Using phase contrast microscopy to measure regular and abnormal flagellar regrowth in deflagellated

Chlamydomonas reinhardii, and to observe the mating behaviors of wild type and mutant strains.

Kirsti Jangaard
 B00505932
BIOL2020 Lab B09
Abstract

Introduction
The unicellular biflagellate eukaryote Chlamydomonas reinhardii is a species of photosynthetic green

algae that is widely studied in molecular and cell biology. It relies on two flagella for motility that are

composed of tubulin and powered by Adenosine Triphosphate (ATP) (Piperno et al., 1977). α - and β - tubulin

form polymers and make microfilaments. These microfilaments form the basic central structure of the

flagellum, the microtubule (Remillard and Witman, 1982). Nine outer doublet microtubules surround two inner

singlet microtubules, forming a structure called the axoneme (Flavin and Slaughter, 1973). This 9-2 pairing

allows for a large range of motility and complex motions (Ringo, 1967).

Chlamydomonas have the ability to regenerate flagella almost immediately after they are amputated

(Weeks and Collis, 1976). Chlamydomonas have precursor pool of tubulin stored that can be used immediately

after deflagellation (Flavin and Slaughter, 1973). This store of tubulin is only sufficient to regrow 80-85% of

the flagella before it is depleted. Polyribosomes bring mRNA that codes for tubulin as early as 15 minutes after

deflagellation, and begins to produce new tubulin (Weeks and Collis, 1976). The flagellum regrows as α - and

β - tubulin heterodimers are added onto the (+) end of the microtubules, bound to GTP. (Tuxhorn et al. 1998).

Certain factors such as gene mutation and inhibitory drugs can be used to inhibit growth of the flagella (Favin

and Slaughter, 1973). In part one of this experiment, the poison Cyclohexamide is introduced to a culture of

Chlamydomonas. Cyclohexamide inhibits biosynthesis of proteins; the purpose of this experiment is to observe

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the effect of cyclohexamide on the regrowth of flagella. The Chlamydomonas were deflagellated by acid shock;

one sample was introduced to cyclohexamide, and the other a fresh culture medium. Phase microscopy was

used to observe and measure the regrowth of the flagella at regular intervals. If cyclohexamide were to inhibit

the production of tubulin in the Chlamydomonas, the regeneration of the flagella would be incomplete.

In part two of this experiment, wildtype (+) and (-) strains of Chlamydomonas were observed

individually, and their mating habits were observed when combined. Chlamydomonas can reproduce sexually

or asexually. In a nitrogen-starved environment, gametes with a haploid number of chromosomes will develop,

and diploid zygotes will be formed when they fuse (Piperno et al, 1977). The purpose of this part of the

experiment was to observe sexual reproduction over a period of time in wildtype Chlamydomonas. If the (+)

and (-) were combined, then sexual reproduction will occur. The purpose of the third part of this experiment

was to observe the behaviour of mutant Chlamydomonas, (+) and (-), both individually and when apart.

Defective genes cause the mutations, and result in the malfunctioning of radial spokes. The radial spoke is

bound to the axoneme of the flagellum, and in healthy individuals plays a crucial role in motility and

motor control (Piperno et al, 1977). The mutants in the experiment are completely immobile. They are first

observed separately, and then mixed together and observed. If two mutants, (+) and (-), successfully mate,

then the zygote formed would have regained motility.

Materials and Methods

Part I: Deflagellation and Flagellar Regrowth

In a 100mL beaker with a magnetic stirrer, 40mL of actively growing Chlamydomonas culture were

transferred. Under constant stirring, pH was lowered to 4.5 (drop-wise 0.5N acetic acid, 30s). Deflagellation

was confirmed under phase microscope after 2 min. pH was restored to 6.8 (dropwise 0.5N KOH). 10mL

deflagellated culture was poured into 15mL centrifuge tubes, and centrifuged (1300g, 5min). Solution was

decanted, and supernatant discarded. 10mL regular media was added to one tube of cells, and 10 mL regular

media + cyclohexamide to the other. Pellets were resuspended. Contents of each tube were poured into 50mL

Erlenmeyer flasks. 10mL of non-deflagellated cells were transferred into 50mL flask. Cotton-plugged flasks

were placed on a gently moving shaker. For each condition, 5 Eppendorf tubes were marked 0, 20, 40, 60, and

80min, and two marked 0 and 80min for the control. One drop of Lugol’s iodine was added to all tubes. At 0
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 min and each following 20min interval, fixed samples from both flasks were pipetted onto separate slides and

observed with an Olympus CX41 microscope (400x total mag., phase contrast). 5≤ cells’ flagellum were

measured in each condition and recorded. The non-deflagellated culture was fixed, prepared as a slide, and

observed and measured at 0 and 80min.

Part II: Wildtype and Mutant Mating

Wildtype (+) strain CC-125 and wildtype (-) strain CC-124 were observed separately with phase

contrast (400x total mag.). They were then mixed together and observed (at 0min, 10min, 30min). Mutant (-)

strain CC-602 and mutant (+) strain CC-1032 were observed separately under phase contrast (400x total mag.).

They were then combined in a centrifuge tube and observed.

Results
Flask 1 and flask 2 show significant overall difference in flagellar regrowth (fig 1A). At 0min, both

samples are completely deflagellated (avg. 0.00µ m). Both show growth at approximately the same rate at 20

min (see table one). After this time point, flagellar growth in flask 2 slows, plateauing just below 7µ m.

Flagellar regrowth in flask 1 reaches a peak of 9.6042µ m, comparable to the average length of non-

deflagellated controls, 9.30µ m (fig 1A). Flagella were completely regrown in Flask I, and had arrested growth

in Flask II. Flask 1 was determined to contain Medium I, and flask 2 Medium I + cyclohexamide.

Table 1: Flagellar regrowth in acid-shock deflagellated Chlamydomonas reinhardii in Medium I (flask 1) and in Medium I+cyclohexamide (flask 2) over
time, and flagellar length of control (non-deflagellated culture).
Time Flask 1 Flask 1 Flask 1 Flask 1 Flask 1 Flask 1 Flask 1 Flask 2 Flask 2 Flask 2 Flask 2 Flask 2 Flask 2 Flask 2
(µ m) (µ m) (µ m) (µ m) (µ m) (µ m) (µ m) (µ m) (µ m) (µ m) (µ m) (µ m) (µ m) (µ m)
Bench Bench Bench Bench Bench Bench Average Bench Bench Bench Bench Bench Bench Average
1 2 3 4 5 6 1 2 3 4 5 6
0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0
20 1.25 3.75 2.5 3.125 3.0125 3.3 2.8229 3.5 3.38 3.0 2.1725 4.0 4.5 3.4254
40 5.5 6.5 7.125 6.75 6.0625 7.425 6.5604 5.0 5.0 4.625 4.5 6.75 7.0 5.4792
60 9.0 8 8.125 7.75 8.375 9.25 8.4167 7.5 5.25 4.625 7.5 7.25 9.5 6.9375
80 10.5 9.625 8.625 8.25 10.25 10.375 9.6042 8.5 5.0 3.625 9.13 6.75 8.85 6.9758
Control 7.5 9.25 9.0 8.75 9.875 9.75 9.0208 7.75 8.25 10.125 10.075 9.75 9.5 9.2417
0
Control 8.5 9.375 8.75 8.5 10.125 10.5 9.2917 8.0 8.5 8.375 10.0 10.5 12.5 9.6458
80

A. B. C.
Figure 1: A) Average flagellar regrowth of Chlamydomonas reinhardii in medium I and in medium I + cyclohexamide over time, and average lengths of

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the control (non-deflagellated). B) i) Wildtype strains (+) CC-125 and (-) CC-124 viewed separately under phase contrast. ii) Wildtype strains (+) and (-)
combined, forming a cluster, observed immediately after mixing viewed under phase contrast.. iii) Mated wildtype strains (+) and (-). Four flagella are
visible. Viewed under phase contrast. C) i) Mutant strain (-) CC-602 viewed under phase constrast. No flagella are visible. ii) Mutant strain (+) CC-1032
viewed under phase contrast. Flagella are present but non-functional. iii) Mutant (+) and (-) strains after being mixed, with regained motility and
functional flagella.
In Wildtype mating, the (+) and (-) strains observed individually were motile, with 2 healthy flagellum

(Figure 1Bi). When combined, immediately some cells began to form small clumps of 2 and 3, while most

remained moving (Figure 1Bii). After 10min, almost all cells had formed clumps and were no longer moving,

and a number were beginning to intertwine flagella and fuse. At 30 minutes, many of the cells had died due to

dehydration, however there were a small number of cells that had 4 flagella instead of 2 (figure 1Biii).

Mutant strain (-) observed individually showed complete lack of flagella and is immotile (figure 1Ci).

Mutant strain (+) has flagellum present, but they are broken and of no use (figure 1Cii). This strain is also

immotile. When mixed, the majority of cells remained immotile, however a very small number of cells

regained motility and appeared normal (figure 1Ciii).

Discussion

The purpose of Part I of this experiment was to observe the effect of cyclohexamide on the regrowth of

flagella in deflagellated Chlamydomonas reinhardii. If cyclohexamide inhibited the production of tubulin in

these cells, then the flagella would not have enough tubulin to regrow completely. The data supported this

result; Flask 1 containing the deflagellated cells and a growth medium showed full regrowth (9.6402µ m),

while Flask 2 containing the deflagellated culture and the cyclohexamide showed incomplete regrowth

(6.9758µ m). The non-deflagellated control had flagella measuring 9.30µ m on average. This occurred because

cyclohexamide inhibited the synthesis of tubulin in the culture cells; the store of tubulin already produced and

available to the cell to regrow flagellum was insufficient to regrow the flagellum entirely. These results show

that drugs or mutations that cause the inhibition of tubulin production in Chlamydomonas are very harmful and

possibly fatal to the organism, which is supported by Flavin and Slaughter’s 1973 article. They observed the

regrowth inhibition that occurs in Chlamydomonas when combined with Antitubulins and other drugs. They

described how some cells became resistant to the Antitubulin, and showed changes in the primary structure of

tubulin assembly. (Slaughter and Flavin, 1973).

The mating of Wildtype (+) and (-) haploid cells was observed in Part II. When they were combined on

a slide, mating and fusion was observed, which supported the hypothesis. The cells are reproducing sexually,

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forming some diploid gametes with four flagella. In nature, this diploid zygote would not be flagellated. When

exposed to light, it would undergo the process of meiosis and release four flagellated haploids (Piperno et al,

1977). This reproduction process may help ensure survival in nature; in the conditions where nitrogen is

unavailable, the diploid zygote forms a solid outer wall to protect from harmful conditions (Forest and

Takasaki, 1975). A possible source of error in this phase of the experiment was that the medium that the wild-

type cells were mounted in while being observed became dehydrated, and could possibly have hindered the

mating process. This could have also been a source of error in Part III. When the mutant (+) and (-) strains were

observed individually, they showed no signs of motility. The (-) strain has no flagella present, and while the (+)

strain has flagella, they are non-functioning. When these two cultures were mixed, the majority of cells

remained immotile, and only the ones brought together by mixing began to fuse. After fusing, the cells regained

mobility, which confirms our hypothesis. In nature, this mutation would have proved deadly, since the cells

have no means of transportation and are defenseless against adverse environmental conditions.

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