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 driven 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. Cycloheximde inhibits biosynthesis of proteins; the purpose of this experiment is to

observe 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. The regrowth of their flagella was measured at regular intervals. If cyclohexamide

were to inhibit the production of tubulin in the Chlamydomonas, one would expect the regeneration of

the flagella to be incomplete. Phase microscopy was used to measure and clearly observe the

specimens.
 In part two of this experiment, wildtype (+) and (-) strains of Clamydomonas 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. Thepurpose of the third part of this experiment was to observe the

behaviour of mutant Chlamydomonas, (+) and (-), both individually and when apart. The mutation

is caused by a defective gene, and causes 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 it would be expected that the zygote 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 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 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 an 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 1 Bench 2 Bench 3 Bench 4 Bench 5 Bench 6 Average Bench 1 Bench 2 Bench 3 Bench 4 Bench 5 Bench 6 Average
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 0 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
Control 80 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

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 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

Bibliography

Flavin, M., and C. Slaughter. 1974. Microtubule assembly and function in Chlamydomonas:
inhibition of growth and flagellar regeneration by antitubulins and other drugs and isolation of
resistant mutants. J. Bacteriol. 118:59-69.

Piperno, G., B. Huang, and D. J. L. Luck. 1977. Two-dimensional analysis of flagellar

proteins from wild-type and paralyzed mutants of Chlamydomonas reinhardtii. Proc. Natl.
Acad. Sci. USA. 74:1600-1604.

Remillard, S. P., G. B. Witman. 1982. Synthesis, transport, and utilization of specific flagellar
proteins during flagellar regeneration in Chlamydomonas. J. Cell. Biol. 93:615-631.

Ringo, D. L. 1967. Flagellar motion and fine structure of flagellar apparatus in

Chlamydomonas. J. Cell. Bio. 33:543-571.

Tuxhorn J, T. Daise, W. L. Dentler. 1998. Regulation of flagellar length in Chlamydomonas.

Cell Motil. Cytoskeleton. 40:133-146.

Weeks, D. P., and P. S. Collis. 1976. Induction of microtubule protein synthesis in

Chlamydomonas reinhardtii during flagellar regeneration. Cell. 9:15-27.