Contribution of cytoskeletal stresses to cell volume regulation
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Cells in the kidney collecting duct experience changes in extracellular osmolarity as urine flows out of the nephron. These changes impart osmotic forces on collecting duct epithelia, causing variations in cell volume. Proper function of the kidney requires control of cell volume, in order to maintain homeostasis. An area of understanding which has in the past been dismissed is the role of mechanical force in maintaining equilibrium across the membrane. In the first part of this thesis, the volume regulatory behavior of cells is evaluated in the adherent and suspended states using impedance-based measurement techniques. Using actin modifying drugs, both swelling and volume recovery were determined to depend on the integrity of the cytoskeleton in adherent cells. However, volume recovery in detached cells was insensitive to actin content, showing that actin content alone was not the important factor. The next section demonstrates sensitivity of the cytoskeleton to fluid shear stress. By utilizing a stress-sensitive probe in an actin-linking host, we visualized spatial distribution of stress in adherent cells cultured in a parallel plate microchannel, in real time. Flow pulses were repeatedly applied, allowing for observation of the time dependent adaptive reorganization at the whole cell and subcellular level. Furthermore, changes in stress were observed with the introduction of actin modifiers as well as in cell detachment, allowing for visualization of tensile changes which preceded our volume measurements. Finally, stress response to hypoosmotic solution was investigated using the stress sensor and microchannel. Varying the osmotic gradient revealed osmotically sensitive tension in the recovery phase, which was borne by filaments that persisted through the swelling phase. Additionally, the initial response to swelling was observed to depend on an intact actin cytoskeleton, but not on the magnitude of the osmotic gradient. The results show actin filaments are tensed in volume recovery, and may add to the balance of forces which resist volume changes.