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

Abstract

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 TpCCTα, TpCCTδ, TpCCTε, and TpCCTη-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 TpCCTδ 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/globularstructures 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.

Revisiting the tubulin folding pathway: new roles in centrosomes and cilia

“…In agreement, in the ciliate Tetrahymena pyriformis, the CCTa, CCT´, CCTd and CCTh subunits localize in basal bodies, the oral apparatus, and contractile vacuole pores (68), all structures known to nucleate/organize microtubules (69). In plants, the nuclear surface actively supports microtubule outgrowth and the CCT´subunit was found at the nuclear envelope of maize coleoptiles (70).…”

“…For example, many CCT interacting proteins contain WD40 domains wfor review see (40)x which are also present in a variety of centrosomal proteins such as the p80 subunit of the microtubule severing protein katanin (71); the centrosomal protein POC1, involved in ciliogenesis and in centriole duplication, stability and length control (72,73); and NEDD1/GCP-WD, a g-tubulin ring complex component (74). CCT subunits, either as free entities or as components of microcomplexes (75), have also been implicated in the regulation of cytoskeleton assembly and dynamics (66,68,76,77), supporting the concept that their function extends beyond the folding assistance or the maintenance of correct tubulin heterodimers concentration.…”

“…Accordingly, CCTa is found in rabbit tracheal cilia and is recruited to cilia during sea urchin embryonic cilia regeneration (93). By contrast, reciliation affects the oligomeric state of CCT subunits with tubulin being preferentially associated with smaller CCT oligomeric species in early reciliation stages (68). A recent study showed that the knockout of CCTa or CCTd in the ciliate Tetrahymena thermophila caused a loss of cell body microtubules, failure to assemble new cilia and cell death (94).…”

Multigenerational effects of tris(1,3-dichloro-2-propyl) phosphate on the free-living ciliate protozoa Tetrahymena thermophila exposed to environmentally relevant concentrations and after subsequent recovery

“…Furthermore, expressions of ten genes were also examined in this study. The ten genes are included in two functional classes and are responsible for assembly and maintenance of cilia (Seixas et al, 2003(Seixas et al, , 2010Avasthi and Marshall, 2012;Lechtreck, 2015). Exposure to TDCIPP significantly up-regulated expressions of all ten genes, and thus the up-regulation was considered as a feedback response since number of cilia and depth and diameter of basal body of cilia were decreased.…”

“…wi.mit.edu) (see Table S1). Expressions of ten genes, which are responsible for assembly and maintenance of cilia (Seixas et al, 2003(Seixas et al, , 2010Avasthi and Marshall, 2012;Lechtreck, 2015), were examined. Expressions of three genes (chlamydial polymorphic outer membrane protein repeat containing protein, ubiquitin carboxyl-terminal hydrolase family protein and Cysteine proteinase 3 precursor were not changed upon exposure to TDCIPP so they were used as housekeeping genes to normalize for variations among quantifications.…”