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P. Deng, J. Qiu, R. Maddala, P.V. Rao; Microtubule Disruption Suppresses –Crystallin Expression in Porcine Primary Lens Epithelial Cells . Invest. Ophthalmol. Vis. Sci. 2005;46(13):3871.
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© ARVO (1962-2015); The Authors (2016-present)
Purpose: To understand the significance of the integrity of actin cytoskeletal and microtubule organization in the regulation of lens fiber cell elongation and differentiation–associated crystallin gene expression. Methods: Primary epithelial cells isolated from porcine lenses were treated for 24 to 72 hours with either cytochalasin D, an actin depolymerizing agent, or with nocodazole, an inhibitor of microtubule polymerization. Soluble cell lysates obtained from these drug–treated cells were examined for changes in ßB2 and γ–crystallin protein levels by immunoblot analysis. In a second set of experiments, we investigated the effects of the microtubule stabilizing agent, taxol, on the levels of ßB2 and γ–crystallin levels in lens epithelial cells pretreated with nocodazole. The status of actin cytoskeletal and microtubule organization in cytochalasin D and nocodazole treated lens cells was assessed by rhodamine–phalloidin staining and tubulin immunofluorescence labeling, respectively. Results: Porcine primary lens epithelial cells treated with cytochalasin D and nocodazole for 72 hours exhibited changes in cell morphology. Cell rounding and cell–cell detachment were evident primarily in cells treated with cytochalasin D, while nocodazole–treated cells remained flat, exhibiting a spread–out morphology. Immunoblot analysis of lens epithelial cell lysates revealed a dramatic and significant decrease in the levels of γ–crystallins only in cells treated with nocodazole, while cytochalasin D was without effect. Under similar conditions, the levels of ßB2 crystallin were also reduced by nocodazole but not to the extent as those of γ–crystallin. Neither nocodazole nor cytochalasin D had any effects on levels of αB and αA crystallins. Interestingly, treatment of lens cells with taxol for 30 minutes either prior to, or after, nocodazole treatment, reversed the nocodazole–induced decreases in the levels of γ–crystallin. Lens cells treated with nocodazole for 72 hours exhibited severe disruption of the microtubule network, but a normal actin cytoskeletal organization. Conclusions: Nocodazole–induced decrease in levels of lens epithelial cell γ–crystallin and its restoration by microtubule stabilization is supportive of a potential association between the expression of γ–crystallin and integrity of microtubule organization.
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