In situ demonstration of actin in normal and injured ocular tissues using 7‐nitrobenz‐2‐oxa‐1,3‐diazole phallacidin

Abstract

The fluorescent derivative of the actin-binding toxin phallacidin, 7-nitrobenz-2-oxa-1,3 diazole phallacidin, has been used to cytologically demonstrate the presence of actin in lens epithelium, corneal endothelium, and retinal pigment epithelium. In these noninjured tissues, no stress fibers are observed and fluorescence is confined mainly to an area at or near the cell membrane, although some diffuse cytoplasmic staining can also be seen. However, following injury to either the lens epithelium or corneal endothelium of rats and frogs, stress fibers are detected, but only in those cells that migrate into the wound area. Cells on the periphery of each tissue do not partake in would repair and thus maintain their normal appearance. After the tissue has regenerated, stress fibers disappear, and those cells involved in the injury response return to their normal morphology. When rabbit corneal endothelium is placed in tissue culture, stress fibers are observed as the cells migrate away from the initial explant. Upon reaching confluency, these cells spread out and each is surrounded by thick actin-containing bands. Furthermore, they exhibit some stress cables within their cytoplasm. This is in contrast to their appearance in vivo where stress fibers are absent and fluorescence is limited to a region near the cell membrane.

In vitro reendothelialization. Microfilament bundle reorganization in migrating porcine endothelial cells.

“…17 Similar MF bundles have been observed in lens and retinal pigment epithelial cells and in corneal endothelial cells. 29 We have not seen DPBs in confluent cultures of porcine thoracic aortic smooth muscle cells. The DPBs are not artifacts created by the close contact of MF bundles of two adjacent cells since even after removal of a single cell from the EC confluent monolayer* 5 the DPB is still present along the cell border adjacent to the absent cell.…”

“…Central MF bundles were present in those ECs that appeared to be under hemodynamic stress. Central MF bundles were also observed in in vivo migrating corneal endothelial and lens epithelial cells following injury 29 and in in vivo regenerating rabbit aortic ECs. 13 Gabbiani et al 13 recently reported that, in vivo, actin-containing MF were present at the periphery of aortic rabbit ECs while central MF bundles were present in ECs at sites located below intercostal arteries.…”

“…MF bundles have been shown to be present in situ in vascui ar 9. io. 12-14, M a n c j nonvascular 29 ' M tissue and thus are not artifacts of the tissue culture system. A comparison of the MF bundles that we observed in in vitro porcine thoracic aortic ECs with those described in recent in vivo studies indicate many similarities.…”

A thermodynamically motivated model for stress-fiber reorganization

“…Continuum description of stress-fibers within a cell Contractile stress-fibers comprising proteins such as -actinin, actin, myosin and tropomyosin and showing an alternating periodic arrangement similar to that seen in muscle sarcomeres have been observed in a range of cells including endothelial cells (De Bruyn and Cho 1974), retinal cells (Gordon et al 1982) and fibroblasts (Byers and Fujiwara 1982). We use these observations to describe the structure of stress-fibers with a view to developing a continuum model for their re-organization.…”

Cytoskeletal organization of migrating retinal pigment epithelial cells during wound healing in organ culture

“…Many studies of cell behaviour on glass or plastic have examined changes in the cytoskeleton and the sites of adhesive interactions with the substratum. Prominent among these have been studies examining the role of focal contacts and the associated microfilament bundles (stress fibers) in cell adhesion and migration of cells [Dipasquale, 1975b;Gordon et al, 1982;Soranno and Bell, 1982;Woods and Couchman, 19881. Focal contacts are regions of strong adhesion between cells and their substrata, where the basal plasma membrane comes within 10-15 nm of the substratum (usually plastic or glass) giving a characteristic black image when viewed with surface reflection interference microscopy [Izzard and Lochner, 19761. They are normally found at the ends of linear microfilament bundles or stress fibers, which are prominent in well-spread cultured cells [Heath and Dunn, 19781.…”