Investigations of the assembly and function of the Toxoplasma micropore

*Toxoplasma gondii* is an apicomplexan parasite that can invade all warm-blooded animals. One of the characteristic features of this parasite is its pellicle, a complex three-layer membrane that includes flattened vesicles that run the entire parasite. *Toxoplasma*’s pellicle has conferred the parasite protection, cell shape, and motility, which is important for the parasite’s host invasion and survival. However, this pellicle has created a barrier to material exchange processes such as endocytosis. This has raised the question of how *Toxoplasma* and other members of the same group perform endocytosis with this extra set of membranes. *Toxoplasma*’s endocytosis is not fully understood. The first insights about this process started with the electron-microscopy images of pore-like plasma membrane invaginations with electro-dense material around it, called micropores. Therefore, it was suggested that *Toxoplasma* would have clathrin-dependent endocytosis through this pore. Later studies proved that *Toxoplasma* ingests material from its environment in intra and extracellular stages. Nevertheless, these studies did not necessarily provide evidence that this material uptake was via endocytosis, nor that it happened through the micropores. This thesis has studied the *Toxoplasma* micropore and its relation with endocytosis. The *Toxoplasma* micropore is a fixed structure present in 2-3 dedicated pits in each cell. It is located closer to the anterior end of the parasite, probably due to its early synthesis during daughter cell assembly. Its molecular composition included known endocytosis proteins such as the AP2 adaptor complex, EPS15, and a dynamin-related protein (DrpC). It also included other proteins implicated in endocytosis in other apicomplexan parasites, such as UBP1 and K13. The micropore is an important part of the parasite’s pellicle since the recruitment and assembly of its components start during early budding, and the depletion of some of these components leads to pellicle disruptions. The molecular composition and endocytosis assays developed by collaborators of the Waller lab, conclude that the micropore is the site of endocytosis in *Toxoplasma*. *Toxoplasma*’s endocytosis occurs through a fixed structure that is part of the parasite’s pellicle. Growth plaque assays proved that endocytosis is essential for the parasite’s survival, and depletion of some of its components (K13 and ISAP1) leads to pellicle ruptures. Replication assays showed that endocytosis is not essential for the parasite’s nutrition. Nevertheless, the inhibition of endocytosis led to failure in egress. Phenotypic observations of endocytosis-inhibited parasites provided evidence about the loss of the rosette-like intracellular organisation and plasma membrane-bound cytosolic extensions. These observations suggest that the main use of endocytosis in *Toxoplasma* is plasma membrane homeostasis. Overall, this thesis has provided evidence about the *Toxoplasma* micropore, its assembly, and the importance of endocytosis in this apicomplexan parasite.

Name dc:type.qualificationname

Doctor of Philosophy (PhD)

Level dc:type.qualificationlevel

Doctoral

Grantor dc:publisher.institution

University of Cambridge

Year dc:date.issued

2023

Author dc:creator

• Mercado Saavedra, Brandon

Advisor dc:contributor.advisor

• Waller, Ross

• Endocytosis
• K13
• Micropore
• Toxoplasma

Licence

• https://www.repository.cam.ac.uk/bitstreams/87dd0c4a-153e-49a5-86a1-0c9bee5dbdba/download
• http://purl.org/NET/rdflicense/allrightsreserved

Language dc:language

eng

DOI dc:identifier.doi

https://doi.org/10.17863/CAM.107384

OAI identifier oai:identifier

oai:www.repository.cam.ac.uk:1810/366481