Role of the Cytoskeleton in Tip Growth and Cytokinesis

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PATHWAY D

Part I: Effect of latrunculin B on tip growth and cytokinesis

1. Potentiate a dish of fronds in ASW (artificial sea water) with light from above at 15ºC.

2. Prepare 3 small-sized (30 mm) dishes with one coverslip each attached to the bottom with clay.

3. After 2 h of potentiation, fronds are collected, rinsed with ASW, and placed in the dark at 15ºC for one hour. In the dark, they release eggs and sperm, and fertilization occurs. The fronds are then removed, and zygotes are ready for use. In each dish, plate newly fertilized eggs (zygotes) in unidirectional light at 15ºC.

4. At 10 h AF (after fertilization), shortly before germination and mitosis, the three dishes of zygotes will be treated as follows:
            Dish 1. 1 micromolar latrunculin B in ASW
            Dish 2. ASW alone (a control)
            Dish 3. DMSO in ASW (the same volume of DMSO as for latrunculin B in Dish 1)
DMSO, a solvent, was used to dissolve latrunculin B and make a concentrated stock solution. To ensure that any developmental effects you see are due to latrunculin B and not DMSO, you will need this DMSO control.

Calculate the volume of latrunculin B stock solution to add to Dish 1. Assume that the latrunculin B stock solution is 10 mM, the final concentration of latrunculin B is 1 micromolar, and the final volume of the solution is 5 mls (the latrunculin B will be added to 5 mls of ASW). Type in your answer in the answer sheet, in MICROLITERS, showing your calculation.

5. At 10 h AF, add the appropriate solution (latrunculin B or control solutions) to your three dishes. To do this, add latrunculin B (or DMSO) to 5 mls fresh ASW in a conical tube. Cap the tube and mix well. Gently pour the ASW off the zygotes and replace with the latrunculin B or control solution. Return zygotes to the incubator at 15ºC.

6. By 24 h AF, 95 to 100% of untreated zygotes have germinated and divided. What effect do you expect the latrunculinB to have on germination (initiation of tip growth)? Cytokinesis?
            a) 100% germinated, 100% divided
            b) 100% germinated, 0% divided
            c) 0% germinated, 100% divided
            d) 0% germinated, 0% divided