γ-Tubulin has a conserved intrinsic property of self-polymerization into double stranded filaments and fibrillar networks

Abstract

γ-Tubulin is essential for microtubule nucleation and also plays less understood roles in nuclear and cell-cycle-related functions. High abundancy of γ-tubulin in acentrosomal Arabidopsis cells facilitated purification and biochemical characterization of large molecular species of γ-tubulin. TEM, fluorescence, and atomic force microscopy of purified high molecular γ-tubulin forms revealed the presence of linear filaments with a double protofilament substructure, filament bundles and aggregates. Filament formation from highly purified γ-tubulin free of γ-tubulin complex proteins (GCPs) was demonstrated for both plant and human γ-tubulin. Moreover, γ-tubulin associated with porcine brain microtubules formed oligomers. Experimental evidence on the intrinsic ability of γ-tubulin to oligomerize/polymerize was supported by conservation of α- and β-tubulin interfaces for longitudinal and lateral interactions for γ-tubulins. STED (stimulated emission depletion) microscopy of Arabidopsis cells revealed fine, short γ-tubulin fibrillar structures enriched on mitotic microtubular arrays that accumulated at polar regions of acentrosomal spindles and the outer nuclear envelope before mitosis, and were also present in nuclei. Fine fibrillar structures of γ-tubulin representing assemblies of higher order were localized in cell-cycle-dependent manner at sites of dispersed γ-tubulin location in acentrosomal plant cells as well as at sites of local γ-tubulin enrichment after drug treatment. Our findings that γ-tubulin preserves the capability of prokaryotic tubulins to self-organize into filaments assembling by lateral interaction into bundles/clusters help understanding of the relationship between structure and multiple cellular functions of this protein species and suggest that besides microtubule nucleation and organization, γ-tubulin may also have scaffolding or sequestration functions.

The GTPase domain of gamma-tubulin is required for normal mitochondrial function and spatial organization

“…We and others have recently reported that γ-tubulin forms a cellular meshwork of γ-strings 5–7 and γ-tubules 8 . While γ-tubules are polar cytosolic filaments within the γ-string meshwork, γ-strings are detected in both the cytoplasm and the nucleus and are formed of non-polar protein threads that cross the double membrane of the nuclear envelope.…”

γ-tubulin as a signal-transducing molecule and meshwork with therapeutic potential

“…Part of the cellular pool is folded into γ-tubulin threads called γ-strings by the type II chaperonin CCT. 36 γ-Strings are 4–6 nm in diameter 23 , 28 , 36 , 37 and span from the cytosolic compartment through the nuclear membrane and into the chromatin. A γ-string is very thin and alone can offer little stability.…”

γ-Tubulin–γ-Tubulin Interactions as the Basis for the Formation of a Meshwork

“…A number of studies in recent years have broadened this view of γ-tubulin [ 9 , 10 , 11 , 14 , 23 , 24 , 25 , 26 , 27 , 28 , 39 , 40 , 41 , 42 , 43 ]. In some cell lines and primary cells, a pool of γ-tubulin in the nuclear compartment [ 27 , 44 , 45 ] has been detected and found to be associated with Rad51, C53, GCP2, GCP3, E2F1, and lamin B1 [ 23 , 24 , 25 , 26 , 27 , 28 ].…”

“…An in vivo analysis of zebrafish embryos and in vitro experiments have shown that part of the cellular γ-tubulin pool is folded into γ-strings by the chaperonin CCT [ 39 ]. Experiments on the plant Arabidopsis thaliana have confirmed and further clarified the structure of γ-strings [ 43 ]. γ-Strings are 4 to 6 nm in diameter [ 14 , 28 , 39 , 43 ] and, in animal cells, span from the cytosolic compartment through the membranes and into the chromatin [ 14 , 28 ].…”