Role of Cytoskeletal Proteins in Apicoplast Vesicle Trafficking and Apicoplast Division in Toxoplasma gondii

Toxoplasma gondii (T. gondii) contain an essential plastid organelle known as apicoplast which is
necessary for fatty acid, isoprenoid, and heme synthesis, and any perturbations affecting the
apicoplast can lead to delayed death of parasites. Considering its importance in parasite survival,
apicoplast division and inheritance must occur with high accuracy. Apicoplast division is a multistep
process with elongation being one of the key steps wherein the apicoplast doubles in size to
ensure that the daughter parasites acquire a copy of each of the apicoplast. This increase in the size
would require an additional supply of lipids and proteins that need to be trafficked to the apicoplast
presumably through vesicles in a process that is not well studied. Two important cytoskeletal
proteins, actin and myosin F (TgMyoF) are required for the apicoplast division. The goal of this
study is to define the roles of these proteins in the apicoplast division and determine the function
of this actomyosin system in vesicle trafficking to the apicoplast. In this study, the localization of
the nuclear encoded protein, apicoplast phosphate transporter 1 (TgAPT1-EmGFP), a marker for
both the apicoplast and apicoplast-derived vesicles, was tracked throughout the cell cycle.
TgAPT1-EmGFP positive vesicle trafficking was found to be increased specifically during the
elongation phase of the apicoplast division cycle. In the absence of actin and TgMyoF the number of TgAPT1-EmGFP vesicles increased in all stages of the cell cycle, suggesting these cytoskeletal proteins were not involved in vesicle formation but required for the transport and/or fusion of the vesicles with the apicoplast. Live cell imaging of control parasites we showed that the apicoplast is highly dynamic during division and that the loss of actin and TgMyoF reduced the dynamics of the apicoplast. Consequently, TgMyoF knockdown resulted in reduced apicoplast length as well as disruption of the apicoplast and centrosome interaction leading to asymmetric localization of the elongated apicoplast to one of the daughter cells. Overall, this study demonstrates that targeting of nuclear encoded proteins to the apicoplast and apicoplast division are integrally linked cellular processes and that TgMyoF and actin play multiple distinct roles in these processes.