Investigations into the biophysical properties of single molecules traditionally involve well defined in vitro systems where parameters such as solvent viscosity and applied forces are known a priori. These systems provide means to develop models describing the polymers response to a variety of conditions, including the entropically driven relaxation of a stretched biopolymer upon release of the tension inducing force. While these techniques have proven instrumental for recent advancements in the fields of polymer physics and biophysics, how applicable they are to life inside the cell remains poorly understood. Here we report an investigation of in vivo stretched polymer relaxation dynamics using chromatin relaxation following the breakage of a dicentric chromosome subjected to microtubule-based spindle forces. Additionally, we have developed an in vitro system used to verify the conformations observed during the in vivo relaxation, including the predicted but previously unidentified taut conformation. These observations motivate our use of existing polymer models to determine both the in vivo viscosity as seen by the relaxing chromatin and the tension force applied by the microtubule-based spindle in vivo. As a result, the technique described herein may be used as a biophysical strategy to probe the intranuclear environment.intranuclear viscosity ͉ stem and flower S ingle molecule analysis of fluorescently labeled DNA under tension has been used in a variety of investigations of polymer dynamics (1-6) in vitro. Traditionally these experiments have been performed using electrophoresis (7) or, more commonly, fluid flow to extend the polymer, with the dynamics of the polymer being investigated at either constant extension [corresponding to a fixed voltage or specific flow rate (2, 5, 8)] or after the flow is stopped (4, 9, 10). In the latter case, the elimination of the stretching force causes the chain to relax from its free end, allowing the relaxation dynamics to be observed. For a relaxing polymer chain, a number of different conformations may be observed as the constraint release propagates throughout the chain (Fig. 1). As a result, relaxation events may be broken up into multiple regimes, each corresponding to a different polymer conformation, with the expressions used to describe the length of the polymer over time yielding information about either polymer specific properties, such as the tension force applied immediately before recoil, or the viscosity of the surrounding environment.While much is known about the mechanism of sister chromatid separation, surprisingly little is known about fundamental quantities such as the forces that are generated by microtubule-based spindles under physiologically relevant loading (i.e., what forces are generated during sister separation) and the intranuclear viscosity. A lack of noninvasive in vivo data collection techniques provided the impetus for developing alternative in vitro or in silico experiments, but very few opportunities to confirm that the assumptions made ...

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