Date(s) - 07/23/2015
The nucleus is the largest and stiffest organelle of the cell. Dynamic positioning of the nucleus is an important biological process that can be observed during cell migration. Dynamic changes in nuclear shape are also observed during cell spreading. Defects in nuclear positioning and nuclear shaping are associated with a host of pathologies such as cancer, laminopathies and aging. We do not currently understand the molecular mechanisms by which the nucleus is positioned and shaped in the cell, and how these mechanisms become abnormal in disease.
The nucleus is not free to ‘float’ in the cell, but rather is anchored through molecular linkages to the cellular cytoskeleton. These linkages are thought to allow mechanical force transfer between the nucleus and the cytoskeleton resulting in dynamic nuclear positioning and shaping. Our knowledge of nucleo-cytoskeletal force transfer is in its infancy in part due to the lack of techniques to mechanically interrogate this structure in living, adherent cells. In this thesis, we developed a novel, direct force probe to apply controlled, known mechanical forces directly to the nuclear surface in the living cell. We studied the nuclear dynamics under known forces and identified the factors that contribute to its positioning and shaping in living adherent fibroblasts cells. With this method, we estimate that a minimum pulling force of a few nanoNewtons – far greater than typical intracellular motor forces – is required to significantly displace and deform the nucleus. Although F-actin and microtubules are known to exert mechanical forces on the nuclear surface through molecular motor activity, we show that the intermediate filament networks maintain nuclear mechanical homeostasis. We demonstrate that this method is sensitive enough to detect differences between normal and cancerous breast epithelial nuclei. In addition, it is sensitive to molecular perturbations that normalize cancer nuclei. We expect our new technique to be useful in better understanding pathologies associated with abnormal nuclear shaping and positioning.
Finally, we studied the mechanical principles of nuclear shaping in normal breast epithelia. We report that vertical cross-sectional shapes of cells and nuclei in epithelial monolayers are remarkably uniform compared to those in isolated cells. Our results suggest that competition between cell-cell pulling forces that work to expand and shorten the vertical cell cross-section, thereby widening and flattening the nucleus in the process, and the resistance of the nucleus to further flattening results in uniform cell and nuclear cross-sections. Our results shed light into the mechanical principles of self-organized uniformity in nuclear shapes in epithelial monolayers.