Fall Modeling Chlamydomonas Reinhardtii Mobility

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Chlamydomonas reinhardtii is a unicellular biflagellate eukaryote. It is a model organism because it contains an abundance of conserved genetic traits that extends back to pre-historic times (Schmidt, 2006). Chlamydomonas has an inducible system to utilize varying calcium levels in the environment to alter chemotaxis and motility. By changing calcium levels from 10-4M to 10-7M, the goal was to determine universal calcium based movement patterns. Four strains of C. reinhardtii were utilized: wild type (control), pf14 mt+, p18 mt+, and oda1 mt+. Wild type Chlamydomonas exhibited the greatest movement patterns from addition of calcium. Pf14 mt+, p18 mt+, and oda1 were specifically selected due to their altered motility protein mutations. This experiment will help to establish a specific movement phototactic pattern of C. reinhardtii uses to survive.

Significance:

C. reinhardtii is a classic model for gene analysis. Despite it's seemingly simplistic nature, the eukaryotic algae has basal bodies and flagella comparable to mammalian centrioles and cilia (León, Rosa, et al., 2007). Cilopathies, or flaws in ciliary motility and assembly in mammals, can effect: sperm movement (because male sperm is the closest human homolog to C. reinhardtii, sharing 75% of the same genes), respiratory tract waste elimination, left-right body orientation during embryosis, and coordinated movement of fluids over epithelial cells (including respiratory tract, female reproductive system, and brain ventricles) (Sidflow and Lefebvre, 2001 and Smith, 2007). Unraveling the mystery behind C. reinhardtii motor proteins would advance a variety of topics including: cancer treatment, respiratory treatment, conception and pregnancy research, genetic obesity, and kidney polycystic research (D'Souza, J. S., 2009). Cilia genes have been conserved since pre-historic times, so it's not a surprise that many of the illnesses above would benefit from uncovering cilia signaling pathways. Realizing that a seemingly simplistic eukaryote can determine the fate of so many, it makes one think that green algae is significant. By determining the movement patterns of C. reinhardtii, higher beings everywhere might potentially benefit. Principles of flagella function in C. reinhardtii can be extended to human homolog. C. reinhardtii wild type and pf14, pf18, and Oda1 motility mutants will be compared to determine the relationship between structure and motility in C. reinhardtii and perhaps one day, humans.

Introduction

Environmental calcium concentration determines the movement of Chlamydomonas. There are five types of movement algae can exhibit: linear forward, linear backward, helical, clockwise circular, and counterclockwise circular and three possible waveform conformations: symmetric, quiescent, and asymmetric (Bessen, M. R., et al. 1980.) Research varies as to what calcium concentrations induce what specific motion. Each cell type (mutant or wild) has the potential to rapidly undergo one of these five types of movement once environmental calcium levels are varied.

Testing nutrient limitation responses would reveal the mechanisms that permit C. reinhardtii, and perhaps organisms with similar genes, to endure nutritionally scarce environments. The purpose of this experiment would to start developing a unified scientific movement model of C. reinhardtii in response to changes in environment, specifically calcium levels.

Background

To cope with low nutrient environments, organisms have inducible systems that enable them to scavenge and efficiently utilize limiting nutrients. Survival is dependant on the ability to adjust. Chlamydomonas reinhardtii displays extensive metabolic flexibility that allows it to inhabit distinct environmental niches and to survive fluctuations in nutrient availability. It is a classical model due to the cell's inherent abundance of evolutionally conserved genes that mirrors plants and non-photosynthetic organism responses. Testing nutrient limitation responses would reveal the mechanisms that permit C. reinhardtii, and organisms with similar genes, to endure scarce environments. The purpose of this experiment would to effectively describe the movement patterns C. reinhardtii and its mutants exhibit in according to changes in environment, specifically calcium levels. Variations in environmental calcium concentrations control C. reinhardtii movement, but prevailing research has failed to provide agreeable data on the movement specific calcium concentrations induces.

Chlamydomonas reinhardtii as a Model Organism

Over a billion years ago, Chlorophytes (green algae, including Chlamydomonas) deviated from Streptophytes (land plants). C. reinhardtii is one of the only surviving organisms that still contain conserved genes that trace back to precursor plant-animal cells. 109 years of evolution separate C. reinhardtii and mammals, yet C. reinhardtii flagella are extremely similar in structure and function to mammalian cilia and flagella. A number of the flagella proteins in C. reinhardtii exhibit 75% identity and similarity to proteins with similar function in mammalian sperm (Merchant, et al., 2010).

C. reinhardtii is a model organism, despite being a unicellular algae protozoa comprising of mitochondria, eyespot, two anterior flagella, and a chloroplast (Harris, 2001). In an optimal environment (light, O2, CO2, and mineral salts), the alga behaves like a phototrophic plant to produce its own carbon. However, C. reinhardtii also has the capacity to survive in less optimal surroundings (high salt, low sulphur, no light). The C. reinhardtii can act as a heterotrophic, like bacteria, and utilize surrounding carbon sources such as acetate for energy (Merchant, 2010, Harris, 2001). Additionally, this cell is can be cultured and maintained with ease (Harris, 2001). With such a short life span, it is easy to force the organism to produce large quantity of protein of interest or grow in a variable environment. Even though it has similarities to mammalian cells it is not nearly as susceptible to infection. The wild type cells genetic material can be manipulated to observe changes in normal function or structure.

Flagella movement

C. reinhardtii uses a pair of anterior flagella, which is a bending organelle that protrudes from the cell wall to swim and sense environmental conditions. Light perception is necessary for phototaxis, but the cell also need to respond to intense light source to prevent too much light absorption, or photoshock. Therefore, the organism's ability to move towards or away from light by flagellated system is essential for survival (Silflow and Paul Lefebvre, 2001).

Central Pair

Flagella have the "9+2" axonemal structure, where nine outer doublet microtubules surround two interior singlet microtubules. The nine doublet microtubules each consist of a complete A tubule fused to a partial B tubule in a stretched ring conformation that forms the walls of the basal body (Alberts, et al., 2008). Dyneins are located on the A tubule consisting of an inner and outer dynein arms that respectively control waveform and force (Movassha, 2010). Dyneins act as an interface between the outer to inner flagella structures because are connected to radial spokes and the A tubule.

Dynein Arms

The inner and outer dynein arms are forces responsible for the sliding motion between outer-doublets that eventually results in flagella movement. Inner dynein arms control the type of movement; outer dynein arms control produce the beat force, or how fast flagella move (Movassha, 2010). In the presence of ATP, the dyneins bind the A tubule of the adjacent outer doublet and move along the B tubule toward its minus end, resulting in sliding between pairs of outer doublets. The sliding motion cannot persist due to the proximity with other doublet microtubules and the basal body. This creates potential energy buildup that is converted into flagella bending motion allowing wave-like movement (Alberts, et al., 2008).

The inner dynein arms are located along the A microtubule. They are comprised of eight heavy chains, each heavy chain has the ability to induce different confirmations that control motility and wave changes. One heavy two-headed chain is connected to six-single headed heavy chains to form a network with intermediate and light chains and change the waveform pattern. One of the important light chains in the inner dynein arm is centrin, is a light chain that binds calcium. The various and variable interactions between the light, intermediate, and heavy chains induce the different types of waveform the flagella exhibits (Movassha, 2010).

The outer dynein arm's three heavy chains: α, β and γ transfer potential energy, ATP, into kinetic energy, movement so the flagella will reposition according to inner dynein waveform changes. α, β and γ contains hexametrical arranged ATP-binding molecules, once filled, they hydrolyze ATP to provide the heavy chains with the ATP energy to induce beat force (Movassha, 2010). Mutations in α, β or γ heavy chains on the outer arm will alter the velocity of outer dynein arms along the microtubules since ATP hydrolysis will be reduced, preventing energy to enter the dynein complex (Heuser, 2009). A specific mutation is oda1 mt+, the ODA1 protein is absent from the outer dynein because the coding gene encodes a stop downstream of the initiator, preventing a docking protein to be formed. The docking protein acts as an anchor for the outer dynein arm onto the microtubule doublet (Takada, 2002).

Radial Spokes

Motile flagella contain radial spokes, that consist of two parts: a "stalk" and "spoke head". The stalk portion is positioned next to the inner dynein arms, while the stalk's head extends into the microtubule doublet. Being near the inner dynein arms and stretching to the inner microtubules facilitates signal transduction (Yang, et al., 2006). Mutants such as pf14, that lack radial spokes exhibit abnormal motility or are paralyzed, since it relays the binding signals from the inner dynein arms to the microtubule doublet. Without signal transduction, dynein can no longer propel the microtubule to the axenome so any variations in environmental Ca2+ concentration would not affect movement since none can be propagated with damaged/absent radial spokes or dyneins (Witman, et al., 1978).

Effects of Environmental Calcium on Chlamydomonas reinhardtii

For heterotrophic photosynthetic organisms, exposure to light is necessary for survival. Plants optimize their exposure to light by growing towards it, but flagellated organisms must swim toward it. C. reidhardtii cells swim to optimal light environments by regulating flagella beating with environmental calcium. They have a Rhodesian-like photoreceptor eyespot to perceive light intensity for phototaxis (Suzuki, et al., 2003). When light is perceived by the photoreceptor, a current propagates from the eyespot to the plasma membrane by signal transduction. The signal transduction induces changes in the intraflagella free calcium levels in the inner dynein arm (Mitchell, 2000). The increase in calcium concentration depolarizes the cell membrane, voltage-dependant calcium ion channels open in response, and the flow of environmental calcium rises in the cell. If the depolarization is great enough, the voltage-gated Ca2+ channels in the flagellar membrane open and Ca2+ permeates.

Calmodulin binds the excess intraflagella Ca2+ concentration which induces a conformational change in the radial spoke stalks that allows signal transduction from the central microtubules to the inner dynein. The inner dynein arm contains centrin, another protein that binds calcium (Silflow and Lefebvre, 2001). Bound centrin forms a subcomplex to an adjacent light chain 4 protein which coordinates tight calcium binding with the centrin. The light chain 4 interacts with the N-terminal stem domain of the γ HC, giving the heavy chain on the outer dynein access to centrin, and therefore calcium. The shape and the size of the waveform are determined by the inner arms (increasing Ca2+ is detected by centrin binding which results in changing flagellar waveform) (Movassha, 2010) and can be powered by the outer dynein to increase beat frequency resulting in photoshock, swimming away from light, (Sakato and King, 2001). After a short period of time, Ca2+ is returned to the phototaxis level by the action of Ca2+ pumps and the cell returns to normal swimming towards light (Mitchell, 2000).

The focus in this study is to accurately describe changes in Chlamydomonas beating patterns when specific amounts of calcium are introduced in the global environment. The moving pattern research is vague and/or conflicting, and the aim of this experiment is to attempt to ratify the ambiguity. The following is comparison of theories of three Chlamydomonas experts chosen based on the amount of papers they wrote/quality of their work. Additionally, only one paper implicitly states the quantitative ratios of movement, we want to note the number of cells that demonstrate different patterns. It does not seem logical that all the cells would exist in only one type of movement at that particular environment. The following (Table 1) is a quick overview of the types of calcium driven phototaxic movement patterns explained in the M. Bessen's, et al., literature entitled "Calcium Control of Waveform in Isolated Flagellar Axonemes of Chlamydomonas," and Sakato and King's research entitled "Calcium Regulates ATP-sensitive Microtubule Binding by Chlamydomonas Outer Arm Dynein."

Author

Cell movements

Indicated amount of environmental calcium.

10-6-10-9 M Ca2+

10-6 M Ca2+

10-6 M Ca2+ -10-5 M

10-4 M Ca2+

10-3 M Ca2+

Sakato and King

Waveform

(S and K)

Asymmetric

Asymmetric waveform.

Quiescent.

Symmetric waveform.

N/A

Propagation of Movement

(S and K)

1 flagella moves.

move in a forward straight line.

No movement cells stayed still in V-shape configuration

Move in a backwards line.

N/A

Bessen, et al.

Waveform

(B., et al.)

None

Asymmetric waveform.

Asymmetric to Symmetric

Symmetric

Symmetric

Propagation of Movement

(B., et al.)

Inhibited

90% cells swam in circle. 10% in corkscrew pattern.

90% cells switch from a circular to straight path. The other 10% swim in a stretched "corkscrew" configuration,

Straight, parallel line forward.

No movement but stayed in straight line.

Table 1. Comparison environmental induced calcium levels according to research.

Sakato states that cells in 10-4 M Ca2+, swim with a synchronized symmetric waveform in a backward direction. Thus, the Chlamydomonas flagellum must contain at least two Ca2+ sensors that respond to different metal concentrations and control flagellar waveform conversion through alterations in dynein motor function (Sakato and King, 2003). Bessen asserts at 10-4 M Ca2+ axenomes moved in straight parallel lines from base to tip. At 10-3 M free Ca2+, a large percentage of the axonemes propagated apparently symmetrical bends, but the beat frequency was very slow (approximately 1 Hz) and no swimming was observed (Bessen, et al., 1980).

During normal forward swimming, the two Chlamydomonas flagella beat with an asymmetric waveform. Sakato states, At 10-6 M Ca2+ two Chlamydomonas flagella beat with an asymmetric waveform to result in forward swimming (Sakato, et al., 2006). Bessen asserts that the asymmetric waveform instead of a swimming in a straight line, that cells in 10-6 M Ca2+, 90% of Chlamydomonas swam in a circle with a diameter of 4μm with a speed of 2-5 rps. When viewed from above, 90% of the cells that touched the glass slide swam counterclockwise, and 90% of cells in contact with the coverslip swam clockwise. The remaining cells swam in a helical "corkscrew" fashion (Bessen, et al., 1980).

Sakato states as intraflagellar Ca2+ increases from below 10-6 M Ca2+ to 10-5 M Ca2+, cells cease to swim and flagella is quiescent in a V-shaped configuration (Sakato and King, 2003). Bessen asserts flagella never pauses its beating rythym, but over 90% of Chlamydomonas switches from an asymmetrical to symmetrical waveform somewhere between 10-5 M Ca2+ and 10-6 M Ca2+. The other 10% swim in a stretched "corkscrew" configuration, but the slope was longer than the cells than the "corkscrew" confirmation at cells in 10-6 M Ca2+ (Bessen, et al., 1980).

At 10-6-10-9 M Ca2+ axenomes did not swim in any specific direction (Bessen, et al., 1980). flagellar movement is apparently inhibited at such low concentrations of Ca2+. Yet, Sakato and King, asserted that in free calcium concentrations between 10-7-10-9M Ca2+, there is only enough free calcium for one flagella to be activated and swim towards light (Sakato and King, 2003).

Chlamydomonas reinhardtii displays extensive metabolic flexibility that allows it to inhabit distinct environmental niches and to survive fluctuations in nutrient availability. The cells have developed a phototactic calcium controlled system to ensure survival. Waveform pattern of Chlamdymonoas in various concentrations of Calcium needs to be ascertained, there should be a method to distinguish if cells move helically or linearly. It does not seem logical that all the cells would exhibit only one type of movement, previous studies fail to mention the speed the cell can move or amount of cells in a particular pattern of movement. Video microscopy allows us to track varying calcium concentrations in wild type and mutant Chlamydomonas reinhardtii, so a motion pattern should be ascertained.

Proposed Research

In order to establish if phototactic movement has specific patterns in varying amounts of environmental calcium, four strains of Chlamydomonas reinhardtii; wild-type, pf14 mt+ (mutated radial spokes), oda1 mt- (dynein arm mutation), and pf 18 mt- (central microtube mutation) will be cultured. All mutants were chosen for their motility defects, because observation of knockout traits are required to compare to wild-type uptake of environmental calcium and therefore flagellar movement. All cells will be cultured at the same density, 1x105cells/mL, then all environmental calcium will be subsequently eliminated by three washes in non-calcium TAP medium. Specific aliquots of calcium be reintroduced into cell cultures and incubated for specific time intervals. To effectively monitor Chlamydomonas reinhardtii movement patterns, a Leica DM1000 inverted compound microscope will capture all cellular activity.

Movement patterns will be determined by counting the frequencies of cells that moved in a particular waveform and direction over a particular time frame. A successful experiment will determine if introducing limiting supplies of the environmental factor, calcium, does have promote moving patterns in Chlamydomonas reinhardtii phototaxis. We will also be determining if mutations that affect normal flagella mobility have the same limited movement in the varying calcium mediums. Comparing the variant samples to the control samples will qualitatively determine effectiveness of the experiment.

C. reinhardtii

C. reinhardtii is an optimal organism to study motility. Its genome is reminiscent of ancestor plant-animal predecessors as well as genes prevalent in a variety of organisms in the present day. C. reinhardtii can survive in a variety of environments from soil to snow on mountaintops, and can survive by phototropic or heterotrophic means (Merchant, 2010, Harris, 2001). Additionally, this cell is extremely uncomplicated to culture, its full life cycle, with such a short life span, it is easy to force the organism to produce large quantity of protein of interest or grow in a variable environment. Even though it has similarities to mammalian cells it is not nearly as susceptible to infection, and small amounts of amphicillian can be introduced to cultures, ensuring survival (Silflow and Lefebvre, 2010). Wild type C. reinhardtii genetic material can be manipulated to observe changes in normal function or structure.

C. reinhardtii Mutants

C. reinhardtii mutants for this experiment will be: pf14 mt+, oda1 mt-, and pf18 mt- (central microtube mutation) each exhibit malfunctions in flagellar structure. Pf14 mt+ contains a mutation within the radial spokes, resulting in the lack of signal transduction from the dynein indicated by the rising or falling concentrations of environmental calcium (Smith, 2002 and Witman, et al., 1978). Since dynein is responsible for driving the movement/force of the flagella, failure of the radial spokes to signal to microtubules would cause the sliding of the microtubules to remain unchanged by any variations in environmental Ca2+ concentration (Witman, et al., 1978).

The oda1 mutant specific mutation is on the outer dynein arm, preventing optimal movement. The ODA1 protein is absent from the outer dynein because the coding gene encodes a stop downstream of the initiator, preventing a docking protein to be formed. The docking protein would normally act in complex anchoring the outer dynein arm onto the microtubule doublet (Takada, et al., 2001). The outer dynein arm generates the movement and acceleration flagella force, by hydrolyzing ATP. Therefore any retardation/loss of the outer dynein arm should decrease swimming speeds even in high calcium levels. An oda1 mutant lacks its outer dynein arm, which is responsible for how quickly the flagella movement is produced. Theoretically, flagella force/speeds should remain similar at all calcium concentrations, since it cannot change speeds. Literature denoted that waveforms/speeds of the oda1 mutant could not mimic wild-type activity in high Ca2+ photoshock patterns, but it can model the wild-type activity in low Ca2+ (Wargo, et al., 2004). Theoretically, this is logical, all dynein arm activity will not be ceased, but just slowed. The outer arm dynein controls speed and force of flagella movement, a mutation would slow the flagella so that any concentration of calcium would mimic the slow, symmetrical movements of wild-type in low calcium environments.

Pf18 mutant lacks the central pair microtubules that make up the axoneme, this defect induces flagella paralysis. In theory, motility should be completely impaired. The sliding of the microtubules in this mutant will be slower than wild type at low calcium level. However, the sliding motion will increase similar to wild type movement at high calcium levels. According to Wargo, et al., the sliding velocity of axonemes from pf18 increased as environmental calcium concentrations rose. Evidently, dynein activity cannot be ceased by the lack of the microtubule doublet in the presence of high concentrations of calcium. However, the calcium-induced dynein activity fails if the C2 microtubule of the central apparatus is present or if radial spoke components are lacking (Brokwaw and Kamiyra, 1987). These results provide evidence that dynein activity is modulated by calcium and indicate that dynein activity is regulated by the response of a particular enzyme to increasing concentrations of free calcium (Wargo, et al., 2004).

Experiment

Four strains of Chlamydomonas reinhardtii; wild-type, mutant pf14 mt+ (mutated

radial spokes), mutant oda1 mt- (dynein arm mutation), and pf 18 mt- (central microtube mutation) will be cultured until they reach the same density of 1x105cells/mL in a standard Tris-Acetate-Phosphate (TAP) Medium. Cells will be centrifuged, supernatants removed, and cells resuspended three times in TAP medium without any calcium. Specific calcium aliquots ranging from 10-3 to 10-7M will be reintroduced into cell cultures, incubated for specific times, and assayed. To effectively assay Chlamydomonas reinhardtii movement patterns, a Leica DM1000 inverted video recording compound microscope will capture all cellular activity. The recorded video will be reviewed in five second increments using MacBook iMovie to ensure all cells will be accounted for. Movement patterns will be determined by counting the frequencies of cells that moved in a particular waveform (symmetrical or asymmetrical) and direction over a particular time frame (linear forward, linear backwards, clockwise, counterclockwise, or spiral). The experiment needs to be performed at least twice per trial for accuracy.

Calcium Levels Tested (M)

Incubation Time (minutes)

Control (wt)

0, 1mM, 10 mM, 100mM, 1µM, and 100µM

20, 100 minutes

pf 14

0, 1mM, 10 mM, 100mM, 1µM, and 100µM

20, 100 minutes

Oda 1

0, 1mM, 10 mM, 100mM, 1µM, and 100µM

20, 100 minutes

pf 18

0, 1mM, 10 mM, 100mM, 1µM, and 100µM

20, 100 minutes

Table 1. Sample testing parameters: time increments for varying amounts of calcium will be incubated into the each culture.

Controls

All cells will be incubated in the same TAP medium until reach maturity at 1x105cells/mL. All the assays will be performed with the same methods. The positive control is the wild type strain of Chlamydomonas reinhardtii exposed to every environmental calcium aliquots for each time increments. The negative control is the pf14 mutant. It is expected these cells will exhibit little to no motility with any amount of calcium or incubation time.

Variables Tested

The calcium environment levels and the time intervals the Ca2+ is allowed to incubate will be the variable factors. Movements (forward, backward, clockwise, counterclockwise, and corkscrew), waveform (asymmetrical, symmetrical, and quiescent), and frequency will vary from type to type. Mutant strains of Chlamydomonas reinhardtii with knockout motility factors will be tested to understand how environmental calcium prevents/enables flagellular mobility as well as justify the Chlamydomonas reinhardtii wild type results.

Expected Results

The results that would support the hypothesis would be varying environmental calcium levels shows a specific pattern for mobility. Since the published data was extremely contradictory on what shapes and waveforms would appear in Chlamydomonas reinhardtii in varying amounts of calcium, the results might be able to support/deny earlier estimations of modeling. Wild type C. reinhardtii should exhibit the most prominent flagella components and exhibit the greatest motility patterns and movement rates.

Results that would reject our hypotheses/experimental methods include calcium levels not affecting cell motility, no motility changes over a period of time. Since we understand how calcium activates dyenin this would oppose our suppositions. If the mutants were able to move exactly the same as the wild type strain this would disprove the flagella functionality.

In order to ascertain this, all free calcium ions in C. reinhardtii wild type and mutated cells will be removed then subsequently reintroduced to specific calcium ion aliquots. The information from this study on Chlamydomonas phototransduction should further our understanding of these homologous events in higher organisms.

pf14 has a mutation within the radial spokes, this results in a lack of dynein-driven microtubule connection to the axoneme. Dynein is responsible for driving the movement of the flagella and in this mutant it would be hard to transfer the potential of dynein throughout axoneme, and therefore perhaps remain paralyzed despite the amount of calcium levels (Smith, E. F., 2002). At low calcium levels, the Oda1 mutant should exhibit similar asymmetric waveforms as the wild type, but will not be able to produce other types of moving due to its missing outer dynein arm (Wargo, M. J., 2004). The pf 18 has mutated central microtubules located within the core axoneme. The mutants will most likely exhibit slower sliding of microtubules than the wild type strain at lower calcium levels, (opposite of Oda1) but at a high calcium concentration (10-4M) the mutant exhibit symmetric waveforms at a similar rate as the wild type (Smith, E. F., 2002).

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