Anaphase in vitro.

Abstract

Despite their varied appearances, all mitotic spindles have a similar architecture: they are organized as two half spindles, and the microtubules in each half spindle are of the same polarity, with many of the plus ends of the microtubules that grow out from the poles interacting with chromosomes or the microtubules of the other half spindle. During anaphase, the chromosomes move to the spindle poles as the kinetochoreattached microtubules depolymerize (anaphase A), and the spindles elongate due to the presence of pushing forces in the spindle midzone and pulling forces acting on the spindle poles (anaphase B). Although Mazia and Dan (1) first isolated the mitotic apparatus from sea urchin zygotes over 40 years ago, still left unanswered are many questions regarding the identities, locations, and rearrangements of the structural, mechanochemical, and regulatory molecules involved in chromosome separation. Even with a plethora of genetic mutants in various organisms, to determine the nature of these mutants and the relative roles of the affected gene products during mitosis, it is essential to have functional in vitro model systems that can be used to dissect and then reconstitute those elements of the spindle responsible for chromosome movement. The mitotic spindle has many unique features as an organelle that make it difficult to analyze with biochemical techniques. First, the spindle is a dynamic structure; the assembly and disassembly of microtubules contributes to the force generation mechanism responsible for chromosome separation (2). As an analysis of the history of mitosis in vitro shows, media that stabilize spindle structure against the rigors of organelle isolation often inactivate the motile apparatus and render an analysis of the role of microtubule dynamics moot (3). A second problem is that multiple mechanisms of force generation contribute to chromosome separation. Furthermore, redundant motor proteins may participate in any one mechanism of force generation. This has come as a shock to many cell biologists, especially those who tried to explain how chromosomes move by evoking just one mechanochemical system as responsible for all aspects of chromosome movement (reviewed in ref. 4). The current inventory of microtubule motors in the cell is very large, and many of them appear to contribute toward driving the assembly of the spindle apparatus and the subsequent chromosome movement it supports. Thus, spindles are much more complex mechanically than we ever suspected, making a biochemical dissection of spindle function difficult.