Dis Model Mech
November 1, 2009;
Learning about cancer from frogs: analysis of mitotic spindles in Xenopus egg extracts.
The mitotic spindle
is responsible for correctly segregating chromosomes during cellular division. Disruption of this process leads to genomic instability in the form of aneuploidy, which can contribute to the development of cancer. Therefore, identification and characterization of factors that are responsible for the assembly and regulation of the spindle
are crucial. Not only are these factors often altered in cancer, but they also serve as potential therapeutic targets. Xenopus egg
extract is a powerful tool for studying spindle
assembly and other cell cycle-related events owing, in large part, to the ease with which protein function can be manipulated in the extract. Importantly, the spindle
factors that have been characterized in egg
extract are conserved in human spindle
assembly. In this review, we explain how the extract is prepared and manipulated to study the function of individual factors in spindle
assembly and the spindle
checkpoint. Furthermore, we provide examples of several spindle
factors that have been defined functionally using the extract system and discuss how these factors are altered in human cancer.
Dis Model Mech
Disease Ontology terms:
[+] show captions
References [+] :
Fig. 1. Generating CSF extract. Xenopus eggs are dejellied, washed and added to a centrifuge tube containing Nyosil or Versilube oil (gray). To pack the eggs, they are spun at top speed in a clinical centrifuge for 15 seconds. During this spin, the oil pushes the buffer surrounding the eggs to the top of the centrifuge tube (blue). Once the excess oil and buffer are removed, the packed eggs are spun in an ultracentrifuge to crush the eggs. This results in the formation of three layers: a lipid layer at the top of the tube (yellow), the soluble CSF extract (pale yellow), and the bottom layer containing yolk, pigment granules and cell debris (brown). The CSF extract is collected using a needle that is inserted through the side of the tube.
Fig. 2. Types of spindles formed in CSF extract. (A) During CSF spindle assembly, sperm chromatin (blue) is added directly to the CSF extract. Within 15 minutes, microtubules (red) are polymerizing from the centrosome (green) that is attached to the sperm chromatin. By 30 minutes, the asters are organized into a half-spindle structure that, by 60 minutes, progresses into a bipolar spindle. (B) If calcium chloride is added to the CSF extract along with the sperm chromatin, the extract shifts into interphase, where nuclei form and both the centrosomes and the chromatin replicate. Addition of fresh CSF extract shifts the extract back into mitosis and a bipolar spindle forms. (C) Magnetic beads that are coated with plasmid DNA promote spindle assembly when added to CSF extract. Image reprinted by permission from Macmillan Publishers Ltd: Nature (Heald et al., 1996) copyright 1996. (D) Ran asters/spindles are formed by the addition of non-hydrolyzable RanGTP to the CSF extract. RanGTP causes the release of spindle assembly factors that, together with molecular motors and other microtubule-associated proteins (MAPs), organize small bipolar spindle-like structures. Bars, 10 μm.
Fig. 3. Spindle assembly pathways. (A) Chromatin-driven pathway. The RanGEF, RCC1, localizes on chromatin (blue) and generates a gradient of RanGTP. RanGTP induces the release of spindle assembly factors, which nucleate and organize microtubules (red) into a bipolar spindle structure around the chromatin. (B) Centrosome-driven pathway. Microtubules (red), nucleated at the centrosome (green), alternate between growth and shrinkage (forward or reverse arrows). Upon binding to a kinetochore, microtubules are stabilized and bundled into K-fibers. Other microtubules are linked to each other by motors to form overlapping microtubules within the spindle structure.
CENP-E as an essential component of the mitotic checkpoint in vitro.