Lab 2

Flagella Regeneration
in Chlamydomonas reinhardtii
or how simple microscopy can be used to
calculate rates of assembly of
macromolecules into cellular structures

Andrew, Jiayun, Michael, Madeline

Fri
Allens sec.

Bharat, John, Russell & Fahd

Fri Kais sec

Rubi, Michelle, David & Raj

Fri Kais sec

Chlamy fixed in Lugols Iodine

1000X phase contrast

experiment
Remove flagella with a pH shock, pH 7.5 to 4.5 for 60s
then back to pH 7.5
In teams of 4, design an experiment to determine the
effect of a drug on flagella regeneration.
Two flasks, one control, one with a drug.
Possible drugs are calcium, lithium, cycloheximide,
actinomycin D or caffeine.
Illuminate for one hour (or more if needed), taking and
fixing samples every 15 min.
Mount samples and measure flagella lengths.
Determine rate of flagella growth in micrometers per
minute.

the flagella

Electron Micrograph

Diagramatic Drawing

The axoneme is composed of a 9 + 2

structure of microtubules.
Each of the nine outer are partial doubles,
one complete microtubule with 13
protofilaments and one partial microtubule
with 10 protofilaments.
The two inner are complete microtubules.

From Iowa State University Agronomy 317

 Based on your rate of flagella

regeneration, calculate the number of
tubulin monomers assembled per minute.
How would you make this calculation?

Actinophrys,
a heliozoan protist.
The heliozoan has a spherical
cell body with thick radiating
strands of cytoplasm called
axopodia, which are supported
by microtubules. When smaller
organisms bump into the
axopodia, they stick to the
axopodia and are then moved
down the axopodia to the cell
body, where they are
phagocytosed.

http://www.microscopy-uk.org.uk/mag/imgfeb02/acti3web.jpg

Chlamydomonas adheres to the axopodia by its flagella but not its cell body,
and has evolved the ability to escape by shedding its flagella. As a result,
the heliozoan has flagella for dinner, while the Chlamydomonas cell floats
away and lives to grow new flagella.

Ref:

Pickett-Heaps, J. and Pickett-Heaps, J. (1996). Predatory Tactics: Survival in the

Microcosmos. NTSC videocassette, 42 minutes. Cytographics, Ascot Vale, Australia.

Actinophrys

axopodia

Chlamydomonas
flagella

Ref.: http://www1.umn.edu/news/prod/groups/ur/@pub
/@ur/documents/asset/ur_87651.jpg

finis


Example: 3 um/15 min. = 0.2 um/min = 2 x 10^-7
m/min.
one 9+2 axoneme = 233 protofilaments/flagella
one tubulin monomer/4 x 10^-9 m
2 x 10^-7 m/min. x one tubulin monomer/4 x 10^9 m = 50 monomers/min.
50 monomers/min. x 233 protofilaments/flagella
= 11,650 monomers/flagella min.
11,650 monomers/flagella min. x 2 flagella/cell =
23,300 monomers/cell min.

 2. Assuming de novo tubulin synthesis,

calculate the number of amino acids
polymerized per minute to make this
tubulin.
23,300 monomers/ min. x 450
aa/monomer = 1.05 x 10^7 aa/min.

 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?
1.05 x 10^7 aa/min. x 3 bases/aa = 3.15 x
10^7 bases/min. for single use mRNA
3.15 x 10^7 bases/min./100 = 3.15 x 10^5
bases/min. for 100x use mRNA

 4. Assuming that every mRNA is

translated 100 times, and assuming that
RNA Polymerase II can polymerize 2,500
bases per minute at maximum, how many
tubulin genes are present in the
Chlamydomonas reinhardi genome?
3.15 x 10^5 bases/min. //2.5 x 10^3
bases/min/RNA Pol II = 126 genes