Dante Moroni

Motility in Chlamydomonas reinhardtii

Introduction

Chlamydomonas reinhardtii is a useful model organism, especially for studying motility. This

unicellular photosynthetic eukaryote has two flagella in which several mutations have been

identified. These flagellar mutations may help us further understand the mechanisms involved in

its motility. Also, an understanding of the proteins involved will help clarify why the flagellum

responds to mutations in these ways.

Comparisons between mutated and wild-type cells can give us insight into what proteins

structures are imperative for flagellar motion. We can also deflagellate the cells in order to study

them. Without their flagella C. reinhardtii cannot move towards areas of light where it can

perform photosynthesis. The need for movement and the energy provided by photosynthesis is

addressed by cells through their ability to re-grow flagella after they have been removed.

Through treatment of the cells with chemicals, we can disrupt their ability to polymerize

microtubules, and to perform translation and transcription. Generally, we see that mutations to

the radial spokes, central pair assembly, or dynein motor proteins cause changes to flagellar

motility (Mitchell and Sale 1999). Analyzing differences between mutant and wild-type strains,

flagellar regeneration, and phototaxis can help us reach our goal of a better understanding of

motility in C. reinhardtii

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Materials and Methods

Flagellar Mutations

Four wet mounts of C. reinhardtii were prepared from 137c, pf-17, sup-pf-1, and sup-pf-1/pf-17

double mutant strains. Strains were obtained from the University of Minnesota-Duluth,

department of biology. These strains were grown in TAP media according to Gorman and Levine

(1965). Observations of each strain’s movements were observed using a light microscope.

Flagellar Regeneration

C. reinhardtii cells were deflagellated using pH shock. The culture grown in TAP media had its

pH lowered to 4.5 within 30 seconds with acetic acid, and then raised to 6.8 with potassium

hydroxide. The cells were then centrifuged and the supernatant was poured off, leaving the cell

bodies behind. Cells were then each transferred into five test tubes and treated with equal parts of

one of five experimental conditions as follows; addition of TAP media, TAP with 2 mg/mL

colchicine, TAP with 0.01mg/mL cyclohexamide, TAP with 0.05 mg/mL actinomycin D, TAP at

4°C. One drop of cells was then transferred to a spot plate well containing one drop Lugol’s

mixture at appropriate time intervals of 0,10,20,30,40,50,60,70,80,90,and 105 minutes. A control

of non-deflagellated cells were also used and only transferred at 0 and 105 minutes. The fixed

cells were prepared on slides and observed under light microscope and flagellar length was

measured.

Phototaxis

C. reinhardtii cells in culture were used with a density of 1.83x106 cells/mL. Two phototaxis

chambers were assembled. Each consisted of three tube segments joined by two rubber

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connecters. Cells were transferred to the chambers. The chambers were then sealed and secured

to a level tabletop. The control chamber was covered completely with tinfoil. The other chamber

had the bottom third segment covered with tinfoil, the middle with one layer of Kimwipe, and

the top was left uncovered. A 60 watt bulb was centered 150mm above each tube for 60 minutes.

Both of the chambers were then clamped between segments to prevent movement of cells. The

cells in each segment were transferred into collection tubes and fixed with Lugol’s mixture. Cell

densities in each tube were measured.

Results

Flagellar Mutations

The 137c wild-type cells moved quickly and frequently. The pf-17 strain mutants were mostly

stationary. The sup-pf-1 mutants just vibrated. Sup-pf-1/pf-17 mutants were able to move

similarly to the wild-type Chlamydomonas.

Flagellar Regenerations

Average flagellae length increased at about 1μm every 10 minutes in cells grown in TAP control

(Figure 1). Cells treated with actinomycin, colchicine, and cyclohexamide initially had their

flagellae increase close to the same rate as the TAP control. However, cyclohexamide and

actinomycin treatments stopped increasing in length and plateaued at 50 minutes. Colchicine

treatment also plateaued, though, earlier at 30 minutes. Cells grown in 4° C TAP did not have

any flagellar growth. The non-deflagellated control’s flagella showed no growth between 10 and

105 minutes.

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0 20 40 60 80 100 1200

2

4

6

8

10

12

14

TAP

Actinomycin

Colchicine

Cyclohexamide

4° C

Non-Deflagel-lated

Time (min)

Flagellae Lenth (μm)

Figure 1. Relationship between time and flagellae length of four experimental and two control assays. Non-deflagellated cells were observed at 0 and 105 minutes.

Phototaxis

Cell incubated under diffuse light had higher density than those incubated under bright light or

no light (Table 1).

Table 1. Cell density per mL in each three segments of the control and experimental phototaxis chambers.

  Segment of Chamber Cells/mLControl: Dark Bottom 3.72 x 106

Middle 3.32 x 106

Top 3.48 x 106

Experimental 

Bright light 2.36 x 106

Diffuse light 4.74 x 106

No light 1.10 x 106

Discussion

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Flagellar Mutations

Mutations in the radial spoke proteins of the pf-17 strain caused paralysis of their flagellae.

Radial spokes must be involved in flagellar movement. The sup-pf-1 strain has a mutation in

their outer dynein arms, though they still showed some vibration, so the mutation may only

partially affect flagellar movement. Sup-pf-1 is somehow able to suppress the pf-17 radial spoke

mutation, resulting in normal motility.

Flagellar Regeneration

The controls showed normal flagellar length to 12μm and linear rate of regeneration.

Deflagellated cells at 4°C were not able to grow back their flagella; this was likely due to the

limited amount of energy available at low temperatures. The treatments of actinomycin and

cyclohexamide inhibited growth after initially growing at the same rate as the control. Colchicine

had its growth plateau at about 30 minutes, and this may be due to the time necessary for it to

affect the cells.

Phototaxis

Cells were most densely located in the diffuse light phototaxis segment. The light from the fully

lighted segment must have been too bright for the cells. The cells may be able to perform their

most efficient photosynthesis under diffuse light and migrated towards it to do so.

Conclusion

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Motility in Chlamydomonas reinhardtii is complex and there are many factors that can affect it.

Flagellar function and growth, motility, and photosynthesis all can be affected by environmental

conditions. Without each motor protein and cellular processes functioning properly, the flagella

have difficulty working correctly and this can influence the survival of C. reinhardtii.

References

Vucica Y, Diener DR, Rosenbaum JL, Koutoulis A (2007) Ultra Structural and biochemical

analysis of a new mutation in Chlamydomonas reinhardtii affecting the central pair

apparatus. J Protoplasma 232:121-130

Mitchell DR, Sale WS (1999) Characterization of a Chlamydomonas Insertional Mutant that

Disrupts Flagellar Central Pair Microtubule-associated Structures. J Cell Biology 144 (2):

293-304

Gorman DS and Levine RP (1965) Culture Media. J Proceedings of the National Academy of

Sciences of the United States of America 54:1665-1669