Putative pacemakers of crayfish show clock proteins interlocked with circadian oscillations.

Although the molecular mechanisms that control circadian rhythms in many animals, particularly in the fly, are well known, molecular and biochemical studies addressing the location and function of the proteins and genes contributing to the cycling of the clock in crayfish Procambarus clarkii are scarce. In this study, we investigated whether three proteins that interact in the feedback loop of the molecular clock described for Drosophila are expressed in the putative circadian pacemakers of crayfish retina, eyestalk and brain and whether their expression cycles in a manner consistent with elements of the circadian clock. Here we identified PER, TIM and CLK immunoreactivity in the cytoplasm and nucleus of cells located in the retina as well as in clusters of cells and neuropils of the optic ganglia, lateral protocerebrum and brain. Brain clusters 6, 10, 9 and 11, in particular, showed Per, Tim and Clk-like immunoreactivity at the perikarya and nucleus, and these antigens colocalized at Zeitgeber time (ZT) 0 and/or ZT 12. A biochemical assay demonstrated circadian functionality of Per, Tim and Clk proteins. Both in the eyestalk and in the brain, these proteins demonstrated apparent daily and circadian rhythms. The presence and colocalization of these clock proteins in the cytoplasm and/or nucleus of several cells of retina, optic lobe and brain, depending on time, as well as their circadian oscillations, suggest interactions between positive and negative transcription factors and clock proteins similar to those forming the feedback loop of the canonical model proposed for different animals.