Most of the work on the molecular mechanism has been done using constant conditions. In the first two lectures we characterized the circadian clock with a collection of properties of which free running rhythm is just one. Two other important hallmarks of the circadian clock are entrainment and temperature compensation. How did they fit into the molecular clock mechanism? In most of the clock mutants that were tested for these properties, they're not normal. So temperature compensation is defective and if entrainment occurs, it's often at the wrong time of day. Another way to approach entrainment is to ask how zeitgebers influence the circadian clock. Which parts of the molecular mechanism are sensing the environment for time cues. It turns out that for light there are pretty direct pathways into the clock mechanism. The most extreme example comes from Neurospora, the fungus. Where one of the transcriptional activators, white collar one, and one of the transcriptional repressors, vivid, are both blue light photo receptors. You might be thinking another transcriptional regulator with a modular functional domain and you would be right. These proteins harvest light via their flavan cofactors. And this changes their activating potential. Light induced transcription in Neurospora is highly regulated with for instance wc-1 and frq RNAs expressed at high levels within minutes of exposure of the fungus to light. This is clearly not the entire story to explain entrainment in Neurospora. If you look at the protein made from the frq RNA and the conidiation that synchronized or entrained by light, the timing of these processes depends not on when light comes on but rather it depends on if a winter or a summer day is simulated. In a summer day, the protein's made only much later, hours after light exposure. In a winter simulation after a long night, the protein is made like the RNA, within minutes. This indicates the many layers and levels of regulation the circadian clock to get just the right timing especially during entrainment. After all, the entrained state is how the clock is functioning in nature. In the case of conidiations, no matter what the structure of the photo period within the 24 hour day. If it's long like in summer or short like in winter or presented as an equinox the conidiation is initiated around midnight. The Neurospora circadian clock can integrate a complex set of light and dark conditions to fine-tune in their FRQ expression and their spore formation. In flies, a blue light photo receptor called CRYPTOCHROME, also using a flavan molecule as a photo receptive cofactor, directly binds the TIMELESS protein on light exposure and degrades it thereby removing half of the HETERODIMER that is involved in the negative feedback mechanism. In the case of Neurospora light induced transcription increases the negative feedback element. In the case of flies, light decreases it. So these systems have developed somewhat different strategies for sensing light but they both depend on flavin mediated blue light sensing. In mice, light comes into the circadian clock from a bit further away from the feedback loop. It is only sensed by photoreceptors in the retina. In addition to some circadian photoreception via the rods and cones, the circadian blue light photoreceptor Melanopsin is expressed in non-visual cells. Cells from the retina extend to the suprachiasmatic nucleus, the SCN, the circadian pacemaker in the brain. And on light exposure, gene expression of the period genes is increased in the SCN. This is time of day specific - PER1 and PER2 are both light induced in the afternoon and evening, causing the circadian clock to be pushed back and delayed. In the end of the night and morning, PER1, but not PER2, is induced, and this results in the circadian clock advancing and moving ahead. So what's the zeitgeber for all of those other cells that have a free running circadian rhythm? An important zeitgeber to entrain many circadian systems and many of our cells is temperature but almost nothing is known as to how this particular signal is integrated by the clock. So I won't discuss that any further here. Another pervasive zeitgeber for mammalian cells is glucocorticoid. If you add a glucocorticoid analog to cell cultures, all the cells synchronize their free running rhythms. Glucocorticoid gives a strong reset. In vivo in a mammal, glucocorticoid receptors are widely expressed and glucocorticoid is clock regulated itself. Glucocorticoid is thus potentially an important endogenous signal for synchronization of the cellular circadian clock. In plants, light has somehow a bigger role in the survival of the organism and perhaps it's appropriate then that they have a large collection of photoreceptors sculpting and regulating the circadian clock. In the case of plants CRYPTOCHROMES and PHYTOCHROMES - so both blue light sensing and red light sensing proteins - are regulating circadian rhythms using flavins and heme molecules as photoreceptive co-factors. A common question that's been asked over the years is where does the 24 hours come from. This is supposed to frame certain tests that might indicate which molecular processes shape the very special kinetic of circadian rhythms. Transcription, translation, RNA or protein stability and so on. Most of these molecular processes can occur with a much higher frequency. For instance Hes1 is a transcription factor that oscillates in its expression each couple of hours. There's no single answer to the question where the 24 hours comes from. The kinetics of induction and destruction of each of the components that are involved in the clock mechanism should play a role in circadian timing. But there are also surprising results from various experiments that change the period of the rhythm little despite big changes in various components through gene manipulation. I think a much more interesting question is which cellular networks and molecules are contributing to transcription of RNA, to the translation of protein and to the degradation of clock gene RNA and proteins. By prediction, these factors should also be involved in the clock timing mechanism. Indeed mutant screens have turned up a number of these, Most notable Fbox proteins which are involved in tagging proteins with ubiquitin, a signal that a protein should be degraded. All of the eukaryotic clock model organisms feature proteins whose career is getting other proteins degraded. One of the next challenges might be to understand how much timing doesn't depend on RNA and protein oscillations. Keeping in mind the clock and the tube, how much of the temporal program could be covered by mechanisms that don't depend on rhythms in transcription or translation? From a molecular perspective, these are very tricky experiments to perform. I've given you a lot of information today about genes and proteins and metabolic processes that control circadian rhythms. For those of you with a scientific background, this might be about the right level. Probably the most important concept is that the transcriptional feedback mechanism is common to circadian clocks in the eukaryotic lineage whether they be plants, animals or fungi. It's also important to see non transcriptional regulation not only downstream of the circadian clock but also feeding back into the molecular mechanism. We addressed some of the molecules that mediate entrainment especially with light. A process that you heard about in the previous lecture. I think it's interesting how the various model organisms have contributed different pieces of the puzzle, according to tools that are available. For the listeners that are not scientists, I think that there are important take home messages that you can also get from this lecture. Probably the most important concept is an understanding of exactly how hardwired the circadian clock is in all of our cells. How feedback systems from various levels shape our daily timing. In the next lecture we'll discuss some of the many processes that are clock regulated from neurotransmitters to behaviors and performance. See you then.