Circadian Rhythm Regulation in Chlamydomonas

by BA

How many times have you stayed up late for a party or, more likely but less fun, for last minute work, sacrificing your usual sleep schedule with the thought that “I can sleep when I’m dead”? You probably found that it’s difficult to catch up on that lost sleep, and sometimes it even takes a couple days to get that sleep-wake cycle back to where it should be.

You can thank your circadian rhythm for that. The circadian rhythm is the 24-hour cycle that regulates your sleep patterns and other physiological processes, like blood pressure and hormone levels, based on your environment. It is regulated by your circadian clock, which makes it possible for you to coordinate your behavior with environmental changes that result from the day-night cycle. Light and temperature can affect it, as evidenced by animal migration and seasonal depression in some people, and it can be located in various parts of the organism’s body. For example, monarch butterflies have circadian clocks in their antenna (1), plants have cells that detect light (known as photoreceptors) to help regulate certain genes, and humans have circadian clocks in a tiny region of the brain called the suprachiasmatic nuclei. Interestingly, unicellular organisms can have circadian clocks too, such as the photosynthetic green algae Chlamydomonas. Why do these little guys need a circadian rhythm, and how have studies on them helped us understand our own?

Chlamydomonas cell with its
components labeled. (8)    

Chlamydomonas has a small, light-sensitive organelle near the middle of the cell known as an eyespot that allows it to move depending on the intensity of the light in its environment. Cells like Chlamydomonas are attracted to sources of light in a phenomenon we call positive phototaxis. (2) This allows cells to find light sources during the day so they can utilize their photosynthetic mechanisms more efficiently. (3) When the night comes and very little light is to be found in the environment, the cell turns to chemotaxis, which is the movement towards or away from certain chemicals or molecules. Chemotaxis allows the cells to find sources of nitrogen, an essential nutrient for photosynthetic organisms. (4) This type of switching behavior allows Chlamydomonas to use its energy in the most efficient way possible; trying to use photosynthesis at night would be useless due to the lack of sunlight, and using chemotaxis to find nitrogen during the day would only waste precious time that would be better spent on photosynthesis. The constant rotation between phototaxis during daylight and chemotaxis at night is indicative of circadian rhythms in the organism, which means there must be a circadian clock. Indeed, many genes for the circadian clock in Chlamydomonas have been identified, including gene families (sets of genes with a similar functions) responsible for phototaxis, and several independent genes attributed to the overall robustness of the circadian clock mechanism. (5)

One gene family in Chlamydomonas called per has been identified as a main regulator of circadian clock periods. Chlamydomonas cells with mutant per genes, wherein the DNA of the genes has been somehow damaged, have been demonstrated to have shortened or lengthened clock periods. (5) By allowing cells with mutant per to grow in culture, waiting an expected number of normal circadian cycles, and testing phototaxis, scientists were able to determine how the mutant genes disrupted the circadian rhythm of the organism. If some cells underwent phototaxis when they weren’t expected to, like during times without sunlight, they were presumed to be mutants. This was confirmed with genetic experiments that involved allowing the mutants to sexually reproduce and examining the new cells’ phototactic movements, which further revealed the existence of multiple genes responsible for the mutants. (5)

Another gene, CK1, encodes a protein kinase, which is an enzyme that changes the function of other proteins by adding phosphate groups to them. CK1 is known to be one of the proteins specific to both the Chlamydomonas eyespot and flagella, a whip-like organelle used for locomotion. (6) The presence of CK1 in both the light-sensing and movement organelles implies that it is important to phototaxis. To test this idea, the functional CK1 gene was prevented from working in the cell, known as knocking out the gene, by a method called RNA interference where RNA was used to specifically block the gene from being expressed. This interference resulted in defects in the formation of flagella, where cells synthesized small flagella or even none at all. (6) RNA interference can be made to be stronger or weaker, and in this case, as interference got stronger, more severe defects in flagella were observed. These CK1 knockout cells were also subjected to phototaxis tests where they were exposed to bright light to mimic the day and darkness to mimic the night. While cells with the normal CK1 gene (wild-type cells) exhibited normal attraction to light during the day and no attraction during the night, the CK1 knockout cells showed significantly lower attraction to the light in the daytime. (6) This can be described by the mutation of the CK1 gene not being able to express its protein kinase. Without the CK1 protein in the Chlamydomonas cells, the eyespot was less able to detect light, and deformed flagella was not sufficient to move the cell towards whatever light it could detect.

ROC genes are suspected to be core genes of the Chlamydomonas circadian clock. They encode transcription factors, which control the expression of other genes in the journey from DNA to proteins. (7) Cells with mutations in their ROC genes display strange circadian cycles, either short, long, or inconsistent. Because these genes control expression, scientists thought they might have a role in feedback mechanisms that regulate the circadian clock, mechanisms in which the product of gene expression determines whether more product is made or not. (3) In particular, two proteins, ROC75 and ROC40, were suspected to have some sort of interaction. This was examined by fusing fluorescent tags to each protein and watching where they went in the cell. T. Matsuo presented his findings at the 14th Annual Chlamydomonas Conference in 2010, explaining that ROC75 was found in around the nucleus of the cell and interacted with the promoter region of the ROC40 gene, part of the DNA that controls the initiation of gene expression. This suggests that ROC40 expression is regulated by ROC75. Levels of expressed ROC genes were then shown to change depending on the phase of the circadian cycle (3). This evidence tells us that genes with feedback mechanisms that respond to day-night cycles form the basis of the circadian clock in Chlamydomonas.

The cool thing is that these genes have homologs in other eukaryotes, including us. (3) Chlamydomonas circadian clock genes have strong homology (similarity) with genes found in both plants and higher animals. Some genes in the ROC family share similarities with the genes responsible for the circadian clock and seasonal flowering in the plant Arabidopsis thaliana. (3) As another example, CHLAMY1, a protein in Chlamydomonas that binds to RNA, has a subunit named C3 that is extremely similar to the protein CUGBP2 in rats. In fact, if an antibody that specifically targets the Chlamydomonas C3 protein is introduced into rats, it will recognize the CUGBP2 protein in the suprachiasmatic nuclei of the rat brain – the same part of the brain that is responsible for the human circadian rhythm. This result suggests numerous similarities between higher animals and the unicellular Chlamydomonas, making it a good model for examination of the human circadian rhythm.

The circadian rhythm allows us to complete our day-to-day tasks as best we can, both conscious (working our jobs and sleeping the proper amount each night) and unconscious (hormone regulation and tissue repair). While Chlamydomonas doesn’t use it for sleeping like humans, it requires functional circadian clock genes to make efficient use of its energy and thrive. By studying this little algae, we can better understand ourselves and the world around us.

References

1. Merlin C, Gegear RJ, Reppert SM. 2009. Antennal circadian clocks coordinate sun compass orientation in migratory monarch butterflies. Science. 325(5948): 1700-1704.
2. Stavis RL, Hirschberg R. 1973. Phototaxis in Chlamydomonas reinhardtii. J Cell Biol. 59(2): 367-377.
3. Matsuo T, Ishiura M. 2011. Chlamydomonas reinhardtii as a new model system for studying the molecular basis of the circadian clock. FEBS Lett. 585(10): 1495-1502.
4. Fernandez E, Galvan A. 2007. Inorganic nitrogen assimilation in Chlamydomonas. J Exp Bot. 58(9): 2279-2287.
5. Matsuo T, Ishiura M. 2010. New insights into the circadian clock in Chlamydomonas. Int Rev Cell Mol Biol. 280: 281-314.
6. Schmidt M, Gessner G, Luff M, Heiland I, Wagner V, Kaminski M, Geimer S, Eitzinger N, Reissenweber T, Voytsekh O, Fiedler M, Mittag M, Kreimer G. 2006. Proteomic analysis of the eyespot of Chlamydomonas reinhardtii provides novel insights into its components and tactic movements. Plant Cell. 18(8): 1908-1930.
7. Brunner M, Merrow M. 2008. The green yeast uses its plant-like clock to regulate its animal-like tail. Genes & Dev. 22:825-831.
8. Chlamydomonas photo courtesy of Connecticut College. Accessed November 12, 2015. http://fmp.conncoll.edu/Silicasecchidisk/LucidKeys3.5/Keys_v3.5/Carolina35_Key/Media/Html/Chlamydomonas_Main.html