| While questions of cell biology are often answered by analyzing the intricacies of protein pathways and signaling events, it is essential to consider the cellular framework and environment in which these interactions and events occur. The cellular cytoskeleton not only confers mechanical integrity to the cell, but also orients cellular organelles and proteins within the cell to regulate metabolic and signaling pathways. Changes in the cytoskeletal organization of cells can thus effect both mechanical and chemical reactions to stresses experienced throughout the body. Perturbations in the proper function of the cytoskeletal network, then, can cause defects in cellular function when responding to environmental stimuli. Cardiovascular disease syndromes, neurodegenerative diseases, cancers, and several skin diseases have been associated with cytoskeletal abnormalities. In this work, the role of the cytoskeleton and in particular, nuclear lamins, in mediating cellular health is investigated using both novel and well-established assays, as laminopathic diseases similarly encompass a broad range of affected organs and tissues. Healthy and laminopathic fibroblasts are characterized by several biophysical properties, including cytoplasmic elasticity, organelle positioning, polarization state, and migration rate, among others. Additionally, protein localization and cytoskeletal structure in laminopathic cells are assessed and compared to their wildtype cells to complement distinguished healthy and disease phenotypes.;First, to quantify cytoplasmic elasticity of individual cells, the method of particle tracking microrheology via ballistic injection of tracer nanoparticles is optimized and validated. Thorough experimentation demonstrates that the method properly assumes that particles undergo Brownian motion under the influence of thermal fluctuations, allowing the Stokes-Einstein relationship be invoked in order to extract rheological information regarding the microenvironment in which tracer particles are embedded. This method is then used to detect changes in mechanical stiffness between healthy fibroblasts and fibroblasts lacking A- type lamins, which serve as a laminopathic mouse model of autosomal dominant Emery-Dreifuss muscular dystrophy. A significant reduction in the mechanical stiffness of A-type lamin-null fibroblasts is observed, in addition to an abrogated ability of wound edge cells to polarize and close a wound in a timely fashion. These results suggest that the loss of A-type lamins from the inner nuclear envelope can impact global cellular behavior and particularly affect cellular mechanics, polarization, and motility. The mechanism behind this linkage of nuclear lamins to cytoskeletal function is further explored in additional laminopathic models, most notably a model of the premature ageing syndrome, Hutchinson Gilford progeria. Laminopathic cells are found to lack proper localization of proteins that physically connect the nuclear lamina to the cytoskeleton, referred to as LINC complex proteins. Results demonstrate that a dysfunctional connection between the nucleus and microtubule and actin networks mediated by these proteins causes a host of cytoskeleton-mediated defects, including loss of mechanical stiffness, an increased separation between the cell's microtubule organizing center, or MTOC, and nucleus, an abrogated ability of cells to polarize, and a reduced ability of cells to adhere to a substrate. The role of the LINC complex in maintaining cellular stiffness is corroborated by results from additional experiments in which fibroblasts arc transfected with a recombinant protein that displaces endogenous components of the LINC complex, namely Nesprin proteins, and the elastic modulus of such cells is significantly reduced compared to cells in which the LINC complex remains intact. Finally, a single-cell micropatterning assay is employed to more quantitatively assess the role of cytoskeletal filaments and specific proteins in the localization of the MTOC and nucleus, the relative positioning of which determines the polarization state of several cell types. Cytoskeletal filaments are found to significantly impact the localization of both MTOC and nucleus, but in a cell shape- and cell-cell contact-dependent manner. Furthermore, specific proteins required for polarization of confluent, wound-edge cells are found to be dispensable for single cell polarization, suggesting a cell polarization pathway that diverges based on the presence of cell-cell contacts.;The results presented here aim to bridge the gap in understanding between the genetic mutations that cause laminopathic disease and the well-characterized phenotypes that are observed in humans. Several biophysical defects, particularly the loss of cellular mechanical stiffness and an impaired ability to polarize, are detected at the cellular level that can contribute to our understanding of the etiology of laminopathies. These results also shed significant light on the function of the ZINC complex as well as cytoskeletal filaments in regulating the biophysical health of cells. In addition, preliminary data indicate another possible defect in fibroblasts lacking A-type lamins which display abnormal localization of a protein implicated in cell polarity, namely Par3, possibly due to a break in signaling in the polarization pathway involving Cdc42. (Abstract shortened by UMI.)... |
