A family of intermediate filament-like proteins is sequentially assembled into the cytoskeleton of Toxoplasma gondii

Abstract

Summary The intracellular protozoan parasite Toxoplasma gondii divides by a unique process of internal budding that involves the assembly of two daughter cells within the mother. The cytoskeleton of Toxoplasma, which is composed of microtubules associated with an inner membrane complex (IMC), has an important role in this process. The IMC, which is directly under the plasma membrane, contains a set of flattened membranous sacs lined on the cytoplasmic side by a network of filamentous proteins. This network contains a family of intermediate filament-like proteins or IMC proteins. In order to elucidate the division process, we have characterized a 14-member sub-family of Toxoplasma IMC proteins that share a repeat motif found in proteins associated with the cortical alveoli in all alveolates. By creating fluorescent protein fusion reporters for the family members we determined the spatio-temporal patterns of all 14 IMC proteins through tachyzoite development. This revealed several distinct distribution patterns and some provide the basis for novel structural models such as the assembly of certain family members into the basal complex. Furthermore we identified IMC15 as an early marker of budding and, lastly, the dynamic patterns observed throughout cytokinesis provide a timeline for daughter parasite development and division.

The Toxoplasma gondii centrosome is the platform for internal daughter budding as revealed by a Nek1 kinase mutant

“…YFP tagging plasmids are based on the ptub-YFP 2 (MCS)/sagCAT plasmid (Gubbels et al, 2003;Anderson-White et al, 2011) and were generated by replacing modular segment, either the a-tubulin promoter (PmeI/BglII), the first YFP (BglII/AvrII), the second YFP (AvrII/EcoRV) or the entire tandem YFP cassette (BglII/EcoRV). The sagCATsag cassette was replaced by the sagBLEsag cassette (van Dooren et al, 2008) by XhoI digest swapping.…”

Emerging roles for protein S-palmitoylation in Toxoplasma biology

“…The IMC family is composed of 14 members in T. gondii and none of them has a predicted TMD, but 10 have putative palmitoylation sites that probably anchor the proteins to the cytoplasmic face of the IMC (Anderson-White et al, 2011). Consistent with these predictions, PfALV4 and PfALV5, corresponding to TgIMC4 and TgIMC15, respectively, were identified in the palmitome of P. falciparum (Jones et al, 2012).…”

“…The cytoplasmic face of the IMC is intimately associated with the basket of subpellicular microtubules that run along the apical two-thirds of the parasite length (Morrissette et al, 1997) through a network of intermediate-like filaments formed by alveolin-containing proteins (named ALVs or IMCs) that are conserved in apicomplexans (Mann and Beckers, 2001;Gould et al, 2008;Anderson-White et al, 2011). The IMC family is composed of 14 members in T. gondii and none of them has a predicted TMD, but 10 have putative palmitoylation sites that probably anchor the proteins to the cytoplasmic face of the IMC (Anderson-White et al, 2011).…”

“…TgIMC11 is restricted to the apical cap, whereas a group of four IMCs are targeted to the basal complex (Anderson-White et al, 2011). For two IMCs that do not contain predicted palmitoylation sites, it is clear that their alveolin repeats are the main but indirect targeting determinants, possibly interacting with a palmitoylated IMC protein (Anderson-White et al, 2011). The targeting of these IMC proteins is tightly regulated in time and space and palmitoylation might play a central role in this orchestration.…”

Targeting Toxoplasma Tubules: Tubulin, Microtubules, and Associated Proteins in a Human Pathogen

“…Following centriole separation, several centriole-associated proteins relocate to emerging daughters. RNG2 shifts from the centrioles to the APR (89), and both IMC15 and Tg14-3-3 move from the centrioles to the IMC of emergent daughters (81,105). Although the timing of bud formation in endodyogeny versus that in endopolygeny is distinct, the cytoskeletal components are likely to be quite similar, with assembly regulated by initiation of key structures, such as the conoid, APR, IMC, and underlying microtubules (69,127).…”