Actin and microtubule filaments are integral structures for cell life. Without these structures, cell structure and organization of the cytoplasm would not be possible. They are intricately formed by the cell during various phases of its life and various drugs can inhibit either of the structures. One of these drugs is taxol, which binds to polymerized microtubules and so prevents microtubule depolymerization (Antin et. al., 1981). Knowing the effect that taxol has on microtubule depolymerization, according to Newton’s Third Law where “Every action has an equal and opposite reaction”, we can then imagine the effect that microtubule depolymerization might play on actin filaments. Using GFP-actin expressing LLC-PK1 cells, we hypothesize that a short term treatment of low taxol concentration (~1µM) will not affect actin depolymerization but at higher concentrations, for the same amount time, this effect will be more prominent.

Investigating the Effect of Taxol on Actin Filament Polymerization in LLC-PK1 cells

Introduction

Antin, P.B., S. Forry-Schaudies, T.M. Friedman, S.J. Tapscott, and H. Holtzer. 1981. Taxol Induces Postmitotic Myoblasts to Assemble Interdigitating Microtubule-Myosin Arrays that Exclude Actin Filaments. J. Cell Biol. 90: 300-308. doi: 10.1083/jcb.90.2.300

References

Acknowledgements

Results

We used three different concentrations of taxol as our treatments which were made using 1mM taxol. We added different concentrations of taxol to GFP-actin expressing LLC-PK1 cells and used untreated LLC-PK1 cell as our control. After each treatments, we incubated the cells at 37°C for 30 minutes. After incubation, we took pictures of each cells using both brightfield and fluorescent microscope to observe the actin cytoskeleton.

We would like to thank Mount Holyoke College Biological Sciences Department for this opportunity and Alivia Price for her guidance in the completion of this project. We would like to thank the members of the Colodner lab for their support and encouragement.

Conclusion

Results (cont’d)

Didjana Celkupa, Kenice Kenlock, Rini Mayangsari, Duong NguyenDepartment of Biological Sciences

Mount Holyoke College, South Hadley, MA 01075

Methods

Figure 1. Flow chart of experimental design of the effect of taxol on actin filaments using GFP-actin expressing LLC-PK1 cells.

Figure 2. Effects of different concentrations of taxol on GFP-actin expressing LLC-PK1 cell line. Control cells had no taxol added (1a and 1b), while others were treated with 1μM, 5μM or 10μM taxol and were incubated at 370C for 30 minutes. Pictures were taken under phase contrast and fluorescence microscopy. Treatment of 1μM taxol did prevent actin filaments from depolymerization (2a and 2b); the effect was more clearly observed with the 5μM treatment (3a and 3b), where the bottom left part of the cell elongated. Highest concentration of 10μM turned out to depolymerize actin filaments and caused cell’s deformation.

The results of our experiment does support our hypothesis because we found that taxol kept actin filaments stabilized and did not cause its depolymerization however at the higher concentrations of taxol there was a more prominent effect in the actin present. The actin filaments in the cells with the ten micromolar concentrations seemed to have changed the morphology of the cell. The cells under this concentration appeared to be much more disorganized than in the control or the lower concentrations which suggests that the higher concentration overstimulated the cells and thus causing actin depolymerization. In order to see a more accurate depiction of the effect of the taxol on actin it would be more beneficial to conduct the experiment for a period of time greater than thirty minutes. It would also be beneficial to use GFP-microtubules expressing cells treated with taxol as another form of comparison to see the effect of taxol on treated cells. 

Short-term treatment of taxol on LLC-PK1 cells did affect actin depolymerization. The effects of the drug could be observed even at the lowest concentration of 1μm taxol, and at higher concentrations of 5μm and 10μm the effects were more prominent.

The control LLC-PK1 cell had a pretty round shape, with a smooth, continuous actin cortex (figure 2.1a and 2.1b). Effects of taxol on actin depolymerization varied depending on the different concentrations.

• Actin filaments of cells treated with 1μM taxol started to depolymerize. Phase contrast image did not really show a noticeable difference (figure 2.2a), but fluorescence image showed that actin filaments were growing out (figure 2.2b).

• At higher concentration of 5μM, the effect of the drug was more prominent. Cell shape was distorted (figure 2.3a) because of actin filaments elongation at the bottom left part (figure 2.3b).

• The highest concentration of 10μM had a severe effect on actin depolymerization and cell was eventually deformed (figure 2.4a and 2.4b). Taxol actually caused actin to depolymerize and thus cell started to shrink.