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

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Part I: Effect of taxol 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. 15 mM taxol in ASW

Dish 2. ASW alone (a control)

Dish 3. DMSO in ASW (the same volume of DMSO as for taxol in Dish 1)

DMSO, a solvent, was used to dissolve taxol and make a concentrated stock solution. To ensure that any developmental effects you see are due to taxol and not DMSO, you will need this DMSO control.

Calculate the volume of taxol stock solution to add to Dish 1. Assume that the taxol stock solution is 10 mM, the final concentration of taxol is 15 mM, and the final volume of the solution is 5 mls (the taxol will be added to 5 mls of ASW). Type 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 taxol 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 submit 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 submit your answers below.

10. What do you conclude from these results?

Part II: Effect of taxol on the cytoskeleton (MTs)

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

Dish 1. 15 micromolar taxol in ASW

Dish 2. ASW alone

Dish 3. DMSO in ASW

Assume that the taxol stock solution 10 mM, the final concentration of taxol is 15 mM, and the final volume of the solution is 8 mls (the taxol will be added to 8 mls of ASW). Type your answer below in MICROLITERS, showing your calculation.

6. At 24 h AF, MTs emanate from daughter nuclei toward the cortex. What effect do you expect taxol to have on microtubule arrays (type your answer)?

17. Describe each result.

18. What do you conclude from your results?