In this last section of this lecture on what and how the circadian clock regulates, I want to touch on three additional examples. In our discussion of circadian organization I told you that the clock is ticking in practically all cells. An interesting class of cells that has a robust and complex circadian program are those of the immune system. Some of them have extremely high amplitude oscillations in gene expression. Others are targeted to specific compartments according to time of day. Hematopoietic stem cells go to peripheral tissues at the beginning of the sleep phase, and they circulate in the blood at the beginning of the activity phase. Immune cell specific factors called cytokines or lymphokines are expressed rhythmically. And this adds up to a defense system that's poised to perform different tasks better at different times of day. This too has been experimentally tested with more or less severe illness resulting from infections or challenges with toxins at different times of day. So the immune system is a major target of the circadian clock. I want to use plants to highlight how the clock is regulating the levels of calcium inside the cell. Calcium is extremely tightly regulated since it is a co-factor for signaling processes and for some molecules and their function. A calcium sensitive reporter protein was transformed into plants and the results showed a striking time of day dependent signal suggesting that the clock is using calcium levels to regulate protein function and cellular signaling potential. In animals, oscillations in calcium channel function, and calcium signaling in neurons has been documented for mammals down to snails, suggesting that this ion is involved in an ancient and shared clock mechanism. The last example of clock regulation that I want to talk about concerns metabolism. The reduced or oxidized state of proteins or co-factors of proteins in the cell also have been followed as clock controlled outputs of the system. Recently we got a new glimpse on this when it was shown that the oxidized form of the molecule peroxiredoxin is rhythmic in clock model organisms from all phyla. This molecule is involved in handling molecular products of oxidation. They act like a sponge allowing a cysteine residue to be oxidized, and then either recycled by reduction or becoming overoxidized to a stable state. Antibodies pick up the stable oxidized molecule of peroxiredoxin and show rhythms in this form of the protein and organisms from all phyla, suggesting that the regulation of redox state with this molecular system is an ancient part of the molecular circadian clock. The puzzling observation is that daily rhythms and oxidized PRX were also observed in mutants that were deficient in clock genes. Many clock mutants - if you get rid of the entire clock gene - become arrhythmic for the phenotype that they were selected on. Mutants that did not show circadian rhythms in most other functions could still support circadian rhythms in oxidized peroxiredoxin. Even human red blood cells show these oscillations, which is remarkable since they have no transcription and therefore no transcriptional feedback loop. There is as yet no evidence that the oxidation of peroxiredoxin has an effect on the clock itself. So it's taken as an example of another major part of the clock representing an output pathway. It also underscores how extensive the clock network is, that there's a molecular clock network ticking even under conditions that might silence the transcriptional machinery. We're now at the end of the fourth lecture. Here is a summary slide and the next video is the wrap-up session. [SOUND]
