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S.C. Chamberlain, R.E. Flynn, R.B. Sacunas; Investigation of Light-Independent Shedding in the Lateral Eye of the American Horseshoe Crab . Invest. Ophthalmol. Vis. Sci. 2003;44(13):2855.
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Purpose: Light-independent rhabdom shedding was recently discovered as a third mechanism for removing light-sensitive membrane from the rhabdomere of horseshoe crab photoreceptors (Sacunas et al., 2002, JCN 449:26-42). This study aims to identify and characterize the mechanism whereby light-independent shedding removes membrane from the microvillar array. Methods: The lateral optic nerves (LON) of some animals were cut bilaterally 2 weeks prior to the experiment to suppress transient rhabdom shedding. Intact and cut LON animals living in natural lighting were sacrificed every two hours for one 24-hr period in late June. The lateral eyes were fixed and processed to preserve the ultrastructure. Thin sections were examined by transmission electron microscopy. Results: Because light-independent shedding, like transient shedding shows immunoreactivity to opsin, but not arrestin, whereas light-driven shedding labels with both anti-arrestin and anti-opsin antibodies, we scrutinized the rhabdom in the animals with cut LON for any evidence of membrane removal by the ballooning of microvillar membrane rather than by clathrin-adaptin coated vesicles. We noted significant numbers of small blebs of rhabdomeral membrane protruding into the cytoplasm at all times of the day in the animals with cut lateral optic nerves. Intact animals showed significantly fewer such blebs, except at dusk (2000 hr). Lumped data for cut LON animals yielded 0.681 ± 0.21 blebs/µm (mean ± s.d.). Data for intact animals has a range from 0.270 ± 0.06 to 0.475 ± 0.13 blebs/µm at all sample times except 2000 where the mean and standard deviation was 0.710 ± 0.19 blebs/µm. Statistical analyses showed that the data for cut LON was different from the data for intact animals (excluding 2000) with <0.01% significance, but that the data for cut LON animals and intact animals at 2000 could not be considered different. Conclusions: Ultrastructurally, light-independent shedding resembles attenuated transient shedding, proceeding by ballooning of microvillar membrane into the cytoplasm. There appears to be a burst of light-independent shedding at dusk (2000) in intact animals. This is consistent with the morphometric data of Chamberlain and Barlow (1984) that showed a reduction in rhabdom area (shedding) in early evening.
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