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Texas Wesleyan University

Department of Biology

Biology 4412 (S24) Laboratory: Regeneration of flagella in Chlamydomonas reinhardtii

Background: Chlamydomonas is a eukaryotic, single-celled photosynthetic organism that is a model system for biological studies of many different processes. The organism has a single chloroplast, and externally as two flagella, which allow the organism to move and sense other organisms.

The flagella are made of microtubules, arranged with a central pair of microtubules surrounded by 9 doublet microtubules. The flagella also contain ciliary dynein, which helps with the movement of the flagella. The flagella are anchored in a basal body that serves as a MTOC from where the microtubules are assembled and can grow. Microtubules are assembled from tubulin protein subunits.

The flagella are remarkably sensitive to external conditions, and also show an ability to regenerate very quickly under the right circumstances. Experimentally, we can induce flagella to fall off with an acid shock, and then we can see flagellar regeneration at certain time intervals after that acid shock.

We will be addressing two questions in the lab over the next two weeks:

· Does inhibiting protein synthesis affect the ability of flagella to regenerate after acid shock?

· Does inhibiting transcription affect the ability of flagella to regenerate after acid shock?

A reminder from last week about how to measure the length of flagella:

Converting ocular micrometer measurements to “real” measurements.

Under 40X, you can use the following relationship: 10 “ocular” units (or 1 unit on our scale)= 0.1 mm (or 100 um)

We will be evaluating the effects of two drugs on flagellar regeneration to help us answer our experimental questions. We will do these experiments over the next two weeks.

Name of Treatment Drug effect Final Concentration of drug

Cycloheximide inhibits translation 10 ug/mL

Actinomycin D inhibits transcription 50 ug/mL

Review from last week:

1. Each of you will have a tube of Chlamydomonas culture (in a glass test tube).

2. Obtain a 1.5 mL plastic microfuge tube, and add 4 uL of Lugol’s iodine to the tube.

3. Then, add 20 uL of Chlamydomonas culture to the plastic microfuge tube.

4. Transfer the 24 uL (culture + Lugol’s) to a microscope slide, and place a coverslip gently over the slide to form a wet mount.

5. Observe the Chlamydomonas under 40X, Ph2 setting. Identify 3 organisms with flagella and estimate their length using the micrometer technique from last week.

Part II: Drug Treatment

--Make sure you record which drug has been assigned to your group on the data sheet provided.

1. Each of you will have a porcelain spot plate with 12 wells. Label the plate as follows (this will make more sense as we go through the procedure):

- Drug + Drug - Drug + Drug

Acid Shock to drop flagella:

2. Transfer 400 uL of the Chlamydomonas culture into a new glass test tube.

3. Add 24 uL of 0.5N acetic acid to the 400 uL of Chlamydomonas. Gently mix by shaking and wait 45 seconds. (If you go longer, you will kill everything. Time this carefully).

4. Quickly add 24 uL of 0.5N KOH to the tube to neutralize the reaction and mix by gentle shaking.

Drug Treatment and Timing

5. You will now split your sample between two new glass test tubes:

· Transfer 200 uL of the sample (from step 4) to a new glass test tube and label that tube - DRUG.

· Now, transfer 200 uL of sample (from step 4) to a new glass test tube and label that tube + DRUG.

· Follow the directions below for the drug that you were assigned:

· Add 2 uL of the stock cycloheximide (1 mg/mL) to the +DRUG glass tube.

· Add 10 uL of the stock actinomycin D ( 1 mg/mL ) to the + DRUG glass tube

6. Quickly: Add 4 uL of Lugol’s iodine to the “Time 0” wells for + Drug and -Drug.

· Immediately remove 20 uL from the + Drug tube and add to the designated well in your spot plate. (Time 0)

· Immediately remove 20 uL from the - Drug tube and add to the designated well in your spot plate. (Time 0)

7. Set a timer for 12 min.

8. Now, during the 12 minute break, you will view the “time 0” samples:

· Transfer 24 uL of the +Drug sample from the plate to a new microscope slide and make a wet mount.

· Transfer 24 uL of the - Drug sample from the plate to a new microscope slide and make a wet mount.

· View each slide under 40X under the Ph2 phase contrast setting.

· For each slide, record the following for 5 organisms on the data sheet

· Whether you see flagella or not (yes or no)

· If yes, estimate if possible how long they are

9. When the 12 minutes are up (when your timer alarm sounds): Quickly: Add 4 uL of Lugol’s iodine to the “Time 12” wells for + Drug and -Drug.

· Immediately remove 20 uL from the + Drug tube and add to the designated well in your spot plate. (Time 12)

· Immediately remove 20 uL from the - Drug tube and add to the designated well in your spot plate. (Time 12)

10. Reset your timer for 12 min.

11. Now, you will view your samples from the “Time 12” wells:

· Transfer 24 uL of the +Drug sample from the plate to a new microscope slide and make a wet mount.

· Transfer 24 uL of the - Drug sample from the plate to a new microscope slide and make a wet mount.

· View each slide under 40X under the Ph2 phase contrast setting.

· For each slide, record the following for 5 organisms on the data sheet

· Whether you see flagella or not (yes or no)

· If yes, estimate if possible how long they are

12. When the 12 minutes are up (when your timer alarm sounds): Quickly: Add 4 uL of Lugol’s iodine to the “Time 24” wells for + Drug and -Drug.

· Immediately remove 20 uL from the + Drug tube and add to the designated well in your spot plate. (Time 24)

· Immediately remove 20 uL from the - Drug tube and add to the designated well in your spot plate. (Time 24)

13. Reset your timer for 12 min.

14. Now, you will view your samples from the “Time 24´wells for + drug and - drug:

· Transfer 24 uL of the +Drug sample from the plate to a new microscope slide and make a wet mount.

· Transfer 24 uL of the - Drug sample from the plate to a new microscope slide and make a wet mount.

· View each slide under 40X under the Ph2 phase contrast setting.

· For each slide, record the following for 5 organisms on the data sheet

· Whether you see flagella or not (yes or no)

· If yes, estimate if possible how long they are

15. When the 12 minutes are up (when your timer alarm sounds): Quickly: Add 4 uL of Lugol’s iodine to the “Time 36” wells for + Drug and -Drug.

· Immediately remove 20 uL from the + Drug tube and add to the designated well in your spot plate. (Time 36)

· Immediately remove 20 uL from the - Drug tube and add to the designated well in your spot plate. (Time 36)

16. Reset your timer for 12 minutes

17. Now, you will view your samples from the “Time 36´wells for + drug and - drug:

· Transfer 24 uL of the +Drug sample from the plate to a new microscope slide and make a wet mount.

· Transfer 24 uL of the - Drug sample from the plate to a new microscope slide and make a wet mount.

· View each slide under 40X under the Ph2 phase contrast setting.

· For each slide, record the following for 5 organisms on the data sheet

· Whether you see flagella or not (yes or no)

· If yes, estimate if possible how long they are

18. When the 12 minutes are up (when your timer alarm sounds): Quickly: Add 4 uL of Lugol’s iodine to the “Time 48” wells for + Drug and -Drug.

· Immediately remove 20 uL from the + Drug tube and add to the designated well in your spot plate. (Time 48)

· Immediately remove 20 uL from the - Drug tube and add to the designated well in your spot plate. (Time 48)

19. Reset your timer for 12 minutes

20. Now, you will view your samples from the “Time 48”wells for + drug and - drug:

· Transfer 24 uL of the +Drug sample from the plate to a new microscope slide and make a wet mount.

· Transfer 24 uL of the - Drug sample from the plate to a new microscope slide and make a wet mount.

· View each slide under 40X under the Ph2 phase contrast setting.

· For each slide, record the following for 5 organisms on the data sheet

· Whether you see flagella or not (yes or no)

· If yes, estimate if possible how long they are

21. Reset your timer for 12 minutes

22. When the 12 minutes are up (when your timer alarm sounds): Quickly: Add 4 uL of Lugol’s iodine to the “Time 60” wells for + Drug and -Drug.

· Immediately remove 20 uL from the + Drug tube and add to the designated well in your spot plate. (Time 60)

· Immediately remove 20 uL from the - Drug tube and add to the designated well in your spot plate. (Time 60)

23. This is the LAST one! Now, you will view your samples from the “Time 60” wells for + drug and - drug:

· Transfer 24 uL of the +Drug sample from the plate to a new microscope slide and make a wet mount.

· Transfer 24 uL of the - Drug sample from the plate to a new microscope slide and make a wet mount.

· View each slide under 40X under the Ph2 phase contrast setting.

· For each slide, record the following for 5 organisms on the data sheet

· Whether you see flagella or not (yes or no)

· If yes, estimate if possible how long they are