Abstract
Introduction: :
Opsin coexpression is more common than previously thought. But the functional relevance of opsin coexpression is mostly unknown as is whether the levels of coexpressed opsins can be differentially regulated. Three opsins are express in Limulus lateral eye (LE): opsins1 and 2, which are 99% identical to one another and called here Ops1-2, and Ops5, which is only 45% identical to Ops1-2. Ops1-2 and 5 are coexpressed in Limulus LE photoreceptors.
Purpose: :
to determine relative levels of Ops1-2 and Ops5 in LE photoreceptors and test whether their relative levels in the photosensitive membrane (rhabdom) change with a diurnal rhythm or are influenced by the circadian clock.
Methods: :
Antibodies specific for Ops1-2 and 5 were applied to Western blots of SDS solubilized LE membranes and known amounts of the antigens against which the antibodies were raised. Immunoreactivity (ir) was visualized with chemiluminescence, and the Ops-ir in the membranes was compared to that of the antigen standards. Immunocytochemistry and confocal microscopy was used to assay relative levels of Ops1-2- and 5-ir in rhabdoms of LEs (1) dissected at three different times of the day from animals maintained in natural illumination. (2) dissected at night from dark-adapted LEs that either received or were deprived of normal circadian clock input.
Results: :
At night, when most Ops-ir in the LE is in the rhabdom, the ratio of Ops5 to Ops1-2 in LE membranes is about 1:5. Immunocytochemistry showed that rhabdomeral Ops1-2-ir falls during the day to about 50% of its nighttime level while the rhabdomeral level of Ops5 is unchanged. Thus, during the day, the ratio of Ops5 to Ops1-2 at the rhabdom is estimated at about 1:2.5. We also found that rhabdomeral Ops1-2-ir is significantly (35%) lower in nighttime LEs deprived of circadian clock input compared to control eyes with clock input, while Ops5 levels are not significantly reduced.
Conclusions: :
In LE photoreceptors Ops1-2 is more abundant than Ops5, but the level of Ops5 is sufficiently high to contribute significantly to the photoresponse, especially during the day. The relative levels of Ops1-2 and 5 at the rhabdom change with a diurnal rhythm and are influenced by the circadian clock. Ops1-2 levels fluctuate more than Ops5. This is the first evidence that the levels of coexpressed opsins can be differentially regulated.
Keywords: opsins • circadian rhythms • photoreceptors