Optical Micromanipulation of Isolated Karyoplasts Reveals Active Chromatin-Driven Dissipation in the Nucleus

We established and optimized a high-efficiency (>80%) karyoplast (nucleus enclosed by the plasma membrane with a thin layer of cytoplasm) isolation protocol for vimentin-null mouse embryonic fibroblasts (mEFs) (1). Cells were cultured on fibronectin-coated coverslips and inverted onto spacers within centrifuge tubes containing serum-free DMEM supplemented with 2 µg/mL cytochalasin D. Centrifugation at 4000 RPM for 40 minutes yielded mechanically intact nuclei (karyoplasts) that were collected in cone-shaped wells. Suspended karyoplasts were significantly smaller than intact mEFs, confirming successful cytoplasmic removal while maintaining structural integrity.

Micromanipulation experiments were conducted using a commercial optical trap system (Lumicks C-Trap). Polystyrene beads were attached as handles to transmit and record pico-Newton-scale forces. One trap was oscillated in a triangular waveform (~0.6 µm amplitude) while forces up to 50 pN were simultaneously recorded. Force–displacement curves revealed an approximately linear elastic response with pronounced hysteresis, distinct from Hertzian contacts. Notably, the hysteresis magnitude remained constant across ramp speeds of 1–10 µm/s, suggesting that energy dissipation is not dominated by viscous drag but rather by active, motor-driven chromatin dynamics.

In addition, a slow creep response reminiscent of a Mullins effect was frequently observed, indicating stress-dependent structural reorganization within the nuclear material. These findings suggest that isolated nuclei behave as active viscoelastic materials, in which internal energy consumption contributes to mechanical dissipation. Extending microrheological approaches traditionally applied to cytoskeletal systems, this work provides new insight into the active mechanical landscape of the genome and its role in nuclear organization.