Spindle Assembly in Xenopus Egg Extracts: Respective Roles of Centrosomes and Microtubule Self-Organization

Abstract

In Xenopus egg extracts, spindles assembled around sperm nuclei contain a centrosome at each pole, while those assembled around chromatin beads do not. Poles can also form in the absence of chromatin, after addition of a microtubule stabilizing agent to extracts. Using this system, we have asked (a) how are spindle poles formed, and (b) how does the nucleation and organization of microtubules by centrosomes influence spindle assembly? We have found that poles are morphologically similar regardless of their origin. In all cases, microtubule organization into poles requires minus end–directed translocation of microtubules by cytoplasmic dynein, which tethers centrosomes to spindle poles. However, in the absence of pole formation, microtubules are still sorted into an antiparallel array around mitotic chromatin. Therefore, other activities in addition to dynein must contribute to the polarized orientation of microtubules in spindles. When centrosomes are present, they provide dominant sites for pole formation. Thus, in Xenopus egg extracts, centrosomes are not necessarily required for spindle assembly but can regulate the organization of microtubules into a bipolar array.

An ATM- and ATR-dependent checkpoint inactivates spindle assembly by targeting CEP63

“…However, mitotic spindles can form in the absence of centrosomes through actions of motor proteins and structural elements (Theirkauf and Hawley, 1992;Bartolini and Gundersen, 2006;Walczak et al, 1998;Compton, 1998;McKim and Hawley, 1995;Matthies et al, 1996;Heald et al, 1997). It is commonly believed that centrosome functions have not been fully elucidated.…”

“…To determine the pathway corresponding to defective spindle assembly, we turned our focus to acentrosomal or anastral selfordering spindle assembly mechanism. An important distinction between spindle assembly mechanisms is that the presence of sperm nuclei promotes the formation of aster/centrosome structures, which act as dominant sites for spindle assembly and organization (Heald et al, 1997). Whereas, anastral spindles formation relies on selfassembly.…”

“…Anastral spindle assembly is prevalent in higher plants, meiotic cells and in some other cell types (Theirkauf and Hawley, 1992;Bartolini and Gundersen, 2006). In the process of spindle self-assembly, chromatin promotes microtubule polymerisation and actions of motor proteins aid sorting and organisation of microtubules into a bipolar spindle array (McKim, and Hawley, 1995;Heald et al, 1997;Theurkauf and Hawley, 1992;Matthies et al, 1996;Khodjakov and Rieder, 2001). …”

“…Anastral spindles form in Xenopus egg extract in the absence of sperm nuclei whose basal body carries a centriole that matures into a centrosome (Heald et al, 1996). Heald et al, determined that centrosomes are not a necessary requirement for spindle assembly, however when present they act as dominant microtubule organisation centres (Heald et al, 1997). The distinctions of normal anastral spindles assembly in the presence of active ATM and ATR provided the focus for an ATM and ATR target within a centrosomes-dependent spindle assembly pathway .…”

Bipolar, anastral spindle development in artificially activated sea urchin eggs

“…The second is a chromosome-dominated pathway found largely in female germline meiotic cells in which the chromosomes contribute to the assembly and stabilization of microtubules (MTs) that are then formed into bipolar, anastral spindles by the action of MT motor proteins. A large number of studies have used the in vitro Xenopus oocyte extract system to characterize both the centrosome-dependent and -independent pathways (Heald et al, 1997;Karsenti and Vernos, 2001; Karsenti and Nedelec, 2004;Mitchison et al, 2004) and the current view is that both of these processes contribute to the formation of the bipolar mitotic spindle in many animal cells (O'Connell and Khodjakov, 2007). In addition, recent studies suggest that the chromosomes may not be the only sites for MT assembly in acentrosomal systems in that MTs have been shown to grow from pieces of the nuclear envelope (Rebollo et al, 2004) as well as from the spindle itself (Mahoney et al, 2006), a process that may be at least partially based on MT severing (Srayko et al, 2006;McNally et al, 2006).…”