Differential effects of inhibitors on the regeneration of flagella Assignment

Flagella are extensions from the cell surface that consist of 9 doublets of microtubules, 2 central microtubules, plus several microtubule associated proteins ( Figures 1B and 1C ). Flagellar microtubules are normally relatively stable structures that grow from the addition of dimers to the distal end of the flagellum. The proximal end of the flagellum is anchored into the cytoplasm via the basal body .

The purpose of this experiment is to study the polymerization of microtubules during the regeneration of flagella in the biflagellate green algae, Gonium sp . The kinetics of microtubule assembly will be examined in cells that have been deflagellated. The deflagellation of cells while leaving them relatively undamaged can be accomplished in a number of ways, such as through pH shock, centrifugation, sonication, and mechanical shearing. In this lab, you will be using mechanical shearing (in a blender) to deflagellate the cells. You will compare the natural rate of flagellar regeneration to that obtained in the presence of two differentially acting drugs: cycloheximide and colchicine . Cycloheximide is a potent inhibitor of protein synthesis , while colchicine binds to free tubulin dimers and prevents additional dimers from binding to the microtubules. Figure 1: Flagella structure. Diagram of the arrangement of alpha and beta tubulin in a microtubule (A). Micrograph (B) and diagram (C) of the structure of a flagellum in cross-section.A B C PROCEDURES:

A. Deflagellation and drug treatments 1. Place 40 ml of the Gonium sp . culture into a blender. Blend on the lowest speed for 15 seconds . Be certain that the blender is set on LO and use the WHIP button. The blending action will deflagellate the culture. Note: the remainder of your culture will act as your non- deflagellated control culture.

2. Label 3 conical centrifuge tubes: Medium, Colchicine, and Cycloheximide. Add 10 mL of the deflagellated culture to each tube. Using the small centrifuge on your bench, centrifuge for 4 minutes at 3700 rpm .

3. Following centrifugation carefully discard the supernatant and add 5 mL of the appropriate medium to each tube. The colchicine and cycloheximide found on your lab bench are already at the proper working concentrations . Very gently resuspend your cells using a separate Pasteur pipette for each tube.

4. IMMEDIATELY following resuspension, proceed to part B to collect your time 0 samples .

CAUTION: Colchicine, cycloheximide, and glutaraldehyde are all extremely dangerous chemicals. ALWAYS wear a lab coat, gloves, and safety goggles when handling them. Dispose of the contents of all culture tubes that contain these chemicals into the appropriately labeled waste containers. There is also a special slide and Pasteur pipet discard container to dispose of these items after you complete your measurements. Be sure to position a snorkel over all areas where glutaraldehyde is present – racks of tubes and microscopes.

B. P reparation of slides Samples of the non-deflagellated, medium, colchicine, and cycloheximide cultures will each be collected at 20 minute intervals from 0 to 120 minutes . It is essential that you collect samples at the appropriate times. As soon as the timer goes off, IMMEDIATELY SET THE TIMER FOR ANOTHER 20 MINUTES!

1. Label collection tubes and glass slides with the treatment and sampling time . Gently resuspend the cells by pipeting up and down with a Pasteur pipette. Fix the sample by transferring 3 drops of culture from the treatment tube into a labelled collection tube. Add 2 of glutaraldehyde and immediately gently resuspend with a Pasteur pipette. Your cells are now fixed and can be examined at your leisure .

2. Transfer one drop of your fixed cells onto the labelled slide and add a coverslip. Please rinse your pipettes in a small beaker of algal medium and re-use them.

3. Repeat this process for the next 20 minute interval . Don’t forget to reset the timer 2 C. Measurement of flagella length Instructions for the use of the phase contrast microscope can be found in Appendix G:

Phase Contrast Microscopy.

Starting with the non-deflagellated culture, observe 10 cells on each slide and use the calibrated computer program to measure the length of one flagellum per cell . Repeat for each treatment and time point.

Note – Occasionally, not all of the Gonium sp. is deflagellated upon blending, so cells that have flagella as long as the normal average length can be IGNORED throughout the experiment. Note – we do not expect all of the cells will actually have flagella! You are observing a random sample of 10 cells. If a cell doesn’t have flagella, put a length of 0 mm in your data table. Please view the Phase Contrast Microscopy video and read Appendix G: Phase Contrast Microscopy prior to the Lab 5 Tutorial.

ASSIGNMENT Your assignment for Lab 5 will include data analysis and answering specific questions and is due by 11:59 PM on November 19 th . The details of this assignment are available in the Lab 5 folder, and the specifics of your data analysis will be discussed during the Lab 5 Tutorial .

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