Abstract
Dynamic changes in the level of chromatin compaction occur throughout the cell cycle, leading to consideration of the potential effects of expansion. Such effects include the fact that when expansion is constrained (by surrounding objects, an external cage, or internal tethers) they may generate pushing forces (compressive stress) on those constraining features. We have developed a Magnetic Force Micropiston, a device that combines magnetic bead manipulation with confinement created by microfluidic channels, to probe the magnitude of, basis for and effects of the forces generated by DNA/chromatin/chromosome expansion and/or compression. This device accommodates objects whose size scales are of the order of bacterial cells and yeast nuclei (1um) and mammalian nuclei (4.5um) and, for some appications, even smaller scales (∼150nm). Objects and changes of interest can be monitored by phase or fluorescence microscopy (presently epifluorescence or TIRF), with effects assessed as a function of time and with real-time monitoring of the effects of changes in the ionic or molecular/biochemical composition of the buffer. Measurable quantities include expansion force, volume changes, mobility of individual loci, hydrodynamic properties of the solvent, changes in the affinities of chromosomal components, and alterations in the organization and order of the confined chromatin. Applications of this system to mammalian and E.coli chromatin will be presented. The device can also be used, without application of force, for multiple parallel single cell analyses, e.g. of vegetative growth and meiosis in budding yeast and of E.coli, as will be illustrated.
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