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

摘要

gamma-Tubulin is essential for microtubule nucleation and also plays less understood roles in nuclear and cell-cycle-related functions. High abundancy of gamma-tubulin in acentrosomal Arabidopsis cells facilitated purification and biochemical characterization of large molecular species of gamma-tubulin. TEM, fluorescence, and atomic force microscopy of purified high molecular gamma-tubulin forms revealed the presence of linear filaments with a double protofilament substructure, filament bundles and aggregates. Filament formation from highly purified gamma-tubulin free of gamma-tubulin complex proteins (GCPs) was demonstrated for both plant and human gamma-tubulin. Moreover, gamma-tubulin associated with porcine brain microtubules formed oligomers. Experimental evidence on the intrinsic ability of gamma-tubulin to oligomerize/polymerize was supported by conservation of alpha- and beta-tubulin interfaces for longitudinal and lateral interactions for gamma-tubulins. STED (stimulated emission depletion) microscopy of Arabidopsis cells revealed fine, short gamma-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 gamma-tubulin representing assemblies of higher order were localized in cell-cycle-dependent manner at sites of dispersed gamma-tubulin location in acentrosomal plant cells as well as at sites of local gamma-tubulin enrichment after drug treatment. Our findings that gamma-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, gamma-tubulin may also have scaffolding or sequestration functions.