migration was significantly influenced by the mechanical force of shear flow. Furthermore, we identified a role for the parasite surface protein MIC2 in the initial phase of adhesion. Our study is the first to document T. gondii interactions with endothelium under shear stress conditions and provides a foundation for future studies on the molecules that mediate parasite interaction with the vasculature.

Project description:UnlabelledKey to the virulence of apicomplexan parasites is their ability to move through tissue and to invade and egress from host cells. Apicomplexan motility requires the activity of the glideosome, a multicomponent molecular motor composed of a type XIV myosin, MyoA. Here we identify a novel glideosome component, essential light chain 2 (ELC2), and functionally characterize the two essential light chains (ELC1 and ELC2) of MyoA in Toxoplasma. We show that these proteins are functionally redundant but are important for invasion, egress, and motility. Molecular simulations of the MyoA lever arm identify a role for Ca(2+) in promoting intermolecular contacts between the ELCs and the adjacent MLC1 light chain to stabilize this domain. Using point mutations predicted to ablate either the interaction with Ca(2+) or the interface between the two light chains, we demonstrate their contribution to the quality, displacement, and speed of gliding Toxoplasma parasites. Our work therefore delineates the importance of the MyoA lever arm and highlights a mechanism by which this domain could be stabilized in order to promote invasion, egress, and gliding motility in apicomplexan parasites.ImportanceTissue dissemination and host cell invasion by apicomplexan parasites such as Toxoplasma are pivotal to their pathogenesis. Central to these processes is gliding motility, which is driven by an actomyosin motor, the MyoA glideosome. Others have demonstrated the importance of the MyoA glideosome for parasite motility and virulence in mice. Disruption of its function may therefore have therapeutic potential, and yet a deeper mechanistic understanding of how it works is required. Ca(2+)-dependent and -independent phosphorylation and the direct binding of Ca(2+) to the essential light chain have been implicated in the regulation of MyoA activity. Here we identify a second essential light chain of MyoA and demonstrate the importance of both to Toxoplasma motility. We also investigate the role of Ca(2+) and the MyoA regulatory site in parasite motility and identify a potential mechanism whereby binding of a divalent cation to the essential light chains could stabilize the myosin to allow productive movement.

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Time-lapse video microscopy of gliding motility in Toxoplasma gondii reveals a novel, biphasic mechanism of cell locomotion.

Publications

Time-lapse video microscopy of gliding motility in Toxoplasma gondii reveals a novel, biphasic mechanism of cell locomotion.

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