Strategies for analysis of acentrosomal spindle assembly and chromosome behaviour
1.4.1. Analysis of spindle assembly in Xenopus egg extracts
Studies in Xenopus egg extracts have paved the way for models of acentrosomal spindle assembly in animal oocytes. They showed that, in the absence of centrosomes, the bulk chromatin mediates the spindle assembly and that it does not require the kinetochore (see 1.1.2.2.; Heald et al., 1996). A number of experiments revealed the role of RanGTP in this process (Kahana and Cleveland, 1999). Further, research in Xenopus egg extract showed that a combination of motor proteins play a role in establishing the acentrosomal spindle bipolarity (see 1.2.2.; Walczak et al. 1998). The advantage of Xenopus egg extract in the research has been the ability to combine microscopy with biochemical experiments.
Nevertheless, it is not entirely clear if the system represents female meiotic or mitotic conditions without the centrosomes. Xenopus egg extracts are prepared from cytoplasm of oocytes arrested in metaphase II, subjected to mitotic cycles in vitro and then, again arrested by addition of meiosis II extract. Additionally, the spindle formation in Xenopus egg extract has often been studied with the use of introduced DNA-coated beads. Therefore, although Xenopus egg extracts undeniably gave an insight into acentrosomal spindle assembly, they do not directly represent the in vivo process in oocytes. They also did not provide answers towards understanding the mechanisms of chromosome positioning within the acentrosomal spindle.
1.4.2. Analysis of spindle assembly in oocytes
Spindle assembly in oocytes has been investigated in several systems; including Drosophila and mouse (see 1.2.1.). In Drosophila, the analysis has mostly been based on identification of female sterile mutants. In mouse, electron microscopy and fluorescence microscopy of the timely arrangement of division apparatus in the wild type provided an insight into the process. Majority of the studies have used fixed oocytes, but some analysis of live samples has also been performed (Theurkauf and Hawley 1992, Jang et al. 2005; Matthies et al. 1996, Skold et al. 2005; Hughes et al., 2009; Colombie et al., 2008; Brunet et al., 1999; Schuh and Ellenberg, 2007; Kitajima et al., 2011). Together, these studies revealed that chromosomes are the leading factor in spindle assembly and that the motors and non-motor proteins sort microtubules to establish a bipolar spindle structure (see 1.2. and 1.3.). Moreover, they indicated that chromosome positioning within the spindle may involve other major mechanisms of chromosome-microtubule interaction than in mitosis.
Although the general principal of chromosome-mediated spindle assembly in oocytes is similar to what is known in centrosome-free Xenopus egg extract, the molecular bases of the process may differ between these systems. Particularly, RanGTP pathway seems to have different contribution to spindle assembly (see 1.3.1.). In oocytes, CPC may have more important role in this process than RanGTP gradient (see 1.3.1.). However, the exact mechanisms of spindle assembly, as well as chromosome positioning within the assembling spindle in oocytes, remain to be elucidated.
The meiotic division apparatus is greatly understudied comparing to in mitosis. This is primarily due to limited number of mutant alleles of essential genes which are, simultaneously, weak enough to allow development of an organism and strong enough to reveal meiotic function of the gene product. Additionally, antibodies commonly used for immunostaining in mitosis often seem not useful for oocytes due to permeability of the oocytes and fixation methods (personal communications).
The study of meiosis is also restricted by availability of very few live imaging tools. In Drosophila, Matthies et al. (1996) performed first live imaging of acentrosomal spindle assembly using rhodamine-tubulin injected into oocytes before NEB. However, this technique is disruptive to oocytes. Skold et al. (2005) developed a method for indirect microtubule visualization, based on Ncd::GFP expression. Only recently a direct visualization of the spindle in Drosophila oocytes has been made possible by Colombie et al., (2008), with the use of GFP::α-tubulin. Nevertheless, tools to visualize other structures of the Drosophila meiotic spindle in live oocytes have not been developed so far.
Analysis of spindle assembly in fixed oocytes has given insights into spindle assembly in Drosophila and mouse female meiosis (see 1.2.1. and 1.3.2.2.; Thekauf and Hawley, 1992; Brunet et al., 1999). Analysis of fixed oocyte stages, however, has a major drawback that it does not show the dynamicity of the processes and does not allow distinguishing oocyte stages due to the procedure of sample preparation.
Oocytes are difficult to examine biochemically. This is primarily because no system to culture oocytes has been established and so oocytes have to hand-picked one-by-one.
1.4.3. Drosophila as a model organism
Drosophila melanogaster is one of the best known organisms in terms of genetics. This mostly determines the usefulness of the fly as a model for studying acentrosomal spindle assembly (Doubilet and McKim, 2007). In particular, mutants affecting molecular processes can be isolated and analysed by genetic and cytological methods. Drosophila genetics offers a number of very sophisticated tools enabling generation of transgenic flies and even analysis of lethal mutations in live tissues.
A study of several chromosome segregation mutants, combined with imaging of the proteins, has led to a model of spindle assembly in Drosophila oocytes. Spindle microtubules first nucleate around chromosomes and then become sorted to produce a bipolar structure (Theurkauf and Hawley 1992). This model is compatible with models for acentrosomal spindle assembly in other studied systems (Doubilet and McKim, 2007). As organisms utilize common molecular mechanisms, understanding the process of acentrosomal spindle assembly in Drosophila may greatly contribute to comprehend the process in higher eukaryotes, including humans.
Drosophila offers greater accessibility to oocytes than humans or mice. The fly breeds easily and produces many eggs that can be quickly extracted. Comparing to a large opaque eggs of Xenopus, Drosophila oocytes allow easy location of the spindle and clear imaging in vivo.
Drosophila oocyte maturates among fifteen nurse cells, which degenerate at the later stages of oocyte development (King, 1970). In early stages of oogenesis, oocyte arrests in a phase equivalent to diplotene/diakinesis. Hormonal signals release the oocyte from the arrest. During most of the oocytes maturation, the chromosomes are compacted in a form of a spherical mass, known as the karyosome. At the later stage of oocyte development, the nuclear envelope breaks down and the spindle assembly begins (Theurkauf and Hawley 1992). By metaphase I the spindle is fully formed. Differently to other systems, female meiosis in Drosophila arrests, for the second time, in metaphase of the first round of division. Activation of the oocyte to enter anaphase I is induced by oocytes passage through the oviduct and meiosis is completed independently of fertilization (see 1; Mahowald and Kambysellis, 1980). In ovaries, oocytes of subsequent stages are arranged into chains, called the ovarioles (King, 1970). The oocytes organized into ovarioles gave the foundation to classification of stages from stage1 to 14. Dissection of an ovary allows identification of the stages based on morphology of the oocytes and surrounding nurse cells. Oocytes before NEB, until stage 13, can be distinguished from stage 14 with fully assembled spindle (King, 1970). Spindle formation can be observed by analysis of oocytes at the appropriate stage. Spindle assembly in Drosophila oocytes is much shorter than in mice, enabling imaging of the full process in shorter time (Brunet et al., 1999). Additionally, arrest in metaphase enables observation of how the spindle and chromosome position within the spindle are maintained over time.
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