The process of
wound repair in monolayers of the intestinal epithelial cell line, Caco-2BBe, was analyzed by a combination of time-lapse differential interference contrast (
DIC) video and immunofluorescence microscopy, and
laser scanning confocal immunofluorescence microscopy (LSCIM).
DIC video analysis revealed that
stab wounds made in Caco-2BBe monolayers healed by two distinct processes: (a) Extension of lamellipodia into the
wounds; and (b) Purse string closure of the
wound by distinct
arcs or rings formed by cells bordering the
wound. The
arcs and rings which effected purse string closure appeared sharp and sheer in
DIC, spanned between two and eight individual cells along the
wound border, and contracted in a concerted fashion. Immunofluorescence analysis of the
wounds demonstrated that the
arcs and rings contained striking accumulations of actin filaments,
myosin-II,
villin, and
tropomyosin. In contrast,
arcs and rings contained no apparent enrichment of microtubules, brush border
myosin-I immunogens, or
myosin-V. LSCIM analysis confirmed the localization of actin filaments,
myosin-II,
villin, and
tropomyosin in
arcs and rings at
wound borders. ZO-1 (a
tight junction protein), also accumulated in
arcs and rings around
wounds, despite the fact that cell-cell contacts are absent at
wound borders.
Sucrase-
isomaltase, an apically-localized
integral membrane protein, maintained an apical localization in cells where
arcs or rings were formed, but was found in lamellipodia extending into
wounds in cells where
arcs failed to form. Time-course, LSCIM quantification of actin,
myosin II, and ZO-1 revealed that accumulation of these
proteins within
arcs and rings at the
wound edge began within 5 minutes and peaked within 30-60 minutes of wounding. Actin filaments,
myosin-II, and ZO-1 achieved 10-, 3-, and 4-fold enrichments, respectively, relative to cell edges which did not border
wounds. The results demonstrate that wounded Caco-2BBe monolayers assemble a novel cytoskeletal structure at the borders of
wounds. The results further suggest that this structure plays at least two roles in
wound repair; first, mediation of concerted, purse string movement of cells into the area of the
wound and second, maintenance of apical/basolateral polarity in cells which border the
wound.