Polar organization of gamma-tubulin in acentriolar mitotic spindles of Drosophila melanogastercells

Abstract

The spindle pole localization of gamma-tubulin was compared in wild type and acentriolar cultured Drosophila cells using polyclonal antibodies specifically raised against the carboxy terminal amino acid sequence of Drosophila gamma-tubulin-1 (-KSEDSRSVTSAGS). During inter phase, γ-tubulin was present in the centrosome of wild type cells and accumulated around this organelle in a cell cycle dependent manner. In contrast, no such structure was observed in acentriolar cells. Wild type mitoses were homogeneously composed of biconical spindles, with two centrosome-associated γ-tubulin spots at the poles. The mitotic apparatuses observed in the acentriolar cells were heterogeneous; multipolar mitoses, bipolar mitoses with a barrel-shaped spindle and bipolar mitoses with biconical spindles were observed. In acentriolar cells, γ-tubulin accu mulation at mitotic poles was dependent on spindle micro tubule integrity. Most acentriolar spindles presented a dispersed γ-tubulin labeling at the poles. Only well polarized and biconical acentriolar spindles showed a strong γ-tubulin polar spot. Finally, acentriolar mitotic poles were not organized around true centrosomes. In contrast to wild type cells, in acentriolar cells the Bx63 centrosome-associated antigen was absent and the γ-tubulin containing material dispersed readily following micro tubule disassembly. These observations confirm that γ-tubulin plays an essential role in the nucleation of micro tubules even in the absence of mitotic polar organelles. In addition the data suggest that the mechanisms involved in the bipolarization of wild type and acentriolar mitoses are different, and that centrioles play a role in the spatial organization of the nucleating material containing γ-tubulin.