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bioRxiv · 10.1101/2025.10.02.679852

Molecular features of Myosin F adapted for driving actin flows in Toxoplasma gondii

Sladewski, T. E.·Heaslip, A. T.
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Abstract

Toxoplasma gondii (T. gondii) is a single-celled Apicomplexan parasite that relies on a highly polarized endomembrane system for its invasion into and survival within host cells. Recent advancements in imaging technologies have revealed that vesicle transport and organization of organelles in the endomembrane pathway requires a highly dynamic actin cytoskeleton. These dynamics in turn rely on the activity of Myosin F (MyoF), a molecular motor unique to Alveolates. The defining characteristic of this molecular motor is a WD40 beta-propeller domain, exclusively found in this class of myosin. To understand the mechanism by which MyoF controls the dynamics and organization of actin, we studied the biophysical properties of the purified motor in vitro. A MyoF construct lacking its WD40 tail domain (MyoF{Delta}tail) is dimeric and can bind and translocate actin in an in vitro motility assay. Single molecule studies show that the dimeric construct is non-processive however small ensembles move inefficiently on single filaments of skeletal actin. In contrast, single molecules of the full-length motor move processively on Toxoplasma actin and jasplakinolide-stabilized skeletal actin bundles. Electron microscopy of negatively stained images of MyoF and quantitative size exclusion chromatography shows that the WD40 domain oligomerizes to form a complex containing multiple dimeric molecules, which provides an explanation for why the full-length motor is processive compared to the dimeric MyoF{Delta}tail construct. Finally, we show that MyoF binds microtubules through its WD40 domain and can slide actin filaments relative to microtubules. We propose a model whereby MyoF oligomers drive actin dynamics by translocating filaments relative to the parasites cytoskeleton. These molecule features provide new insight into how MyoF functions in the cell to regulate actin organization during vesicle transport.

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Sladewski, T. E., Heaslip, A. T.. 2025-10-03. Molecular features of Myosin F adapted for driving actin flows in Toxoplasma gondii. https://doi.org/10.1101/2025.10.02.679852

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