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A. Lichtenstein, P. J. Minogue, E. C. Beyer, V. M. Berthoud; Autophagy: A Degradative Pathway for Lens Connexins. Invest. Ophthalmol. Vis. Sci. 2008;49(13):1523.
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To elucidate the involvement of autophagy, a degradation pathway induced by cellular stresses like starvation, in proteolysis of wild type connexins and a cataract-associated mutant.
Levels and cellular localization of wild type CX50 and the mutant, CX50P88S, were assessed by immunoblotting and immunofluorescence in samples from stably transfected HeLa cells. Autophagosomes were visualized by fluorescence microscopy using monodansylcadaverine (MDC), anti-LC3 antibodies or after transient transfection with GFP-LC3. Lysosomes were detected by fluorescence imaging after transient transfection with LAMP1-YFP.
CX50 localizes to gap junction plaques and in the perinuclear region whereas CX50P88S localizes to cytoplasmic accumulations. Under normal growth conditions, some of the cytoplasmic CX50 spots co-localized with GFP-LC3 or anti-LC3 immunoreactivity. In contrast, most CX50P88S cytoplasmic accumulations co-localized with autophagosomal (MDC, GFP-LC3 and anti-LC3 antibodies) and lysosomal (LAMP1-YFP) markers. Starvation, which induces autophagy, decreased levels of CX50 and decreased anti-CX50 immunoreactivity both at the plasma membrane and at intracellular locations. Similarly, starvation led to a reduction in the amount of CX50P88S as detected by immunoblotting and immunofluorescence. Under normal growth conditions or during starvation, chloroquine, a lysosomal inhibitor, increased levels of CX50 as well as the extent of overlap between LC3-GFP and anti-CX50 immunolabeled structures. Impairment of autophagy by treatment with an Atg5-silencing RNA elevated levels of CX50.
These results indicate that wild type and mutant lens connexins are degraded through autophagy. The association of most CX50P88S accumulations with autophagosomal markers suggests that impaired autophagic degradation may be involved in cataract formation.
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