Date of Award
Joan C. Edwards School of Medicine
Type of Degree
Gary L. Wright
Elsa I. Mangiarua
William D. McCumbee
Active remodeling of the cytoskeleton has been proposed to contribute to the low energy cost and maintenance of the sustained contraction in smooth muscle. Using confocal microscopy and standard immunohistochemical techniques, direct observation of actin remodeling was studied in the contracting A7r5 cell in response to the diacylglycerol (DAG) analog phorbol 12, 13 dibutyrate (PDBu). -actin was shown to exist in the resting cell as parallel stress cables that extend across the cell. Stimulation by PDBu resulted in a sustained contraction that occurred in approximately eighty-five percent of the A7r5 cells. The initial contraction was not uniform, but primarily occurred in the axis parallel to the ?-actin cables. During the latter portion of contraction shortening occurred in all axis. During contraction, the cells shortened without apparent compression of the ?-actin fibers or new cable formation, suggesting that ?-actin fibers undergo active remodeling during contraction. Treatment of the cells with cytochalasin resulted in dissolution of fibers in the central region of the cell and elongation of precontracted cells in the axis parallel to the ?-actin fibers, suggesting that the ?-actin cytoskeleton may serve to hold the cell in the contracted state. In unstimulated cells, ?-actin exists as series of stress fibers similar in appearance to ?-actin fibers. In contrast, within ten minutes of PDBu stimulation ?-actin disassembled and reassembled into intensely-staining column-like structures that extend from the cell?s base. The ?-actin column-like structures demonstrate a strong colocalization with ?-actinin suggesting that they may be homologous to dense bodies in differentiated smooth muscle. Smooth muscle myosin also undergoes remodeling, in a manner very similar to ?-actin. Dual immunostaining for ?-actin and smooth muscle myosin indicate a strong colocalization of the two proteins in the resting cell. However, following PDBu stimulation there was a partial dissociation of myosin from ?-actin. Treatment of cells with cytochalasin B or dissolution of the ?-actin fibers with thapsigargin resulted in similar patterns of staining for ?-actin and myosin. This suggests that myosin is in association with ?-actin and follows changes in ?-actin structure during contraction. This work suggests that the ?- and ?-actin domains in the A7r5 cells undergo specific remodeling in A7r5 cells, and suggests a model in which ?-actin filaments undergo remodeling to hold the cell in the shortened state at low energy cost while ?-actin and myosin remodel into a configuration that allows maximal mechanical advantage for contraction.
Cells - Contraction.
Fultz, Michael E., "Actin and Myosin Remodeling in the A7r5 Smooth Muscle Cell" (2002). Theses, Dissertations and Capstones. 595.