Characterization of gamma-tubulin filaments in mammalian cells

Abstract

Overexpression of γ-tubulin leads to the formation of filaments, but nothing is known about such filaments with regard to possible presence in cells, structure and probable dynamics. Here, we used mammalian cell lines to investigate the ability of γ-tubulin to form filaments. We found that γ-tubulin produces fibers called γ-tubules in a GTP-dependent manner and that γ-tubules are made up of pericentrin and the γ-tubulin complex proteins 2, 3, 5 and 6. Furthermore, we noted that the number of cells with cytosolic γ-tubules is increased in non-dividing cells. Our experiments showed that γ-tubules are polar structures that have a low regrowth rate compared to microtubules. Also, we observed that γ-tubules were disassembled by treatment with cold, colcemid, citral dimethyl acetal, dimethyl fumarate or mutation of γ-tubulin GTPase domain, but were increased in number by treatment with taxol or by stable expression of the γ-tubulin GTPase domain. Our results demonstrate that γ-tubulin forms filaments, and such assembly is facilitated by the GTPase domain of γ-tubulin.

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.…”

“…Human pEGFP-γ-tubulin 334–449 (γ-tubulin (334–449)), pEGFP- sh-resistant TUBG1 gene, pEGFP-A13-γ-tubulin resist , and γTubulin shRNA were prepared as previously described 4, 11, 14 . pSpCas9(BB)-2A-GFP γTubulin sgRNA, and pcDNA3-sg-resistant TUBG1 gene were prepared as previously reported 8 . The γ-tubulin fragment γ-tubulin (336-451) was prepared using a Quickchange Mutagensis Kit (Stratagene) and the primers listed in Supplementary Table 3.…”

“…To remove possible cytoskeletal elements attached to mitochondria, cells were pre-incubated for 15 min with culture medium containing 100 ng/ml colcemid and 5 µg/ml cytochalasin B (37 °C, 5% CO 2 ) 4, 5, 8 . Thereafter, purification of the mitochondrial fraction by a Percoll density gradient centrifugation was prepared as described elsewhere 45, 46 , with the following modifications.…”

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

“…At the cellular level, simultaneous knockdown of both γ-TUBULIN genes is lethal. 32 – 34 γ-TUBULIN 1 knockout mice survive only to the morula/blastocyst stage because redundant function is provided by the activation of γ-TUBULIN 2 during the first stage of embryonic development. 33 In contrast, γ-TUBULIN 2 knockout mice are viable and fertile.…”

“… 34 In contrast to γ-strings, which are static structures, γ-tubules are temperature-sensitive polar structures that vary both in size and location and can emanate from centrosomes. 34 γ-tubules differ from microtubules in that they consist of pericentrin and γTURCs, and they lack α- and β-tubulin heterodimers. 34 Other γ-tubulin-rich structures include the centrosomes.…”