Lab 11  

OBSERVATIONS OF FLAGELLAR ACTION IN CHLAMYDOMONAS  

I. INTRODUCTION  

There ia a basic ultrastructure that is common to both cilia and flagella. The individual cilium or flagellum in eukaryotes contains microtubules, which are unbranched, hollow cylinders, about 24 nm in diameter, composed entirely of protein. The microtubules are almost always observed in a characteristic array of nine outer doublets or pairs of microtubules and two central single microtubules. The "9 + 2" core is referred to as the axoneme. The axoneme is enclosed by an extension of the plasma membrane so that each cilium and flagellum is really protruding region of the cytoplasm.  

At the base of each cilium and flagellum, there is a basal body, a barrel-shaped organelle that has the same basic ultrastructure as a centriole. There is compelling evidence that ciliary and flagellar motion results from the doublets sliding past each other (Satir, 1974). Electron micrographs reveal that the doublets in the inner curvature of the bent cilium are the same length but that the doublets in the inner curvature are always closer to the tip of the cilium.  

Flagellar regeneration in Chlamydomonas is basically a process of microtubule assem­bly. Each microtubule is a hollow, unbranched cylinder composed of globular protein subunits. Perhaps the most intriguing feature of microtubules is their ability to assemble from globu­lar protein subunits in the cytoplasmic pool. During microtubule assembly, the subunits add onto the ends of existing microtubules, thereby increasing their length. Much of the research on this polymerization process has been carried out on regenerating flagella.  

All microtubules have a similar ultrastructure.  In cross sec­tion, the wall of each microtubule is composed of 13 subunits.  The wall thickness is 5 nm, and the total diameter of the microtubule is about 24 nm.  Along the length of the microtubule, which can measure 25 μm or more,the globular subunits form 13 rows, each row termed a protofilament. Ultrastructural analyses of the microtubule have shown that the wall consists of pairs of globular subunits, i.e., dimers.  Biochemical studies have revealed that there are two types of globular subunits in the dimer, so it is appropriately termed a heterodimer.  Each heterodimer is composed of one molecule of α-tubulin and one molecule of ß-tubulin (see pages 559-560 in your text).  

Both are proteins with similar molecular weights (-50,000 daltons) and related amino acid sequences.  The heterodimers in the microtubule wall are arranged in a character­istic way, with the α- and ß-tubulins alternating along the protofilament and slightly staggered in adjacent protofilaments so as to form a helical array. In 1972, it was discovered by R. C. Weisenberg that tubulin extracted from cells could assemble into microtubules in a test tube.  For such in vitro assembly to occur, both guanosine triphosphate (GTP) and Mg²⁺ must be present.   

The process of microtubule assembly is greatly facilitated if microtubule-associated proteins, or MAPs are present.  MAPs are proteins that remain bound to the tubulin after its extraction from cells.  Both   assembly and disassembly can occur in the same in vitro system.  Factors that shift the equilibrium toward disassembly include a relatively high concen­tration of Ca²⁺ and low temperature.  

While the precise conditions that favor microtubule assembly or disassembly in the cell are not known for certain, assembly in vivo probably involves the addition of tubulin dimers onto one end of the microtubule. The process of disassem­bly is believed to occur at the opposite end of the microtubule.  Thus, there is a polarity to the microtubule, with polymerization occurring at one end and depolymerization occurring at the opposite end.   

Flagellar microtubules are relatively stable structures compared, for example, to the microtubules in spindle fibers.  In flagella, there appears to be very little depolym­er­ization at the depolymerization end of the microtubules.  In cells that are regenerat­ing flagella, it has been shown that the tubulin dimers add on at the distal end of the glowing flagellum (Rosenbaum, Moulder, and Ringo, 1969).  

Chlamydomonas is an excellent subject for studying the relationship between flagellar function and growth. This is due to its ability to lose or regenerate flagella depending on culture conditions and experimental manipulations. For example, when grown on solid medium, such as an agar slant, Chlamydomonas lose their flagella and grow as a nonmotile form, When they are returned to liquid medium, the organisms regenerate new flagella and soon become motile. It is this return of motility, which accompanies the flagellar regeneration, that is the focus of todays lab. Specifically you will be determine the minimum length that the flagella must be for the organisms to swim.  

You will be working with normal flagellated Chlamydomonas that have been growing in liquid medium for several days. The first step is the experiment is the removal of the flagella (deflagellation). This is accomplished by subjecting the organisms to a pH shock in the form of a rapid lowering of the pH of the medium. The flagella fall off into the medium (if we are lucky) and the deflagellated cells can be recovered by centrifugation.  

The deflagellated cells are then transferred to fresh liquid medium in which they soon begin regenerating flagella (again if we are lucky). Accompanying the regeneration is a return of motility, which you will monitor by scoring the percentage of motile organisms in a T-25 flask. You will also score the percentage of motile organisms in the original (nondeflagellated) culture as a control. When normal motility has returned to the deflagellated culture (when the percentage of motile organisms is approximately the same as that in the control culture, you will fix a sample and determine the flagellar length at that time.  

II. PROCEDURES  

Microscopic Examination of Chlamydomonas  

1.         On a clean slide, place a drop of the Chlamydomonas culture under a coverslip. Under low power, observe the normal motion of the swimming organisms.  

2.         Make a fresh wet mount with a drop of the Chlamydomonas culture and a tiny drop of protoslo. Mix it in with a toothpick and add a coverslip. 

3.         Examine under low power and High-dry. close the condenser iris until the flagella are visible. Examine some slowed organisms that are moving forward (with the flagella toward the leading edge). Describe the motion of the flagella.  

4.         Now observe the culture in a T-25 under a phase contrast microscope. Whatever swimming motion you see represents 100% motility. 

Experiment on Flagellar Regeneration  

1.         Place four test tubes in you test tube rack and label them as follows:  

1 - Deflagellated/sample 1

2 - Control/sample 1

3 - Deflagellated/sample 2

4 - Control/sample 2  

2.         To each tube, add one drop of Lugol's iodine solution (a fixative and stain) and three drops of culture medium (Medium I of Sager and Granick). These tubes will be used to fix the samples at the onset and at the time when motility has returned.   

Deflagellation Procedure.

1.         From an actively growing Chlamydomonas culture (the control) in a T-25, transfer a one half (7 ml) of it to a clean 50-ml beaker containing a magnetic stirrer. Then add 8 ml of Medium I to bring the final volume to 15 ml.  Save the other half of the remaining control culture for determination of normal flagellar length.  

2*.        Deflagellate the organisms in the beaker by rapidly lowering the pH of the medium.  Monitor the pH with the electrodes of a pH meter.  With constant stirring, lower the pH to 4.5 within 30 sec by adding dropwise 0.25 N acetic acid.  

3*.        As soon as the pH 4.5 has been reached, immediately prepare a slide and check to see if there is any motility. If they are still motile, drop another 0.5 pH units until they are non-motile. Then restore the pH of the culture to 6.8 - 7.2 (depending on the origional pH of the solution) by adding dropwise 0.5 N KOH.  

4.         Pour the 15 ml of the deflagellated culture into a 15-ml conical centrifuge tube, and centrifuge at 3,500 rpm for 5 min.  

5.         Decant and discard the supernatant liquid, which contains the flagella.  To the pellets, which contain the deflagellated cells, add 10 ml of Medium I. Resuspend the cells with a Pasture pipet.  

6.         Pour the contents of the centrifuge tube into a labeled 50-ml Erlenmeyer flask labeled deflagellated. Place both the origional control culture in a T-25 and 50 ml erlenmeyer deflagellated culture with loose T-25 caps in a gently reciprocating shaker that is illuminated with a fluorescent lamp and proceed immediately to step 1 below.  

Determination of the Return of Motility  

1.         As soon as possible, remove three drops from the deflagellated culture with a pasteur pipet, add to the test tube labeled Deflagellated/sample 1, and swirl the tube to fix (kill) the cells rapidly. Using a fresh pasteur pipet, remove on or two drops from the control culture, add to the test tube labeled control/sample 1 and swirl the tube. 

Record the time in your notebook. Put these samples aside until the last two samples have been taken. Then all the samples will be examined for flagellar length.  

2.         Use the low power, 10x objective on the inverted-phase contrast scope and examine the control T-25 to determine the percent motility.   

4.         Repeat the scoring every minute until the percentage of motile cells in the "deflagellated" T-25 is approximately the same as that in the "Control" T-25. Immediately after normal motility has returned to the deflagellated cells, fix sample 2 from the deflagellated and control cultures using the appropriately labeled test tubes and fresh pasture pipets. Record all your data for analysis.  

Measurement of Flagellar Length.*  

1.         With a fresh Pasteur pipet, gently resuspend the fixed sample and place a drop on a clean slide.  Add a coverslip and examine under low power to locate an area with a concentration of cells.  

2.         Using a calibrated ocular micrometer with the oil-immersion objective, measure flagellar length in each of 15 cells from the two cultures.  Select only cells that have at least one fairly straight flagellum.  Since both flagella will be about the same length, just measure the straighter of the two.  Record the measurements for your cul­ture(s) in your laboratory notebook.  

III. DATA ANALYSIS  

1.         From the time you fixed the first samples, how long did it take for normal motility to return to the deflagellated organisms?  

2.         Was the return of motility sudden or gradual?  

3.         What does the graphical relationship of your data look like?  

4.         What does this suggest about flagellar function in these organisms?  

5.         Run t-tests on your data of length measurements.  

6.         What percentage of the length in the controls must be attained before normal motility returns?  

7.         For the time period you studied, what is the mean rate of flagellar regeneration in Chlamydomonas?

 

IV. REFERENCES  

1.         Allen, R.D. 1981. Motility. J. Cell Biol. 91:148s-155s.  

2.         Bergman, A. 1990. Laboratory Investigations in Cell and Molecular Biology. Third Edition, John Wiley and Sons, New York. Much of the material in this lab was adapted from this manual.  

3.         Dustin P.  1980 (Aug.).  Microtubules.  Sci. Am. 243:66-76.  

4.          Lefebvre, P. A. and Rosenbaum, J. L.  1986.  Regulation of the synthesis and assem­bly of ciliary and flagellar proteins during regeneration.  Ann. Rev. Cell Biol. 2:517-546.  

5.         Rosenbaum, J. L., Moulder, L. E., and Ringo, D. L.  1969.  Flagellar elongation and shortening in Chlamydomonas:  The use of cycloheximide and colchicine to study the synthesis and assembly of flagellar proteins.  J. Cell Biol. 41:600-619.  

6.         Satir, P. 1974. How Cilia Move. Sci. Am. 231:45-52

 

V. MATERIALS  

1.         15 ml disposable conical centrifuge tubes  

2.            Compound Microscopes with ocular micrometers, slides and coverslips  

3.         Two pH meters, each with 50 ml 0.25 N and 1.0 N KOH in dropping bottles  

4.         Large Reciprocating Shaker  

5.         Large Centrifuge, clinical centrifuges  

6.         Small test tubes with test tube racks (4 test tubes per group)  

7.         Pasteur pipets, marking pens, Vaseline petroleum jelly (four jars), toothpicks  

8.         T-25 flasks, protoslo, pipettors  

9.         10 ml graduated cylinders, 50 ml beakers  

10.        Fluorescent lamps with two cool-white, 15-watt bulbs  

11.        Four culture of Chlamydomonas reinhardi* in T-25 culture flasks  

12.        Two Magnetic mixer with stirbars  

14.        100 ml Lugol's iodine solution dispensed in four dropping bottles  

Dissolve 6.0 gms KI and 4.0 gms I in 100 ml distilled water    

15.        30 ml 0.25 N acetic acid dispensed in a dropping bottle  

(Dilute 2.9 ml glacial acetic acid to 100 ml with distilled water)  

16.        150 ml of Medium I of Sager and Granick: To a 1000 ml volumetric flask add:  

0.5 gms Na₃ citrate @ 2H₂O

0.1 gms KH₂PO₄

0.3 gms NH₄NO₃

0.3 gms MgSO₄ @ 7H₂O

0.04 gms CaCl₂

0.01 gms FeCl₃ @ 6H₂O

10 mls of trace metal solution described below              

17.        1000 ml of trace metal solution: To 1000 ml distilled water add:  

100 mg H₃BO₃

100 mg ZnSO₄ @ 7H₂O

40 mg MnSO₄ @ 4H₂O

20 mg CoCl₂ @ 6H₂O

20 mg Na₂MoO₄ @ 2H₂O

4 mg CuSO₄  

*           The Chlamydomonas are cultured in Medium I of Sager and Ganick as follows: Using sterile technique, add 10 ml of Medium I to the slat cultures and rinse off as much of the curture as possible. Place this mixture in a T-75 tissue culture flask and bring the total volume to 25 ml.  The culture is grown at room temperature with a constant aeration (cotton filtered air) and constant illumination (fluorescent lamp). The culture is ready for use in the experiment when the absorbance at 675 nm  is between 0.1 and 0.5, a culture density range that is reached 2-3 days after inoculation.  

Commercially available cultures of Chlamydomonas may be supplied growing on agar slants, on which the organisms lack flagella. Thus, even for the initial observations of Chlamydomonas, they must be grown in liquid Medium I for 2-3 days. Long-term cultures are easily maintained on an agar slant of Medium I (10 ml Medium I + 0.15 gms agar). The slant should be kept in a well-illuminated area at room temperature. 

Lab 12 and 13  

     EFFECT OF DRUGS ON MICROTUBULAR REASSEMBLY IN CHLAMYDOMONAS  

I. INTRODUCTION  

The objectives of this project are to study the kinetics of normal flagellar regeneration in Chlamydomonas  and the effects produced by three chemicals, colchicine, cytochalasin-B and cycloheximide. This experiment is based on a study by Rosenbaum, Moulder, and Ringo (1969).  It examines the effects of a number of drugs on flagellar regenera­tion in Chlamydomonas reinhar­di.  Colchicine is known to bind free tubulin dimers to the cytoplasmic pool.  The colchicine-bound dimers are able to attach to the growing ends of the microtubules but, once attached, prevent any further addition of dimers, whether bound to colchicine or not.  Cycloheximide is a potent inhibitor of protein synthesis in eukaryotic cells.   

In the presence of cycloheximide, there is no longer synthesis of a- or ß-tubulin. Cytochalasin-B functions to disrupt actin microfilaments. By exposing the cells to these agents, we will be able to determine if tubulin dimers, protein synthesis or actin microfilaments are necessary for flagellar regeneration. Design and perform an experiment to determine the effects of these three drugs on flagellar regeneration.  

II.  MATERIALS  

1.         Compound Microscopes with ocular micrometers, slides and coverslips  

2.         pH meter, 30 ml of 0.25 N KOH in dropping bottles  

3.          Reciprocating Shaker  

4.          Centrifuge  

5.         Small test tubes with test tube racks  

6.         Pasteur pipets, marking pens, disposable rubber gloves  

9.         10 ml graduated cylinders, 100 ml beakers  

10.        Fluorescent lamp with tow cool-white, 15-watt bulbs  

11.        Culture of Chlamydomonas reinhardi*  

12.         Magnetic mixer with stirbars  

13.        Protoslo  

14.         Colchicine (3 mg/ml)** in Medium I (50 ml Medium I + 150 mg Colchicine)  

15.         Cycloheximide (10 μg/ml)** in Medium I (100 ml Medium I + 1 mg Cyclohexaminde)  

15.5.      Inverted phase contrast microscopes