Biology subjects

Heaslip, A. T.

Publications and source records attributed to Heaslip, A. T..

7 recordsLinked to original sources

bioRxiv2026

TGIF is a golgin-like protein required for Golgi structural maintenance and function in Toxoplasma gondii

The Golgi is an essential organelle that serves as a central hub for endomembrane trafficking. In the protozoan parasite Toxoplasma gondii, a single Golgi stack is essential for parasite survival; however, the molecular determinants governing Golgi structure and function remain poorly understood. Here, we characterize a Golgi-associated protein that is required for Golgi integrity and function, which we named Toxoplasma Golgi Integrity Factor (TGIF). Loss of TGIF disrupts parasite replication and natural egress and is lethal to the parasite. To investigate the impact of TGIF depletion on secretory protein trafficking, we adapted a fluorescence-based pulse-chase assay to monitor the synthesis and trafficking of microneme and rhoptry proteins. We found that loss of TGIF significantly impaired the synthesis and trafficking of microneme and rhoptry neck proteins, whereas trafficking of rhoptry bulb proteins was minimally affected. These findings suggest that rhoptry bulb proteins may traffic independently of canonical Golgi-dependent pathways. Collectively, our study provides new insight into the mechanisms of Golgi-mediated trafficking in T. gondii and identifies TGIF as a critical regulator of parasite secretory pathway organization and function.

cell biology↗

bioRxiv2025

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

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.

biophysics↗

bioRxiv2025

An apicoplast localized GTPase is essential for Toxoplasma gondii survival

The apicoplast is an essential organelle found in Apicomplexa, a large phylum of intracellular eukaryotic pathogens. The apicoplast produces metabolites that are utilized for membrane biogenesis and energy production. A majority of apicoplast resident proteins are encoded by the nuclear genome and are trafficked to the apicoplast, and are referred to as Nuclear Encoded and Apicoplast Targeted (NEAT) proteins. In this study, we characterized a NEAT protein named TgBipA, that is a homolog of the highly conserved prokaryotic translational GTPase BipA. BipA is essential for bacterial survival in stress conditions and functions through interactions with the prokaryotic ribosome, although its role is not fully understood. Through genetic knockouts of TgBipA and immunofluorescence imaging we show that loss of TgBipA results in apicoplast genome replication defects, disruption of NEAT trafficking, loss of the apicoplast, and subsequently parasite death. Furthermore, we show through comparative studies that this phenotype closely resembles the delayed death phenomenon observed when inhibiting apicoplast translation. Finally, we show that TgBipA is an active GTPase in vitro, and its GTP hydrolysis activity is critical for its cellular function. Our findings demonstrate TgBipA is a GTPase which has an essential role in apicoplast maintenance, providing new insight into the cellular processes of the organelle. Importance StatementToxoplasma gondii, and many other parasites in the phylum Apicomplexa, are pathogens with significant medical and veterinary importance. Most Apicomplexa contain a non-photosynthetic plastid organelle named the apicoplast. This organelle produces essential metabolites and perturbation of apicoplast function results in parasite death. The apicoplast contains bacterial-like pathways for apicoplast genome replication and expression. Thus, the discovery of the apicoplast lead to optimism that this organelle would provide a wealth of anti-parasitic drug targets. Therefore, the identification and characterization of new apicoplast proteins could provide new opportunities for therapeutic development. In this study we characterized the function of a protein called TgBipA, a homolog of a highly conserved bacterial GTPase bipA which has been implicated in maturation of the 50S ribosomal subunit and adaptation to cellular stress. We show that TgBipA is essential for apicoplast maintenance and parasite survival.

microbiology↗

bioRxiv2023

Toxoplasma gondii actin filaments are tuned for rapid disassembly and turnover

The cytoskeletal protein actin plays a critical role in the pathogenicity of Toxoplasma gondii, mediating invasion and egress, cargo transport, and organelle inheritance. Advances in live cell imaging have revealed extensive filamentous actin networks in the Apicomplexan parasite, but there is conflicting data regarding the biochemical and biophysical properties of Toxoplasma actin. Here, we imaged the in vitro assembly of individual Toxoplasma actin filaments in real time, showing that native, unstabilized filaments grow tens of microns in length. Unlike skeletal muscle actin, Toxoplasma filaments intrinsically undergo rapid treadmilling due to a high critical concentration, fast monomer dissociation, and rapid nucleotide exchange. Cryo-EM structures of stabilized and unstabilized filaments show an architecture like skeletal actin, with differences in assembly contacts in the D-loop that explain the dynamic nature of the filament, likely a conserved feature of Apicomplexan actin. This work demonstrates that evolutionary changes at assembly interfaces can tune dynamic properties of actin filaments without disrupting their conserved structure.

biophysics↗

bioRxiv2023

F-actin and Myosin F control apicoplast elongation dynamics which drive apicoplast-centrosome association in Toxoplasma gondii

Toxoplasma gondii contains an essential plastid organelle called the apicoplast that is necessary for fatty acid, isoprenoid, and heme synthesis. Perturbations affecting apicoplast function or inheritance lead to parasite death. The apicoplast is a single copy organelle and therefore must be divided so that each daughter parasite inherits an apicoplast during cell division. In this study we identify new roles for F-actin and an unconventional myosin motor, TgMyoF, in this process. First, loss of TgMyoF and actin lead to an accumulation of apicoplast vesicles in the cytosol indicating a role for this actomyosin system in apicoplast protein trafficking or morphological integrity of the organelle. Second, live cell imaging reveals that during division the apicoplast is highly dynamic, exhibiting branched, U-shaped and linear morphologies that are dependent on TgMyoF and actin. In parasites where movement was inhibited by the depletion of TgMyoF, the apicoplast fails to associate with the parasite centrosomes. Thus, this study provides crucial new insight into mechanisms controlling apicoplast-centrosome association, a vital step in the apicoplast division cycle, which ensures that each daughter inherits a single apicoplast.

cell biology↗

bioRxiv2023

Trafficking of nuclear-encoded apicoplast proteins depends on F-actin and Myosin F in Toxoplasma gondii

cell biology↗

bioRxiv2020

Actin and an unconventional myosin motor, TgMyoF, control the organization and dynamics of the endomembrane network in Toxoplasma gondii.

Toxoplasma gondii is an obligate intracellular parasite that relies on three distinct secretory organelles, the micronemes, rhoptries and dense granules, for parasite survival and disease pathogenesis. Secretory proteins destined for these organelles are synthesized in the endoplasmic reticulum (ER) and sequentially trafficked through a highly polarized endomembrane network that consists of the Golgi and multiple post-Golgi compartments. Currently, little is known about how the parasite cytoskeleton controls the positioning of the organelles in this pathway, or how vesicular cargo is trafficked between organelles. Here we show that F-actin and an unconventional myosin motor, TgMyoF, control the dynamics and organization of the organelles in the secretory pathway, specifically ER tubule movement, apical positioning of the Golgi and post-Golgi compartments, apical positioning of the rhoptries and finally, the directed transport of Rab6-positive and Rop1-positive vesicles. Thus, this study identifies TgMyoF and actin as the key cytoskeletal components that organize the endomembrane system in T. gondii. Author SummaryEndomembrane trafficking is a vital cellular process in all eukaryotic cells. In most cases the molecular motors myosin, kinesin and dynein transport cargo including vesicles, organelles and transcripts along actin and microtubule filaments in a manner analogous to a train moving on its tracks. For the unicellular eukaryote Toxoplasma gondii, the accurate trafficking of proteins through the endomembrane system is vital for parasite survival and pathogenicity. However, the mechanisms of cargo transport in this parasite are poorly understood. In this study, we fluorescently labeled multiple endomembrane organelles and imaged their movements using live cell microscopy. We demonstrate that filamentous actin and an unconventional myosin motor named TgMyoF control both the positioning of organelles in this pathway and the movement of transport vesicles throughout the parasite cytosol. This data provides new insight into the mechanisms of cargo transport in this important pathogen and expands are understanding of the biological roles of actin in the intracellular phase of the parasites growth cycle.

cell biology↗