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Dynamin 2 binds γ-tubulin and participates in centrosome cohesion

Abstract

Dynamin 2 (Dyn2) is a large GTPase involved in vesicle formation and actin reorganization1,2,3. In this study, we report a novel role for Dyn2 as a component of the centrosome that is involved in centrosome cohesion. By light microscopy, Dyn2 localized aside centrin and colocalized with γ-tubulin at the centrosome; by immunoelectron microscopy, however, Dyn2 was detected in the pericentriolar material as well as on centrioles. Exogenously expressed green fluorescent protein (GFP)-tagged Dyn2 also localized to the centrosome, whereas glutathione S-transferase (GST)-tagged Dyn2 pulled down a protein complex(es) containing actin, α-tubulin and γ-tubulin from liver homogenate. Furthermore, gel overlay and immunoprecipitation indicated a direct interaction between γ-tubulin and a 219-amino-acid middle domain of Dyn2. Reduction of Dyn2 protein levels with small-interfering RNA (siRNA) resulted in centrosome splitting, whereas microtubule nucleation from centrosomes was not affected, suggesting a role for Dyn2 in centrosome cohesion. Finally, fluorescence recovery after photobleaching (FRAP) analysis of a GFP-tagged Dyn2 middle domain indicated that Dyn2 is a dynamic exchangeable component of the centrosome. These findings suggest a novel function for Dyn2 as a participant in centrosome cohesion.

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Figure 1: Dyn2 localizes to the pericentriolar material and centrioles.
Figure 2: Dyn2 cofractionates and colocalizes with γ-tubulin in centrosomes from rat fibroblasts.
Figure 3: The middle domain mediates Dyn2 centrosomal localization and interacts with γ-tubulin.
Figure 4: Centrosome splitting is induced in Dyn2 siRNA-treated cells.
Figure 5: Dyn2-Mid–GFP is a dynamic component of the centrosome.

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Acknowledgements

The authors thank E.W. Krueger, S.G. Weller and Dr. Y. Yoon for technical advice, and the entire McNiven lab for helpful suggestions and critically reading the manuscript. The authors also thank J.L. Salisbury and S.L. Schmid for anti-centrin and anti-Dyn1 antibodies, respectively, and D.J. Tindall for the scrambled siRNA oligonucleotides.

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Correspondence to Mark A. McNiven.

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Thompson, H., Cao, H., Chen, J. et al. Dynamin 2 binds γ-tubulin and participates in centrosome cohesion. Nat Cell Biol 6, 335–342 (2004). https://doi.org/10.1038/ncb1112

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