Friday, December 29, 2017

When the Sun Goes Down: How Scintillating Dinoflagellates Titillate the Golden State

By Amy

Have you ever heard of places in the world where the water glows at night? In places like Baja and southern California (1), these shining waves are actually illuminated by a type of algae! These algae are members of the dinoflagellates, which are known for their bioluminescence (2). Dinoflagellates also cause red tides, which are sometimes harmful blooms of algae that rise to the surface of the water, conferring a rusty red color; hence the name, red tides. During these red tides, the concentration of algal cells can reach 20 million cells per liter of ocean water (3)!
Lingulodinium polyedrum really lights up the shoreline! (Especially when you have a fancy camera and a really long exposure time.) This picture is from here.   
Left: Here you see a drawing of a Gonyaulax cell. Typical of dinoflagellates, it has several plates with a flagellum circling it (that propels it through the water) and a flagellum that sticks out the end of it (that helps with steering). Right: This beautiful little guy is a Lingulodinium polyedrum cell. So photogenic!   

Many species of dinoflagellates exist, but one of the most well-studied is Lingulodinium polyedrum (formerly known as Gonyaulax polyedra). L. polyedrum has been the focus of many scientific studies because of its bioluminescence (4). L. polyedrum is fascinating because it controls its bioluminescence with a circadian rhythm. You may be familiar with your own circadian cycles that tell you things like when to wake up, when to eat, and when to go to sleep. L. polyedrum likewise has circadian cycles of gene expression that drive its rhythms of bioluminescence (5).

To get us started, there are two big things that we need to discuss: bioluminescence and circadian cycles.

OK… How do these algae glow?
So, let’s talk a little bit about how bioluminescence works. Bioluminescence is caused by an enzyme called luciferase, which oxidizes luciferin (see the diagram below). This is a common chemical reaction that produces flashes of bioluminescence in many organisms (2). You may be more familiar with this chemical reaction than you know: fireflies use the same system to produce light (4)
Here you can see a cartoony explanation of the luciferase-catalyzed luciferin oxidation that produces bioluminescence. The green dots on the hexagon represent luciferin. Little blue pentagons represent luciferase, and the blue circles represent the oxygen that luciferase uses to oxidize luciferin. Once luciferin has been oxidized to oxyluciferin (the teal dots in the diagram), it is no longer able to give off light and is inactive.
As you can see, bioluminescence is essentially a chemical reaction. It happens when luciferase oxidizes luciferin that has been released from a protein that binds luciferin, aptly named Lucifer Binding Protein (or LBP for short). While LBP is bound to luciferin, luciferase cannot catalyze the oxidation of luciferin, and light is not produced. So, how does luciferin escape from LBP? This is where it starts getting more complicated. Inside of scintillons, which are little compartments in the cell that house the bioluminescence machinery, changes in pH alter the binding of LBP (6). The pH is a measure of acidity, where a higher number of hydrogen ions (protons) means a higher acidity and therefore a lower pH. In acidic conditions, LBP does a poor job of holding on to luciferin, and luciferase becomes more active. This means that luciferase can only oxidize luciferin in acidic scintillons (6). There are actually proton channels, which are proteins that let protons flow into the scintillons when activated, that acidify the scintillons before each flash of bioluminescence (6). The proton channels are activated through something called a signal transduction pathway which is actually started with mechanical forces acting on the cell. From the time this signaling pathway is activated, it only takes 15-20 milliseconds for the cell to produce a flash—in other words, bioluminescence really does happens in a flash (2).

This form of bioluminescence, called “flashing,” occurs in response to mechanical stimulation, but there is also another type of bioluminescence pattern called “glowing” which occurs more randomly and is solely regulated by the circadian clock (2). “Flashing” and “glowing” are both under circadian control, thus they both only happen at night. “Flashing” also requires physical stimuli, such as those caused by predators and kayak paddles, but “glowing” seems to happen randomly (2). It is still unknown whether glowing has a specific cause or whether it truly happens randomly throughout the night.

Does bioluminescence have any use?
Bioluminescence actually helps L. polyedrum cells not to get eaten. There are two current theories for how this works. The first theory is that flashes of bioluminescence startle predators away (I call this the Warning Light hypothesis), and the second theory is that the light actually attracts predators that eat the predators of the dinoflagellates, sort of like how a burglar alarm attracts cops to dispel robbers (I call this the Bigger Fish hypothesis). As it turns out, bioluminescence in L. polyedrum is used as a Warning Light when algal cell concentrations are low, but during red tides, when cell concentrations can get very high, the Bigger Fish hypothesis probably dominates (7).

Copepods provide an example for how L. polyedrum uses its bioluminescence to keep from getting eaten. Copepods are tiny crustaceans that prey on dinoflagellates. If there are few L. polyedrum cells and few copepods, L. polyedrum will produce dim flashes that startle copepods away (8). During red tides, when there can be very high concentrations of L. polyedrum cells in the ocean, the combined flashing produces enough light to attract fish such as the Flame Cardinalfish, which will eat the copepods, keeping the L. polyedrum cells safe from predation. Additionally, molecules produced by the copepods can actually trigger more bright bioluminescence to occur in L. polyedrum (8), so when there are many copepods around, even a small number of dinoflagellates can attract Flame Cardinalfish.
Copepods are tiny little crustaceans that are only about a millimeter long. That’s one shrimpy shrimp! They feed on dinoflagellates as prey, shown in this picture. The dinoflagellate pictured above is Alexandrium fundyense, a red tide species that can be found on the East Coast of the United States. A. fundyense and L. polyedrum both face predation by copepods. 
There’s always a bigger fish, right? As it turns out, in this case, the bigger fish is
the Flame Cardinalfish, which is only a couple inches long.
So… Getting back to circadian cycles… How do those work, exactly?
Circadian cycles (also called circadian rhythms) are biological cycles that occur over an approximate 24-hour period (4). In order for a biological cycle to be considered a “true” circadian rhythm, it must have certain properties. One such property of a circadian cycle is that it must occur on a 24-hour basis without external stimuli (4). This means that, while a circadian cycle may be synchronized to an external stimulus (i.e. sunlight), if this stimulus is taken away (i.e. the organism is placed in constant light conditions where sunlight does not fluctuate), the circadian cycle will continue regardless on an approximate 24-hour schedule. Another property of a circadian cycle is that it can be entrained (4). This is essentially what happens to us when we experience jet lag. Entering a new time zone means that we are now on a different day-night cycle than before, but we are only jet lagged for a day or two until our circadian cycles are able to adapt and entrain to this new schedule. One other interesting property of circadian cycles is that they function at a single-cell level, even in multicellular organisms like humans (4).
You have a circadian rhythm too! This diagram highlights the sleep-wake cycles seen in humans.    

What else does the circadian rhythm control?
The most well-studied circadian system in L. polyedrum is its cycle of bioluminescence, but this is not the only circadian cycle in this dinoflagellate. Many other processes, such as photosynthesis, cellular aggregation and movement, cell division, and protein synthesis are also controlled by circadian rhythms (5). Photosynthesis is essentially the process of turning sunlight and carbon dioxide into energy and food so, as you might imagine, the light-dependent reactions of photosynthesis occur during the day, not at night. The circadian control of aggregation and movement helps with this too. During the day, the algae tend to clump together (aggregate) and float to the top of the water where it is easier for sunlight to reach them (4). On the flip side, it is not very useful for the cells to be at the very surface of the water at night when there is not sunlight anyway. They exit their clumps and sink a little lower in the water where they can find other nutrients that don’t depend on sunlight, such as nitrogen (4). Yet another circadian rhythm controls cell division. L. polyedrum is most likely to divide around dawn (4). Finally, protein synthesis is also controlled by a circadian rhythm. This is the famous one, and the one that is also involved in bioluminescence (more on the specifics of that later). The short explanation is that different proteins are actually translated at different times of day in a circadian manner, including the proteins needed for bioluminescence (5).

Wait—hold up! Protein translation? Can we go over that quickly before moving on?
Oh yes, gladly! If you’d like a quick little refresher (or crash course!) on the central dogma, here it is: DNA sits in the nucleus of a cell, holding the genetic information. DNA doesn’t do much though, besides sit around and store information. In order for the information stored in DNA to have any effect on the cell, this information has to be converted into a form that can actually affect the cell, such as a protein. Proteins are actually what carry out most of the main functions of cells, doing the dirty work of the DNA. To get the information into protein form, messages encoded in messenger RNA (mRNA) are transcribed from DNA in the nucleus, exported out of the nucleus, and translated into proteins by large molecular machines called ribosomes. These proteins are now available to perform their designated function in the cell.
This is a diagram showing the central dogma. What we’re concerned with here is “GENE 1” which codes for an mRNA that makes a protein (polypeptide). Transcription is the process of converting the information in DNA into RNA, and translation is the process of converting the message in mRNA into a protein. There are other types of RNA molecules that are encoded by DNA, but for our purposes here, we only really need to worry about mRNA, which is translated into protein. Image from here.
But getting back this circadian rhythm business…
Right, back to the circadian cycle. For this part, I’m going to focus on the cycle of bioluminescence because that’s the most well-known circadian cycle in L. polyedrum. As mentioned previously, luciferase oxidizes luciferin, giving off light. This reaction occurs at night because during the day, luciferase cannot oxidize luciferin for three main reasons: 1. The scintillons, which are the compartments in the cell where luciferase and luciferin are kept at night to produce bioluminescence, are degraded in the morning, 2. LBP blocks the interaction between luciferase and luciferin in alkaline environments, and 3. Luciferase, luciferin, and LBP are actually not being made during the day, and their levels in the cell drop dramatically as the scintillons and the molecules inside are degraded (4). These mechanisms effectively shut down bioluminescence during the day. At night, the scintillons are re-formed, LBP releases luciferin inside the scintillons, and luciferase is able to oxidize luciferin, giving off light (4). I included a picture below that outlines these three controls on bioluminescence to help visualize what is going on.
I drew out this diagram to help visualize the controls on bioluminescence. #1 shows that at night, each cell contains hundreds and hundreds of scintillons, but during the day, they are degraded, and each cell is left with about forty (4). #2 shows that, in an alkaline scintillon (pH 7.5), LBP is holding on to luciferin. Protons (H+) must be pumped into scintillons in order to create a more acidic environment (pH 6.5) so that LBP will let go of luciferin. Luciferase is also more active in acidic environments, where the pH is less than 7. This means that outside of scintillons, or inside of alkaline scintillons, luciferin will not be oxidized, and no light will be produced (6). #3 shows that the ribosome is unable to bind to the mRNA of bioluminescence-related proteins (LBP pictured above) during the day, so these proteins (LBP and luciferase) are made during the night.

Interestingly, the mRNA molecules that encode the proteins involved in bioluminescence are constantly being transcribed, so the message is always present. It is protein translation that fluctuates on a circadian cycle, not RNA transcription. Circadian control of translation is probably mediated by proteins that bind to the ends of the mRNA molecule (called Circadian-Controlled Translation Regulator proteins, or CCTR for short) and prevent proper interactions with the ribosome (4). (Remember, the ribosome is the molecular machine that makes proteins.) When CCTR is blocking the mRNA, the ribosome is unable to attach to the mRNA, and therefore it cannot make proteins such as LBP or luciferase. So, while the message is constitutively transcribed, it is only translated at night (4). This is why the bioluminescence proteins are not made during the day, as mentioned above in #3.

How do we REALLY know that these cells have circadian cycles, and aren’t just sensing sunlight?
Great question! We know this because if L. polyedrum cells are brought indoors and left in a room without fluctuating light, they retain their circadian cycle (5). This means L. polyedrum actually has a built in “clock” that makes certain processes happen at different times, characteristic of a true circadian cycle. This is, of course, synchronized to the sun in nature, but in a lab setting with constant light, the cycle will actually continue by itself. Even more fascinating, if you have two L. polyedrum cultures that are not in sync (their cycles peak at different times: think jet-lagged), they will actually synchronize to each other, even with constant light (5)! How can this happen? The cells must be communicating somehow with each other so that they can be on the same time schedule (5). In fact, L. polyedrum cells are indeed communicating. They accomplish this by secreting molecular signals that diffuse through the liquid surrounding the cells. They use a molecule called gonyauline to essentially communicate the lengths of their circadian cycles with surrounding cells (9). This is really an amazing feat, considering the incomprehensible number of cells that must exist out in the ocean off the coast of Mexico and California!
This is a diagram of gonyauline I made. Gonyauline is secreted by L. polyedrum cells, diffuses through the water, and allows these cells to synchronize their circadian clocks to each other. 

Conclusion
Lingulodinium polyedrum is a fascinating microorganism that can be found in the ocean, glowing at night according to its circadian cycles. Research on circadian cycles is important and fascinating because circadian clocks exist in almost all organisms. Plants, animals, and microbes alike all have circadian rhythms that control their behavior at different times of the day, influencing their health and wellbeing. Research on the mechanisms that control these “clocks” even won the Nobel Prize in Physiology or Medicine this year (10). Not only does L. polyedrum provide a beautiful display in the oceans at night, its circadian cycles can also provide insight into circadian cycles in other organisms, with potential applications in human health.

And now I leave you with a little song parody I wrote for the amazing dinoflagellate we all now know and love, Lingulodinum poloedrum.

“Hey polyedrum”: an ode to our good friend, Lingulodinium polyedrum    



Works Cited

1.        J. W. Hastings, Circadian Rhythms in Dinoflagellates: What Is the Purpose of Synthesis and Destruction of Proteins? Microorganisms. 1, 26–32 (2013).
2.        K. Jin, J. C. Klima, G. Deane, M. D. Stokes, M. I. Latz, Pharmacological Investigation of the Bioluminescence Signaling Pathway of the Dinoflagellate Lingulodinium polyedrum: Evidence for the Role of Stretch-Activated Ion Channels. J. Phycol. 49, 733–745 (2013).
3.        Latz Laboratory, Dinoflagellates and Red Tides. Scripps Inst. Oceanogr., (available at https://scripps.ucsd.edu/labs/mlatz/bioluminescence/dinoflagellates-and-red-tides/).
4.        M. Mittag, Circadian Rhythms in Microalgae. Int. Rev. Cytol. 206, 189–212 (2001).
5.        J. W. Hastings, The Gonyaulax clock at 50: Translational control of circadian expression. Cold Spring Harb. Symp. Quant. Biol. 72, 141–144 (2007).
6.        J. D. Rodriguez et al., Identification of a vacuolar proton channel that triggers the bioluminescent flash in dinoflagellates. PLoS One. 12, 1–24 (2017).
7.        K. D. Cusick, E. A. Widder, Intensity differences in bioluminescent dinoflagellates impact foraging efficiency in a nocturnal predator. Bull. Mar. Sci. 90, 797–811 (2014).
8.        J. Lindström, W. Grebner, K. Rigby, E. Selander, Effects of predator lipids on dinoflagellate defence mechanisms - Increased bioluminescence capacity. Sci. Rep. 7, 1–9 (2017).
9.        H. Nakamura, M. Ohtoshi, O. Sampei, Y. Akashi, A. Murai, Synthesis and absolute configuration of (+)-Gonyauline: A modulating substance of bioluminescent circadian rhythm in the unicellular agla Gonyaulax polyedra. Tetrahedron Lett. 33, 2821–2822 (1992).
10.      The 2017 Nobel Prize in Physiology or Medicine - Press Release. Nobel Media AB (2017), (available at http://www.nobelprize.org/nobel_prizes/medicine/laureates/2017/press.htm).

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