Abstract
In vitro studies have identified LIMK2 as a key downstream effector of Rho GTPase-induced changes in cytoskeletal organization. LIMK2 is phosphorylated and activated by Rho associated coiled-coil kinases (ROCKs) in response to a variety of growth factors. The biochemical targets of LIMK2 belong to a family of actin binding proteins that are potent modulators of actin assembly and disassembly. Although numerous studies have suggested that LIMK2 regulates cell morphology and motility, evidence supportive of these functions in vivo has remained elusive. In this study, a knockout mouse was created that abolished LIMK2 biochemical activity resulting in a profound inhibition of epithelial sheet migration during eyelid development. In the absence of LIMK2, nascent eyelid keratinocytes differentiate and acquire a pre-migratory phenotype but the leading cells fail to nucleate filamentous actin and remain immobile causing an eyes open at birth (EOB) phenotype. The failed nucleation of actin was associated with significant reductions in phosphorylated cofilin, a major LIMK2 biochemical substrate and potent modulator of actin dynamics. These results demonstrate that LIMK2 activity is required for keratinocyte migration in the developing eyelid.
Highlights
Regulation and remodeling of the actin cytoskeleton are critical events affecting cell-cell and cell-extracellular matrix interactions during tissue morphogenesis and wound repair [1,2]
embryonic stem (ES) cell clone OST80053 was selected for further characterization based on the sequence similarity of its 39 RACE tag with exons that code for the kinase domain
Keratin 6 expression in the tip of the emerging eyelid epithelium was similar in LIMK2-deficient and wild type mice (Fig. 7 E, F). These results suggest that the classical ligand-receptor pathways associated with eyes open at birth (EOB) phenotypes remain intact in LIMK2-deficient mice and that keratinocytes in the eyelid tip exhibit a pro-migratory phenotype
Summary
Regulation and remodeling of the actin cytoskeleton are critical events affecting cell-cell and cell-extracellular matrix interactions during tissue morphogenesis and wound repair [1,2]. Molecular signaling pathways that control actin dynamics are highly conserved among species and similar phenotypes are often observed in genetic models [1]. Closure of the eyelid in mammals occurs during embryogenesis. Eyelid closure initiates on the tips of the eyelid folds at embryonic day 15 (E15) and is completed approximately 24 hours later, on E16. Fusion of opposing eyelids occurs through extension of the eyelid fold in the form of a sheet of migrating keratinocytes and surrounding periderm cells. Fusion begins in the temporal and nasal canthi and progresses towards the center of the eye [3,4,5]
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