Lab 10: Flagellar Regeneration in Chlamydomonas reinhardtii

Rates of flagellar and microtubule assembly

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Introduction
The flagella of sperm and protozoa are long, whip-like threads built of microtubules and dynein.
Cilia have a similar composition and are shorter structures. In our lungs, cilia constantly beat, sweeping
mucus containing trapped dust, bacteria and fungal spores up to our throats where the mass is either
swallowed or expectorated. For those who smoke cigarettes, the inhaled smoke paralyzes the cilia, and
the lungs can no longer be cleansed, hence smokers cough and blackened lungs. The unicellular green
alga Chlamydomonas reinhardtii uses its flagella to move toward light or nutrients or away from predators
or harmful chemicals.
On the molecular level, both flagella and cilia share a common core structure, the axoneme, a 9
outer + 2 inner arrangement of microtubules. Each of the outer 9 are doublets, one complete microtubule
and one partial, sharing a common tubule wall. Each complete microtubule has 13 protofilaments, and
each partial microtubule has 10. The inner 2 are two separate, complete microtubules. In total, each
flagellum contains 9 outer doublets with 13+10 protofilaments and 2 inner microtubules with 13
protofilaments. Each protofilament is a chain of dimers of alpha-tubulin and beta-tubulin, each monomer
450 amino acids and 4 nm in diameter. There are some 200 different proteins in a normal flagellum, one
of which is dynein, but the major protein is tubulin.
The majority of what is known about flagella has come from the study of Chlamydomonas
reinhardtii. A simple shift in pH from 7.5 to 4.5 by the addition of acetic acid will cause
Chlamydomonas to lose both flagella. We will use this property to study the rate of flagellar growth, and
by inference, the rate of microtubule assembly.

Experiment Design
As part of preparation for this lab, you and your partner are asked to formulate a hypothesis about
the molecular process of flagella regeneration and design an experiment which will test that hypothesis.
To formulate a hypothesis, just ask What if....? After removing the flagella, what if we cut off the cells
energy supply? What if we inhibited protein translation? What if we prevented RNA transcription?
What if we blocked second messenger production? What if we prevented second messenger removal?
Once you have formulated a question of interest, you should use your Cell Biology training to formulate a
hypothesis. For instance, the question What if we inhibited protein synthesis? might lead to this
hypothesis, After removing the flagella, if we inhibited protein synthesis, flagella would still regenerate
using tubulin from the existing microtubule network. (There are other possible hypotheses related to
this what if question.)
Any experiment measuring the effect of some treatment must have a control. If we measure the
rate of flagella regeneration in the presence of caffeine, a control would be measuring the rate of flagella
regeneration in a set of cells treated in all ways like the experimental sample except that caffeine is absent.
A process like flagella regeneration can be affected by many variables, and without the control, we would
not be able to conclude anything about effect of caffeine.
We will have the following drugs available for your use in this experiment:
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Drug Effect Stock Solution Working Concentration

cycloheximide translation inhibition 2 mg/ml in ethanol 10 micrograms/ml

actinomycin D transcription inhibition 5 mg/ml in ethanol 50 micrograms/ml

calcium second messenger 100 mM in water 1 mM

lithium chloride IP3 production inhibitor 1 M in water 20 mM

caffeine cAMP phospho- 66 mM in water 6.6 mM

diesterase inhibitor

Experiment
1. Teams of 4 will work together. One person will record all data and monitor so that no time points are
missed. A second person should be time keeper, and fix all aliquots at the proper time. The third and
fourth persons should measure the lengths of the fixed flagella.
2. Label 6 eppendorf tubes 0, 15, 30, 45, 60 and 75, and 6 eppendorf tubes C0, C15, C30, C45, C60 and
C75. Pipet into each 50 microliters of Lugols iodine (6% w/v KI; 4% w/v I 2). Cap the tubes to contain
the iodine. Lugols iodine will both fix the cells and stain the flagella.
3. After the Chlamy have been deflagellated by pH shock and stored in the dark to suspend growth, two
aliquots of 5 mls each will be placed in two small erlenmeyer flasks, labeled experimental and control.
The drug you chose to affect various cell processes will be added to the experimental flask, and an equal
volume of the drugs solvent is added to the control flask. (Remember that the control must be treated
exactly as the experimental except for the presence of the drug.) A 50 microliter sample will be taken
from the experimental flask and placed in the tube labeled 0 for the time 0 sample. A 50 microliter
sample will be taken from the control flask and placed in the tube labeled C0 for the time 0 control sample.
This sampling process will be repeated at 15, 30, 45, 60 and 75 minutes. Between sampling, the flasks
will be placed on a rotary shaker under illumination.
4. To measure the length of the flagella, gently tap the eppendorf tube to resuspend the fixed cells, and
withdraw 10 microliters. Pipet the 10 microliters in the center of a clean microscope slide, and cover with
a 22 x 22 mm cover slip. Place the slide on the microscope stage, and locate the cells first at 200X phase
contrast and then 400X phase contrast. Place a drop of immersion oil on the cover slip, and focus at
1000X phase contrast. The microscope should be fitted with an ocular micrometer, a ruler 10 cm in
virtual length.
5. Select 10 cells which have one fairly straight flagella, and measure and record the length of each.
Measure only one flagella per cell. You should record 10 measurements for each experimental time point
and 10 for every control time point. For both experimental and control samples, calculate an average
length at each time point, and calculate the rate of flagella regeneration in micrometers per minute.

Three caveats:
First, the pH shock may not remove all flagella from all cells. If you observe cells with full length
flagella in your samples, ignore them, and measure only those cells with short flagella.
Second, after the cells have begun to regenerate the flagella, classify any cell without flagella as
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dead and do not include the zero length in your observations.

Third, calculate the rate of flagellar growth after flagella have begun to regenerate. Do not
include the lag phase when no growth is observed. Of the two graphs below, which of the two dotted
lines is a true representation of the rate of flagellar growth?

Length (micrometers)
Length (micrometers)

Time (min.) Time (min.)

Make a sketch of a fixed Chlamydomonas cell with flagella. Include a 10 micrometer scale bar.

Calculation
Using the structure and dimensions of microtubules and tubulin given in the introduction,
we will perform a series of calculations.
1. Based on your rate of flagella regeneration, calculate the number of tubulin monomers
assembled per minute.
2. Assuming de novo tubulin synthesis, calculate the number of amino acids polymerized per
minute to make this tubulin.
3. Assuming that all mRNA for tubulin synthesis is being made de novo and that each mRNA is
translated only once, calculate the number of RNA bases transcribed per minute. What if every
mRNA were translated 100 times?
4. Assuming that every mRNA is translated 100 times, and assuming that RNA Polymerase II
can polymerize 2,500 bases per minute at maximum and that there is only one RNA Polymerase II
on any one tubulin gene, how many tubulin genes are present in the Chlamydomonas reinhardtii
genome?
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Goals:
1. Describe the microtubule structure of the flagellar core, the axoneme.
2. Measure flagella length using an ocular micrometer.
3. Demonstrate teamwork in experimental design, lab work, and data collection.
4. Make a testable working hypothesis on the molecular mechanism of flagellar regeneration, and design
an experiment to test this hypothesis.
5. Design a control for your hypothesis.
6. Calculate of the rates of flagellar regeneration in micrometers/min.; and from this rate, calculate the
rates of tubulin assembly in monomers/min., of amino acid polymerization in a.a./min., and of mRNA
transcription in nucleotides/min. Calculation of the number of tubulin genes.
7. Cellular and molecular targets of cycloheximide, actinomycin D, calcium, lithium, and caffeine.
8. Explain the experimental results shown in figures 1-5 in Farrell, K.W., Flagellar Regeneration in
Chlamydomonas Reinhardtii: Evidence That Cycloheximide Pulses Induce a Delay in Morphogenesis, J.
Cell Science, vol 20, pp. 639-654, 1976

Lab Report
1. Statement of the experimental hypothesis your group tested.
2. Table of average flagella lengths per at each time for experimental and control samples.
3. Rate of flagellar regeneration for experimental and control samples.
4. Conclusion and discussion about your hypothesis.
5. Sketch of a single Chlamy cell.
6. Results of the calculations 1-4 on p. 4.
7. Farrell, K.W., Flagellar Regeneration in Chlamydomonas Reinhardtii: Evidence That
Cycloheximide Pulses Induce a Delay in Morphogenesis, J. Cell Science, vol 20, pp. 639-654, 1976
a. In Fig. 1, what would the graph of [3H]-lysine-incorporation into protein versus time in the
cycloheximide-pulsed culture () have looked like if the protein synthetic machinery had been damaged
by the cycloheximide pulse?
b. From Fig. 2, what can one conclude about protein synthesis and the extent of flagellar
regeneration?
c. What did this author measure to quantitate the cellular pool of flagellar precursors in figs. 4 & 5?

Sources:
1. This lab is based on the work of Dr. Malcolm Campbell of Davidson College, Davidson, N.C. who
adapted the work of Vanderwalle and Heyes, Journal of Biological Ed., (1993) 27(2): 125-129. Dr.
Campbell is a distinguished alumnus of the graduate program of the Biology Department of the Johns
Hopkins University.
http://www.bio.davidson.edu/Courses/Bio111/Bio111LabMan/Lab%205.html
2. Gasque, C. Edward, A Manual of Laboratory Experiences in Cell Biology, Wm. C. Brown, Dubuque,
Iowa, 1989, pp.263-267.