Measuring Actomyosin Function In A Living Parasite Using A Laser Trap

The pathogenic parasite Toxoplasma gondii invades host cells as part of its life cycle. Forward motion of the parasite during invasion is driven by rearward motion of adhesion receptors through the parasitic plasma membrane. These adhesion receptors are coupled to a unique actin (TgACT1) that forms very short filaments (∼100 nm) for which there is no structure to lend them polarity. These are driven rearward by a fast, single-headed, class XIV myosin (MyoA) that is attached to an inner membrane complex. It is unknown how this system of un-oriented, short actin filaments and membrane-associated myosin can result in directional motility. Here we show that the motile apparatus of Toxoplasma is not pre-organized for directional motion, but rather becomes directional after a period of randomly oriented force generation. A laser trap was used to position microspheres on live Toxoplasma and to measure the transduction of force from TgACT1 and MyoA through cell surface adhesion receptors. We found that a ∼50 second period of randomly oriented bead movement was followed by the force becoming oriented toward the rear (basal end) of the cell. The stall force was only 5.6 pN, and we see occasional series of 5 nm steps that may represent the activity of single MyoA. Force becomes directional at the basal end of the cell approximately 3 seconds later than at the apical end of the cell, but the magnitude of force generation was independent of location on the parasite. Addition of the actin filament stabilizer jasplakinolide abrogated directionality. These data suggest that MyoA is activated in Toxoplasma soon after receptor ligation, but that actin filament dynamics are critical to direction finding, and consequently to the regulation of host cell invasion.