Reversible and Irreversible Unfolding of Mitotic Newt Chromosomes by Applied Force

Abstract

The force-extension behavior of individual mitotic newt chromosomes was studied, using micropipette surgery and manipulation, for elongations up to 80 times native length. After elongations up to five times, chromosomes return to their native length. In this regime chromosomes have linear elasticity, requiring ϳ1 nN of force to be stretched to two times native length. After more than five times stretching, chromosomes are permanently elongated, with force hysteresis during relaxation. If a chromosome is repeatedly stretched to ϳ10 times native length and relaxed, a series of hysteresis loops are obtained that converge to a single reversible elastic response. For further elongations, the linear dependence of force on extension terminates at a force "plateau" of ϳ15-20 nN, near 30 times extension. After Ͼ30 times extensions, the elastic moduli of chromosomes can be reduced by more than 20-fold, and they appear as "ghosts": swollen, elongated, and with reduced optical contrast under both phase and differential interference contrast imaging. Antibody labeling indicates that histone proteins are not being lost during even extreme extensions. Results are interpreted in terms of extension and failure of chromatin-tethering elements; the force data allow estimates of the number and size of such connectors in a chromosome.

Micromechanical studies of mitotic chromosomes

“…Our experiments on newt and Xenopus mitotic chromosomes removed from cells and manipulated with micropipettes showed that mitotic newt chromosomes could be doubled in length by roughly 1 nN forces (Houchmandzadeh et al 1997, Poirier et al 2000, in good accord with Nicklas (1963). We also found stretching force to vary nearly linearly with extension for elongations up to four times the native length, allowing us to summarize the elastic response with a number, the Fforce constant_ or slope of the force-versus-elongation curve.…”

“…Methods have been developed to isolate and to carry out stretching experiments on single prometaphase chromosomes, using glass micropipettes (Houchmandzadeh et al 1997, Poirier et al 2000, Pope et al 2006). The method is broadly similar to microneedle-based manipulation of meiotic metaphase chromosomes inside grasshopper spermatocytes (Nicklas 1963(Nicklas , 1983, as well as to classic studies of lampbrush chromosome mechanics (Callan 1954).…”

“…This calibration is done by pushing the floppy pipette against a stiffer pipette with known force constant; this calibration pipette has itself been calibrated in the same way. Through a sequence of three or four calibrations against progressively stiffer pipettes, one obtains a pipette that can have its stiffness measured by pushing it against an analytical balance with a micrometer (Poirier et al 2000). Calibration of the force-measuring pipette stiffness allows the deflectionYextension data to be converted to forceYextension.…”

“…Mitotic chromosomes have robust elasticity, returning to native length even after 5-fold extensions (Nicklas 1983, Houchmandzadeh et al 1997, Poirier et al 2000. During mitosis, chromosomes are often doubled in length by spindle-generated forces on the order of 1 nN in large animal or insect cells (Nicklas 1983).…”

“…This expresses what stress (force per area) would be required to double an object_s length, if the initial linear elasticity were extrapolated. For a mitotic chromosome, this stress is about 500 pascals (Nicklas & Staehly 1967, Poirier et al 2000 (1 pascal (Pa)=1 newton/meter 2 is the SI unit of pressure and stress). A 500 Pa modulus is low, even for a very loose highpolymer gel: 1% agarose gels have moduli of about 10 kPa (10 000 Pa), plexiglass and folded biomolecules (B-DNA and globular protein domains) have moduli near 1 GPa (10 9 Pa), and covalently bonded materials (metals, glasses) have moduli of about 10 GPa.…”