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Elasticity measurements show the existence of thin rigid cores inside mitotic chromosomes.
Houchmandzadeh B
,
Dimitrov S
.
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Chromosome condensation is one of the most critical steps during cell division. However, the structure of condensed mitotic chromosomes is poorly understood. In this paper we describe a new approach based on elasticity measurements for studying the structure of in vitro assembled mitotic chromosomes in Xenopus egg extract. The approach is based on a unique combination of measurements of both longitudinal deformability and bending rigidity of whole chromosomes. By using specially designed micropipettes, the chromosome force-extension curve was determined. Analysis of the curvature fluctuation spectrum allowed for the measurement of chromosome bending ridigity. The relationship between the values of these two parameters is very specific: the measured chromosome flexibility was found to be 2,000 times lower than the flexibility calculated from the experimentally determined Young modulus. This requires the chromosome structure to be formed of one or a few thin rigid elastic axes surrounded by a soft envelope. The properties of these axes are well-described by models developed for the elasticity of titin-like molecules. Additionally, the deformability of in vitro assembled chromosomes was found to be very similar to that of native somatic chromosomes, thus demonstrating the existence of an essentially identical structure.
Figure 3. Successive micrographs of typical forceâextension measurement. A chromosome was suspended between two micropipettes and stretched with the upper one. The lower micropipette (prepared in order to have K â 2â4 à 10â4 N/m spring constant) was calibrated after each measurement. The length of the chromosome and the force applied to it were measured after digitization of the recorded images. Bar, 10 μm.
Figure 1. Structural changes of Xenopus sperm nuclei in mitotic egg extract; control sperm nuclei (a), decondensed sperm after 10 min of incubation in the extract (b), chromosomal structures (câg) found after 30, 60, 90, 120, and 150 min, respectively. Upon 180 min of incubation (h) well-separated individual chromosomes were observed. Bars, 5 mm.
Figure 4. The force per section versus deformation curve for two different chromosomes. Y1 = 840; Y2 = 1,350. For simplicity, the error bar for one point only is shown. Chromosome diameter is 0.8 μm (section 0.5 μm2). The initial length of chromosome 1 was 9.5 μm, and that of chromosome 2 was 8.7 μm.
Figure 5. Forceâextension cycles of a single chromosome (initial length 7 μm) for high deformations at 1 μm/s. The solid lines represent the numerical resolution of equations of 45 parallel titin-like molecules. 25% of the domains are supposed to be irreversibly unfolded after the first cycle, 40% after the second one, and 60% after the third one. The parameters used to solve the kinetic equations were: lunfold = 29 nm, lfold = 4 nm, A = 2 nm, Ea = 22 pN nm, Îx = 0.1 nm, Îxâ² = 2 nm, Ï0 = 1 sâ1. The initial chain deformation, due to chromatin pressure, is taken as z/L = 0.5.
Figure 6. (a) Apparition of plateau regime before the breakdown of the chromosome for high extension regime (initial chromosome length: 7.8 μm). (b) If the extension is stopped at the middle of the plateau regime, domains of thick chromosome connected by thin filaments are observed. Three successive micrographs of a partially unraveled chromosome are shown, where one micropipette moves to show the existence of the connecting thin filament.
Figure 7. Schematic representation of mitotic chromosome structure based on elastic measurements. The chromosome is proposed to be formed of a few rigid axes, surrounded by a soft envelope of chromatin attached to the axes. The rigid axes are built of titin-like molecules, formed of repetitive domains, which can be unfolded upon application of force.
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