Subunits of the chaperonin CCT are associated with Tetrahymena microtubule structures and are involved in cilia biogenesis

The cytosolic chaperonin CCT is a heterooligomeric complex of about 900 kDa that mediates the folding of cytoskeletal proteins. We observed by indirect immunofluorescence that the Tetrahymena TpCCTalpha, TpCCTdelta, TpCCTepsilon, and TpCCTeta-subunits colocalize with tubulin in cilia, basal bodies, oral apparatus, and contractile vacuole pores. TpCCT-subunits localization was affected during reciliation. These findings combined with atomic force microscopy measurements in reciliating cells indicate that these proteins play a role during cilia biogenesis related to microtubule nucleation, tubulin transport, and/or axoneme assembly. The TpCCT-subunits were also found to be associated with cortex and cytoplasmic microtubules suggesting that they can act as microtubule-associated proteins. The TpCCTdelta being the only subunit found associated with the macronuclear envelope indicates that it has functions outside of the 900 kDa complex. Tetrahymena cytoplasm contains granular/globular-structures of TpCCT-subunits in close association with microtubule arrays. Studies of reciliation and with cycloheximide suggest that these structures may be sites of translation and folding. Combined biochemical techniques revealed that reciliation affects the oligomeric state of TpCCT-subunits being tubulin preferentially associated with smaller CCT oligomeric species in early stages of reciliation. Collectively, these findings indicate that the oligomeric state of CCT-subunits reflects the translation capacity of the cell and microtubules integrity.

Besnoitia besnoiti and Toxoplasma gondii: two apicomplexan strategies to manipulate the host cell centrosome and Golgi apparatus

Besnoitia besnoiti and Toxoplasma gondii are two closely related parasites that interact with the host cell microtubule cytoskeleton during host cell invasion. Here we studied the relationship between the ability of these parasites to invade and to recruit the host cell centrosome and the Golgi apparatus. We observed that T. gondii recruits the host cell centrosome towards the parasitophorous vacuole (PV), whereas B. besnoiti does not. Notably, both parasites recruit the host Golgi apparatus to the PV but its organization is affected in different ways. We also investigated the impact of depleting and over-expressing the host centrosomal protein TBCCD1, involved in centrosome positioning and Golgi apparatus integrity, on the ability of these parasites to invade and replicate. Toxoplasma gondii replication rate decreases in cells over-expressing TBCCD1 but not in TBCCD1-depleted cells; while for B. besnoiti no differences were found. However, B. besnoiti promotes a reorganization of the Golgi ribbon previously fragmented by TBCCD1 depletion. These results suggest that successful establishment of PVs in the host cell requires modulation of the Golgi apparatus which probably involves modifications in microtubule cytoskeleton organization and dynamics. These differences in how T. gondii and B. besnoiti interact with their host cells may indicate different evolutionary paths.