Abstract
Purpose::
Myosin III (myo3) may mediate some of the circadian changes in Limulus photoreceptors that lead to a nighttime increase in visual sensitivity. Clock input to eyes at night causes the PKA-mediated phosphorylation of the actin-binding region of myo3 which may reduce the affinity of myo3 for actin. Consistent with this hypothesis, we found that during the day in the light, when clock input to the eye is silent, myo3 concentrates over the actin-rich rays of the photosensitive rhabdom. However, in response to clock input, the concentration of myo3 over rhabdom decreases. The current study tested whether the change in myo3 distribution is caused by activation of receptors for the biogenic amine octopamine (OA), which is released onto photoreceptors at night from clock-driven retinal efferent neurons, and a subsequent elevation of cAMP in photoreceptors.
Methods::
Slices of lateral eyes (LEs) dissected from light-adapted animals two hours after sunrise were incubated in organ culture medium (OCM) for 2 hr in the dark at 150 C then for 6 hr in the dark in OCM containing no or one of the following additions: OA (40 uM), a phosphodiesterase inhibitor (1 mM IBMX), 1 mM IBMX plus 40 uM OA, or 8 Bromo cAMP (40uM - 500 uM). Tissues were fixed, frozen sections were immunostained for myo3 and actin, and the distributions of antigens were examined with confocal microscopy.
Results::
Compared to control LE slices from the same animal, those incubated with IBMX plus OA or OA alone consistently showed a lower concentration of myo3 over the actin-rich rhabdom. In these tissues, myo3 often concentrated at the edges or between the rays of rhabdom; actin also concentrated near the edges of the rhabdom in these tissues. In eyes of some animals, IBMX alone caused similar changes in myo3 and actin distributions. Surprisingly, incubations with even a high concentration of 8 bromo cAMP were relatively ineffective at changing the distributions of myo3 and actin in photoreceptors.
Conclusions::
We conclude that the reduction of myo3 over the rays of the actin-rich photosensitive membrane is caused by the circadian efferent neurotransmitter OA. Since incubations of LE slices with IBMX alone and IBMX plus OA produce a 2-fold and a 10-fold increase in cAMP levels, respectively, our findings also suggest that the activation of a cAMP cascade is involved. Our finding that high levels of 8 bromo cAMP have little or no effect is, on the surface, inconsistent with this idea. However, the changes in myo3 and actin distributions may require a local change in cAMP levels in the photoreceptor. We speculate that a reduction in myo3 at the rhabdom reduces actin stability in rhabdomeral microvilli.
Keywords: circadian rhythms • cytoskeleton • photoreceptors