The Role of cytoskeleton in tip growth and cytokinesis in Silvetia compressa

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PATHWAY D

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

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 mM 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 mM, and the final volume of the solution is 5 mls (the latrunculin B will be added to 5 mls of ASW). Submit your answer, below, in MICROLITERS, showing your calculation.

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

100% germinated, 100% divided

100% germinated, 0% divided

0% germinated, 100% divided

0% germinated, 0% divided

7. At 24 h AF examine the zygotes under the microscope at 10X. Count the number of zygotes that have germinated for each treatment. Determine the percent germination and type your answers below.

8. Label the membranes with the fluorescent lipid, FM 4-64, to a final concentration of 5 mM. Remember that your dishes contain 5 mls of ASW. The stock solution of FM 4-64 is 5 mM. Determine the volume of FM 4-64 stock solution to add to each dish and type your answer, below, in micro liters, showing your calculation.

9. For each treatment, examine the zygotes under the eppifluorescent microscope at 10X using the Texas Red filter cube. Count the number of zygotes that have divided at least once for each treatment. Determine the percent that have completed cytokinesis and type your answers below.

10. What do you conclude from these results?

Part II: Effect of latrunculin B on the cytoskeleton (actin filaments)

4. As for Part I, calculate the volume of latrunculin B to add to Dish 1.

Dish 1. 1 micromolar latrunculin B in ASW

Dish 2. ASW alone

Dish 3. DMSO in ASW

Assume that the latrunculin B stock solution 10 mM, the final concentration of latrunculin B is 5 mM, and the final volume of the solution is 8 mls (the latrunculin B will be added to 8 mls of ASW). Submit your answer, below, in MICROLITERS, showing your calculation.

6. At 24 h AF, a cone of filamentous actin extends from the rhizoid nucleus to the subapex of the tip, and a plate of actin forms, predicting the position of the new membrane and wall that will be formed during cytokinesis. What effect do you expect latrunculin B to have on actin arrays (type your answer below)?

17. Describe each result.

18. What do you conclude from your results?