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Reasons to Rejoice in Green Algae

By Lynne Quarmby

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Every once in awhile you get shown the light

In the strangest of places if you look at it right

– The Grateful Dead

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We’ve had three hundred years of microscopy and some of us are still fascinated with the beautiful creatures that swim in pond water. To the naked eye, to the unpracticed observer, they look like cloudy, icky scum and we don’t want to swim with them. But they are also delightfully alive, they congregate, they swim (and wouldn’t care if we swam with them), they even “see” or at least sense light. And under the microscope, in the lab, in experiment after experiment, these tiny green algae are yielding discoveries that are important to you and me, in terms of health and the environment and, yes, in the revelations they bring of the wondrous reality of the molecular world.

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In which wondering how Chlamydomonas navigates led to a revolution in the field of neuroscience.

Chlamydomonas swims by the synchronized beating of two flagella that pull the cell forward – think of a breast-stroke: a wide sweep that pushes back against the water and a narrow recovery stroke that repositions the flagella for another power stroke. My colleague Pinfen Yang has used a very high speed camera to capture the flagellar beat of these cells. Note especially the cell that comes up from the bottom right.

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Using a behavioural response aptly named phototaxis, Chlamydomonas swims to position itself where the sunlight is just right, not so bright that the sensitive molecules that capture photons will be damaged yet bright enough to provide sufficient photons for photosynthesis and growth. How does it find this sweet spot?

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The movie starts with cells being unstimulated and the time is displayed at the top in hours:minutes:seconds (00:00:00). When the hours go out, the cells are being stimulated from the left, when the minutes go out, the cells are stimulated from the right. After chasing the cells back and forth, they are flashed a few times with bright light that causes them to stall –- Greg Pazour, U. Massachusetts, USA

In order to phototax, the cell must sense the intensity and location of the light source. Chlamydomonas does this by way of an “eyespot” on one side of the cell, built such that it receives light only from the one side. The eyespot can provide information about the brightness of the light. In order to discern the direction of the light source, Chlamydomonas spins as it swims. This is achieved by a slight offset in the positioning of the two flagella. Imagine yourself as a Chlamydomonas cell, on your right hip is your eyespot; your arms are your flagella. Now, as you beat your arms in a breaststroke pattern, imagine being propelled towards the ceiling. Shift your right shoulder a little forward and your left one a little back, and continue beating your arms. Now you are spiraling up to the ceiling. Every time your right hip is facing that sunny window, there is a beep. Ah … radar. If the light is too bright, a loud beep causes your left arm to beat less effectively than your right arm and your spiral path arcs away from the light. A dim light has the opposite effect on your two arms. Cool, but how does the cell accomplish this?

A perk of science is the friendships built with people scattered around the globe. One such friend is Peter Hegemann with whom I have a special bond. Peter and his family generously hosted my son Jacob on the Berlin leg of Jacob’s solo post-high school backpacking trip. Peter works on phototaxis and his particular fascination is the relationship between light detection and a set of ion channel proteins.

Proteins are chains of amino acids that fold into characteristic three-dimensional shapes. What proteins do and how they interact with one another is determined entirely by their shapes – shapes that “breath” and can change dramatically in response to interactions with one another or with small molecules or with the physical world, including light..

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The sequence of a stretch of DNA, a gene, dictates the sequence of amino acids that in turn determines the shape of the protein. Ion channels are proteins that span the cell’s envelope (or membrane) and form pores that can be opened and closed by changes in shape, thereby controlling the flow of ions such as Ca2+, K+, Na+ and Cl-. (It is the tight control of ion channels that carries electrical impulses down the axons and dendrites of our neurons.) Gradually, Peter’s group revealed that communication between the eyespot and the flagella was via the gating of ion channels, not unlike what happens in our eyes.

In our eyes, light is detected in a specialized domain of rod and cone cells known as the outer segment. Outer segments contain the Rhodopsins – molecules that undergo changes in shape when hit by a photon. The change in shape of Rhodopsin triggers a cascade of shape changing by other molecules, culminating in the gating of ion channels and the sending of an electrical signal to the brain.

Peter’s quest was to identify the molecule in the eyespot that was responding to light and to learn how that molecule then generated the electrical signal that is transmitted to the flagella. His work had revealed that the time interval between light signal and electrical response in Chlamydomonas was much shorter than in our eye. The response was so fast that he speculated that the light receptor might itself be the ion channel. Eventually Peter’s group identified two proteins, Channel Rhodopsin-1 & -2 (ChR1 & 2) each of which behaved as both light sensor and ion channel. This work was published in 2002 and 2003.

About 250 scientists attend the biannual Chlamydomonas conference where we talk excitedly about green algae for six days straight. Occasionally there will be some yodeling or dancing, but mostly we share data and ideas. About 30,000 people attend the annual Neuroscience meeting and lately one topic dominates: using the Chlamydomonas ChR proteins to light up brains. This new technology is so hot it was recognized as Method of the Year for 2010.

As you know, the human brain is incomprehensibly complex: One hundred billion neurons, each one talking to 200,000 others, friending and unfriending with millisecond resolution. It is so complex that even formulating the questions is a challenge. One major hurdle has been the ability to precisely stimulate specific neurons with extreme rapidity and precision. It had been speculated for decades that a highly focused beam of light would be one way to accomplish this, but no one knew how to make that work.

In 2004, Karl Deisseroth, a bright and well-resourced young Stanford psychiatrist and bioengineer assembled a team to take on this high-risk project. The theory was straightforward: use genetic engineering to modify the Chlamydomonas gene so that it would only be turned on in neurons of a defined type (in mice). With ChR present in only a few select neurons, a beam of light could be used to activate those cells on cue. Although the experiments were difficult, in Deisseroth’s words, they worked “shockingly well.” Since 2006 there has been an explosion of new applications, refinements, and improvements. Peter Hegemann remains engaged in this effort, engineering ChR to a variety of specifications, thus further broadening the applicability of the technique.

Parkinson’s Disease, Narcolepsy, Depression, and Schizophrenia are early targets of this new technology. The approach is being applied with such intensity and breadth that we are on the threshold of numerous discoveries many of which may lead to the alleviation of human suffering, not only relating to brain function, but also the heart and other tissues as scientists take this new ball and run with it. It is stunning how quickly this new tool has stimulated a flurry of previously unthought-of questions.

Serendipitous discoveries with relevance to human and environmental health often surprise us, but in the field of cell biology, they are inevitable. The story of Channel Rhodopsins is an example of one reason this is so: Occasionally, powerful new tools arise from discoveries in unusual organisms because extremely bright scientists with wide-ranging open minds make novel connections. However, there is a more profound and fundamental reason that curiosity about the mysteries of life inevitably yields surprising and relevant outcomes.

The major route from the science equivalent of poetic musings to medicine and back again arises because of the inter-relatedness of all life. The next story is dedicated to Canada’s Science Minister, Gary Goodyear who, by the way, doesn’t believe in evolution (and was nevertheless recently re-elected).

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In which wondering how flagella are built led to a surprising discovery about Polycystic Kidney Disease

Although its flagella are important in nature, Chlamydomonas doesn’t need them to survive in the lab. Because of this we can isolate mutants with defects in their flagella: too long, too short, three instead of two, paralyzed, or cells that cannot build flagella at all. This alone makes Chlamydomonas a excellent organism for learning about flagella, but something additional makes Chlamydomonas over-the-moon wonderful for this work: when the cells are chemically tickled in just the right way, the flagella pop off yielding purified preparations of flagella that are a key resource for biochemical and structural studies. Happily, the cells grow new flagella, providing an opportunity for studying the process of assembly. It is a wonderful package of attributes that made Chlamydomonas the premier organism for scientists interested in flagella.

At the core of the flagella is a set of tubes (microtubules) that are crucial for structure and motility but also, it turns out, for assembly and maintenance. The microtubules serve as tracks for a miniature train that chugs up and down, taking building blocks to the tip and worn out bits back into the cell body. Since the discovery of this process of Intraflagellar Transport (or IFT) in 1995, several labs have been working to understand how it works.

There is a great deal of research to be done in order to understand how the train cars of IFT are built, loaded with cargo, hooked together and moved along the microtubules into and out of the flagella. An important aspect of the project is to learn the identity of the components of the train cars (IFT particles) and how they are put together. Three labs were collaboratively engaged in finding ways to isolate the particles and identify their components when something unexpected happened.

One of the people involved in this project was my friend, Greg Pazour, at the University of Massachusetts. When Greg and I go out for a beer at a conference he reminds me to take off my conference badge, as if so doing would disguise what is revealed by the drawings on napkins, strange gesturing explanations and obliviousness to our surroundings. Talking science with Greg is fun.

The unexpected happened when Greg & company compared the DNA sequence for a gene encoding one of the Chlamydomonas proteins with all of the gene sequences from a wide range of species in a large publicly available database. Greg discovered that a group of scientists interested in Polycystic Kidney Disease were studying disease progression and testing interventions in a mouse with cystic kidneys caused by a mutation in an unknown protein – a protein that Greg recognized as being one of the IFT components. It had never occurred to the kidney researchers that flagella might play a role in the disease that they were studying. Kidneys don’t even have flagella, do they?

The structures that I have been calling flagella are known as cilia in most cells (Chlamydmonas and sperm cells are two common exceptions). At the time of Greg’s observation there was little awareness of cilia beyond the obvious ones that keep our respiratory tract clear, propel sperm or those fascinating pond water creatures. It was known that small “vestigial” cilia were found on most human cells, but they were not thought to be important.

Greg and his colleagues discovered that the strain of mutant mice with the defective IFT component could not assemble proper cilia on their kidney cells, suggesting for the first time that defects in ciliary function might cause kidney cysts. The publication launched what became known as the ciliary hypothesis of Polycystic Kidney Disease..

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Since Greg and his colleagues published the ciliary hypothesis paper, cilia research has become one of the hottest things going. Within ten years it was established that there is a large family of diseases caused by defects in ciliary assembly or function. The so-called ciliopathies encompass a wide spectrum of diseases and syndromes that includes certain forms of obesity, blindness, mental retardation, developmental defects such as too many fingers and toes, as well as cystic diseases of liver and kidney. From vestigial structures not even mentioned in textbooks, cilia are now well respected and have dedicated chapters, alongside mitochondria and the nucleus.

The current intensive effort to understand ciliary assembly and function is providing a plethora of new targets for the design of treatments and cures. It is also providing a new paradigm for understanding how we develop from embryos and function as organisms. Greg Pazour is now working to understand the roles of cilia in mammalian development and he no longer works on Chlamydomonas. He remains a good friend nonetheless and when he visits Vancouver this fall we’ll drink some beer and talk science and hopefully get up into the mountains for a hike.

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In which deflagellation is my fascination.

I mentioned above that if Chlamydomonas cells are tickled just right their flagella pop off. For many decades, this response has been invaluable to researchers, but the behaviour itself had been pretty much ignored. I don’t think that people gave it much thought at all, or if they did, they assumed it was a pathological response and not an interesting biological phenomenon. Fortunately, I was naïve.

One piece of information gave me the confidence to make this flagellar shedding response the focus of my independent research career: a strain of Chlamydomonas that seemed otherwise normal, but it’s flagella would not pop off, no matter how vigorously tickled. I reasoned that if a mutation could prevent the response, then there was at least one gene dedicated to the process and if there was a dedicated gene, then there was a reason and a mechanism and I wanted to know what they were.

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After a few years working on the problem of how flagella are shed, my lab made a serendipitous discovery that took us into medically relevant work. One of the proteins that we identified as essential for shedding was a member of a poorly understood family of regulatory proteins. We identified this protein and its gene the same year that Greg and his collaborators published the breakthrough ciliary hypothesis paper. At that time there were eight mouse strains being studied as models for polycystic kidney disease. One of them carried the mutation that Greg had been studying and two of the others carried mutations in genes in the same family as our flagellar shedding gene. Suddenly my lab was studying mouse cells.

In collaboration with human geneticist Friedhelm Hildebrandt at the University of Michigan, we discovered that mutations in the human form of one of these genes causes a rare, but very severe form of juvenile cystic kidney disease. Our discovery is not of direct benefit to these children, but it does define another component of the pathways that go wrong when kidneys form cysts instead of tubules. The work will play its small part in the search for improved treatments for the 1 in every 600 people who will develop late onset PKD with no current hope of treatment beyond dialysis or kidney transplant.

While other labs continue with the kidney research, I’ve brought the focus of my group back to Chlamydomonas and flagellar shedding (known as deflagellation). Last year we published intriguing observations that suggest we may at last be on the way to discovering the reason why cells shed their flagella. It turns out that virtually all cells with cilia or flagella reabsorb these structures before they divide. We have evidence that the molecular machinery involved in making flagella pop off is also involved in reeling in the flagella before cell division.

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It is impossible to know when or even if the next serendipitous discovery will thrust us back into the fray of direct medical relevance and that suits me just fine. I am happy out here on the fringes watching my fascinating pond creature under the microscope and learning how it accomplishes bizarre feats of no obvious relevance.

— Lynne Quarmby

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Several friends and colleagues enthusiastically provided images to support this essay. I am grateful to Laura Hilton, Moe Mahjoub, Ichiro Nishii, Greg Pazour, Pinfen Yang and especially Edgar Young who generously took the time to produce the drawing of an ion channel, open and closed, using bona fide scientific structural coordinates (PDB:3FB8 & 1BL8). Also Alan Shinn, from whose webpage I lifted the picture of a van Leeuwenhoek microscope. DG helped with readability & spin.

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