Chromosomes (white) segregated by microtubules (stained with anti-tubulin, (red)), illustrating the dynamics of mitosis that Inoue deduced from the birefringent observation of spindle fibres. Courtesy of Z. Yang and C. L. Rieder, Wadsworth Center, Albany, NY, USA.
The fundamental question of how the mitotic spindle forms and functions to capture, align and segregate chromosomes into two daughter cells dates back to 1882 and the microscopic observations that were made by Walther Flemming of the changes in spindle morphology seen at different stages of mitosis. Despite their fundamental importance, these findings were limited by the use of fixed cell preparations, which would not allow detailed analysis of the mechanisms that are involved in spindle assembly or its ability to control chromosome behaviour.
A decisive step towards the solution to this problem was realized in the 1950s, when polarized light microscopy and live-cell imaging allowed the limitations of fixed samples to be overcome, and opened the way for a dynamic view of biological processes. During the following two decades, Shinya Inoue and his co-workers pioneered live-cell imaging by developing microscopes that allowed them to visualize parallel spindle fibres that polarized the light thanks to their birefringent properties. Crucially, birefringence could be measured and correlated with structural alterations occurring in the fibres during mitosis or in response to given experimental conditions.
In 1967, in a seminal paper based on the discussion of their own observations as well as those of several other investigators, Inoue and Hidemi Sato laid a fundamental cornerstone by presenting a model of mitotic spindle dynamics and their role in chromosome movements. They proposed that the birefringent fibres could reversibly polymerize and depolymerize during normal mitosis. In their 'dynamic equilibrium model', the spindle fibres were described as orientated polymers in equilibrium with a pool of 22S particles that were found around that time, by Robert Kane, to be the major proteins extractable from an isolated spindle. Tubulin was then identified as the protein that comprises the spindle fibres or microtubules (see Milestone 6). Inoue showed that the equilibrium could be shifted towards depolymerization by low temperatures and colchicine, or towards polymerization by treatment with heavy water, and that fibre reassembly from the soluble pool occurred in the absence of de novo protein synthesis. Inoue proposed that, throughout mitosis, the fibre dynamics were controlled by the activity of 'orientating centres' (centrioles, kinetochores and the cell plate) and by the concentration of the free subunits.
"...protein polymerization dynamics drive morphogenesis of, and force production by, the mitotic spindle..."
Tim Mitchison
A second fundamental observation made by Inoue and co-workers was that the chromosome movements during mitosis were controlled by the shortening and lengthening of the fibres. Experiments involving fibre depolymerization by treatment with colchicine or gradual cooling led them to the groundbreaking hypothesis that spindle dynamics can generate forces capable of pushing and pulling chromosomes. It took another 20 years, and the development of biochemical assays and labelling techniques in the 1980s, to start to uncover the mechanistic details of how the spindle assembles and controls chromosome movements (see Milestone 14). Nevertheless, the work of Inoue was an early description of a new form of biological motility driven by the assembly and disassembly of a biological polymer. Inoue and Sato further speculated that such motility could be used to move organelles other than chromosomes or to deform the cell surface, paving the way for the modern view of the crucial role of cytoskeleton polymerization dynamics in cellular morphogenesis and force generation.
