Interactions between the actin filament capping and severing protein gelsolin and the molecular chaperone CCT: evidence for nonclassical substrate interactions.

CCT is a member of the chaperonin family of molecular chaperones and consists of eight distinct subunit species which occupy fixed positions within the chaperonin rings. The activity of CCT is closely linked to the integrity of the cytoskeleton as newly synthesized actin and tubulin monomers are dependent upon CCT to reach their native conformations. Furthermore, an additional role for CCT involving interactions with assembling/assembled microfilaments and microtubules is emerging. CCT is also known to interact with other proteins, only some of which will be genuine folding substrates. Here, we identify the actin filament remodeling protein gelsolin as a CCT-binding partner, and although it does not behave as a classical folding substrate, gelsolin binds to CCT with a degree of specificity. In cultured cells, the levels of CCT monomers affect levels of gelsolin, suggesting an additional link between CCT and the actin cytoskeleton that is mediated via the actin filament severing and capping protein gelsolin.

Subunits of the chaperonin CCT interact with F-actin and influence cell shape and cytoskeletal assembly.

The integrity of the cytoskeleton is closely linked to the oligomeric chaperonin containing TCP-1 (CCT) via the folding requirements of actin and tubulin, but the role of CCT in cytoskeletal organization remains unclear. We address this issue by analyzing the effects of targeting CCT subunits via siRNA and assessing their location/assembly state in cultured mammalian cells. Reducing levels of individual CCT subunits implicates CCTepsilon in influencing cell shape and reduced levels of this subunit limit the cells' ability to recover from microfilament depolymerization. Conversely, cells displayed enhanced microtubule regrowth when CCT subunit levels were altered by siRNA. Some CCT subunits co-localize with F-actin, whilst all are predominantly monomeric in extracts enriched for the cytoskeleton. This provides compelling evidence that some CCT subunits as monomers can influence cytoskeletal organization/polymerization. Therefore the activity of CCT may well extend beyond the folding of newly synthesized polypeptides, representing a novel function for CCT subunits distinct from their role in the CCT oligomer.

Activities of the chaperonin containing TCP-1 (CCT): implications for cell cycle progression and cytoskeletal organisation.

The chaperonin containing TCP-1 (CCT) is required for the production of native actin and tubulin and numerous other proteins, several of which are involved in cell cycle progression. The mechanistic details of how CCT acts upon its folding substrates are intriguing: whilst actin and tubulin bind in a sequence-specific manner, it is possible that some proteins could use CCT as a more general binding interface. Therefore, how CCT accommodates the folding requirements of its substrates, some of which are produced in a cell cycle-specific manner, is of great interest. The reliance of folding substrates upon CCT for the adoption of their native structures results in CCT activity having far-reaching implications for a vast array of cellular processes. For example, the dependency of the major cytoskeletal proteins actin and tubulin upon CCT results in CCT activity being linked to any cellular process that depends on the integrity of the microfilament and microtubule-based cytoskeletal systems.

The inter-ring arrangement of the cytosolic chaperonin CCT.

The eukaryotic cytosolic chaperonin CCT (chaperonin containing TCP-1) is the most complex of all chaperonins-an oligomeric structure built from two identical rings, each composed of single copies of eight different subunits. The arrangement of the eight subunits within each ring has been characterised for some time, but the phasing between the two rings remains unknown. Here, three-dimensional reconstructions generated by cryoelectron microscopy of complexes between CCT and either of two different monoclonal antibodies that react specifically with the CCTepsilon and CCTdelta subunits have been used to determine the phasing between the two chaperonin rings. The inter-ring arrangement is such that up/down inter-ring communication always involves two different CCT subunits in all eight positions, and the group of subunits concerned with the initiation and completion of the folding cycle cluster together both in the intra- and inter-ring arrangement. This supports a sequential mechanism of conformational changes between the two interacting rings.

Substantial CCT activity is required for cell cycle progression and cytoskeletal organization in mammalian cells.

The chaperonin CCT hexadecamer is required for the folding of non-native actins and tubulins in eukaryotic cells. Among the consequences of greatly reducing CCT holocomplex levels in human cell lines by siRNA targeting are growth arrest and changes in cell morphology and motility. Less extensive reduction of CCT activity via microinjection of an inhibitory anti-CCT epsilon subunit monoclonal antibody, which alters the rates of substrate processing by CCT in vitro, causes a delay in cell cycle progression through G1/S phase in synchronized Swiss 3T3 cells. The degree of growth arrest strongly correlates with the extent of CCT depletion, indicating that full CCT activity is required for normal cell growth and division. Depletion of CCT does not affect actin polypeptide synthesis but causes a reduction in levels of native actin and perturbation of actin-based cell motility in BE cells. There are no large-scale effects on cytoplasmic protein synthesis or a general heat shock response during periods of low CCT activity.