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

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PATHWAY B

Part I: Effect of oryzalin 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. 5 mM oryzalin in ASW

Dish 2. ASW alone (a control)

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

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

Calculate the volume of oryzalin stock solution to add to Dish 1. Assume that the oryzalin stock solution is 10 mM, the final concentration of oryzalin is 5 mM, and the final volume of the solution is 5 mls (the oryzalin 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 the oryzalin 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 oryzalin on the cytoskeleton (MTs)

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

Dish 1. 5 micromolar oryzalin in ASW

Dish 2. ASW alone

Dish 3. DMSO in ASW

Assume that the oryzalin stock solution 10 mM, the final concentration of oryzalin is 5 mM, and the final volume of the solution is 8 mls (the oryzalin will be added to 8 mls of ASW). Submit 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 oryzalin to have on microtubule arrays (type your answer below)?

17. Describe each result.

18. What do you conclude from your results?