| In many species, meiotic spindles assemble microtubules in the absence of centrosomes. An acentrosomal mechanism has been proposed where the chromosomes initiate spindle assembly by nucleating and capturing microtubules. While preliminary evidence has supported this model in Drosophila, very little is actually known about how a bipolar spindle is initiated and maintained in the absence of centrosomes.;RanGTP is a small GTPase which has been shown to be important for chromosome-dependent spindle assembly in Xenopus extracts. I investigated the effect of manipulating the Ran pathway on spindle assembly in Drosophila oocytes and embryos. Surprisingly, I found that RanGTP is not essential for the formation of the acentrosomal spindle near the chromosomes. However, I discovered that RanGTP is essential for completing chromosome-independent microtubule assembly, such as pro-nuclear fusion. Thus, RanGTP may be required for microtubule assembly when chromosomes are absent, but redundant with the chromosomal passenger complex when chromosomes are present.;Another contributor to acentrosomal spindle assembly is Subito, a member of the kinesin 6 superfamily. Subito binds and bundles microtubules at the central spindle in both centrosomal and acentrosomal systems. We have previously shown that expression of Subito with an N-terminus deletion results in the formation of chromosome-independent spindles, or ectopic spindles, in the Drosophila oocyte. By continuing the mutational analysis within the N-terminus, I have further characterized its regulatory role. We have shown that two conserved serines negatively regulate the motor activity of Subito, while other domains within the N-terminus are essential for positive regulation. So while originally believed to be simply a negative regulator, the N-terminus is actually a complex region of negative and positive regulators.;My studies have helped to clarify the requirements for the initiation of acentrosomal spindle assembly in Drosophila. RanGTP is essential for spindle assembly in a chromosome independent manner, suggesting that chromosomal passenger complex is necessary for chromosome dependent microtubule assembly. Furthermore, we suggest a model of auto-inhibition for Subito with the N-terminus interacting with the C-terminus, preventing the binding of microtubules. Together, these results have provided new insights into acentrosomal spindle assembly. |
