Intracellular Cargo Transport in Toxoplasma Gondii

University Of Connecticut Storrs, Storrs-Mansfield CT

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Abstract

PROJECT SUMMARY Intracellular cargo transport is a vital and ubiquitous cellular process that occurs in all eukaryotic cells. However, the mechanisms of cargo transport have only been extensively investigated in a small number of “model” species, leaving a large knowledge gap in our understanding of the mechanisms in other eukaryotic groups. Studies in a wider range of species will ultimately lead to a more comprehensive understanding of the mechanisms, functions and evolutionary origins of these cellular processes. The overall goal of this proposal is to characterize how the actin cytoskeleton controls cargo trafficking in Apicomplexan parasites, a large phylum that contains over 5,000 species, many of which have significant medical relevance, using Toxoplasma gondii as a model organism. T. gondii is a genetically tractable organism with straight-forward cell culture and amenable to live-cell and super- resolution microscopy, so it is well suited for the proposed studies. We will employ a range of intradisciplinary methods including parasite cell biology and genetics, live cell microscopy and in vitro single molecule biophysics to study this cellular process. This study builds on previous data demonstrating that the organization of the endomembrane system in T. gondii is regulated by filamentous actin and an unconventional myosin motor, MyoF. Of relevance for the current study, we showed that vesicle formation at the trans-Golgi network and vesicle transport are dependent on this acto-myosin system, despite the parasite having a simplified repertoire of actin- binding proteins (ABPs) and is missing several conserved actin regulators, including Arp2/3 complex, WASP- family proteins and bundling proteins, such as fascin. Three lines of evidence strongly indicate that MyoF and actin execute these cellular processes using a novel mechanism of action: (1) In vitro studies of actin’s polymerization properties show that this actin isoform is tuned for rapid disassembly and turnover; (2) Live cell imaging demonstrated that F-actin in the parasite cytosol is highly dynamic and undergoes continual rearrangement and does not to form stable tracks on which MyoF could transport cargo; and (3) MyoF does not associate directly with membrane-bound cargo. Instead, MyoF is an organizer of the actin cytoskeleton and loss of this protein results in the aberrant accumulation of actin adjacent to the Golgi. Based on this data, we propose that both vesicle formation and transport are powered by a membrane-associated actin-nucleator and that MyoF and other newly identified actin bundling proteins control actin organization. We will test this model by creating inducible knockdown parasite lines, and defining the roles of these ABPs in vesicle formation and transport, and actin organization. We will reconstitute the activity of these proteins in vitro and determine how each protein influences actin filament polymerization and bundling, both individually and in combination. Defining how this actin isoform, with novel polymerization properties and a reduced set of ABP’s, mediates protein trafficking will be highly valuable in characterizing protozoan specific aspects of this cellular process and will lead to a more comprehensive understanding of general eukaryotic cargo transport mechanisms.