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A. Kim, W.M. Petroll; Microtubule Disruption Induces Corneal Fibroblast Contraction and Extracellular Matrix Compaction in 3–D Culture . Invest. Ophthalmol. Vis. Sci. 2006;47(13):1816.
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© ARVO (1962-2015); The Authors (2016-present)
Microtubules play an important role in regulating the morphology and motility of a variety of cells on 2–D substrates. The purpose of this study was to investigate the role of microtubules in modulating corneal fibroblast structure and mechanical behavior using 4–dimensional imaging of cells in 3–D culture.
Rabbit and human corneal fibroblasts transfected to express GFP–zyxin (to label focal adhesions) or GFP–tubulin (to label microtubules) were plated at low density inside 100 µm thick type I collagen matrices. Using confocal microscopy, fluorescent (for GFP) and reflected light (for collagen fibrils) 3–D optical section images were acquired at multiple points beginning at 3 hrs or 24 hrs after plating gels. During time–lapse image, fresh media buffered with HEPES was continuously perfused through the dish and temperature was maintained at 37°C. After 1–2 hours, cells were treated with cytochalasin D (to disrupt f–actin) and/or nocodazole (to depolymerize microtubules).
This experimental model allows dynamic 3–D visualization of both GFP–tagged proteins within cells and the fibrillar collagen surrounding them. At 3 hours, focal adhesions were not well developed, microtubules were weakly labeled, and little matrix compaction was observed surrounding the cells. In contrast, at 24 hrs cells had well organized microtubules and prominent focal adhesions, and significant cell–induced matrix compaction was observed. Nocodazole induced rapid microtubule disruption which resulted in cellular contraction at both 3 hours and 24 hours. The matrix was pulled inward and compacted by retracting cell processes; this process was mediated by focal adhesions, when present. Overall, the effects of nocodazole were much more dramatic at 24 hours. Cytochalasin D induced cell elongation, ECM relaxation and disassembly of focal adhesions at both time points evaluated; however, the response was significantly greater at 24 hours, suggesting greater tension within the f–actin cytoskeleton.
Overall, the data suggest that microtubules play an important role in regulating cell tensegrity (e.g. by counteracting tension within the f–actin cytoskeleton) and/or contractility (e.g. by sequestering Rho–GEF) in corneal fibroblasts within 3–D collagen matrices. Overall, the effect of microtubule disruption appears to be enhanced in cells with a well organized cytoskeleton and greater pre–existing contractile forces.
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