Cytoskeletal Membrane Dynamics - Mark A. McNiven

Dynamin 2 Binds γ-tubulin and Participates in Centrosome Cohesion

Nature Cell Biology, Volume 6, Number 4, APRIL 2004 pp335-342

Heather M. Thompson, Hong Cao, Jing Chen, Ursula Euteneuer and Mark A. McNiven

Dynamin 2 (Dyn2) is a large GTPase involved in vesicle formation and actin reorganization1–3. 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. (a–i) FR cells were costained for endogenous Dyn2 using either the antipan- dynamin antibody MC-63 (a) or the anti-Dyn2 antibody (d, g) and the centriolar protein centrin (b, e) or the centrosomal protein γ-tubulin (h). Overlays are also shown (c, f, i). Insets in c, f and i show enlargements of the boxed centrosomal regions. Note that Dyn2 (green) is aside the centrin-positive spots (red, f), whereas in i there is extensive colocalization between Dyn2 (green) and γ-tubulin (red). Arrows indicate the centrosomal localization of Dyn2, centrin and γ-tubulin. (j–l) HeLa cells were fixed and processed for immunoelectron microscopy using the MC-65 anti-pan-dynamin antibody (j, k) or pre-immune serum (l) as a control. In j, a lower-magnification electron micrograph of a mitotic cell shows numerous 10-nm gold particles (arrows) in the pericentriolar material, as well as on the centrioles themselves. In k, a higher magnification image of centrioles in an interphase cell shows that Dyn2 localizes along the length and at the ends of the triplet microtubules that compose the centrioles (arrows). In l, very few gold particles were present on centrioles from interphase cells stained with pre-immune serum from an MC-65 peptide-injected rabbit. Scale bars represent 10 μm in a–i and 200 nm in j–l.

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Figure 4 Centrosome splitting is induced in Dyn2 siRNA-treated cells. Treatment of FR cells over 96 h with scrambled siRNA (a) or Dyn2 siRNA (b). Splitting of centrosomes was detected in b, but not in a, as shown by γ-tubulin staining. Insets show enlargements of boxed regions of γ-tubulin staining, emphasizing the splitting of centrosomes in Dyn2 siRNA-treated cells. (c) Quantification of the distance between centrosomes in controltreated (n = 543) and Dyn2 siRNA-treated (n = 471) cells after at least 96 h indicated a decrease in the percentage of cells containing centrosomes separated by less than 1 μm and a subsequent increase in the percentage of cells containing centrosomes separated by more than 3 μm, when comparing Dyn2 siRNA-treated cells to control-treated cells. The separation in centrosomes could be partially rescued by expressing Dyn2-Mid–GFP while treating cells with Dyn2 siRNA (n = 211). Centrosome separation of more than 1 μm was also noted in cells microinjected with the antipan- dynamin antibody MC-63 (n = 343). Error bars indicate the standard deviation of three experiments. Scale bar for a and b represents 10 μm.