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In an experiment by Pollard, T.D. and R.R. Weihing, amoeba cells injected with Cytochalasin B were inhibited of movement, while the control group amoeba cells did move. A hypothesis was made that Cytochalasin B blocked polymerization of actin monomers into actin filaments, thus preventing the cell from moving. To validate this hypothesis the experiments were made again with cycloheximide (inhibits new protein synthesis), and dinitrophenol (inhibits new ATP formation), and colchicine (inhibits polymerization of microtubules Here are the results:$\\$

$\begin Condition: % of cells that didn't move No drug 3 Cycochalasin B 95 Colchicine 4 Cycloheximide 3 Cycloheximide + Cycochalasin B 94 Dinitrophenol 5 Dinitrophenol + Cycochalasin B 85$ $\\$

Question: Why were these control important? How did they help validate the hypothesis? Note: this is for self-study.

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The other inhibitors work on other processes, as you already noticed. There is almost no difference between the conditions when they are added and the corresponding condition when they are missing, suggesting those other processes play no role in the observed phenomenon. Eliminating those other processes makes it more believable that actin polymerization is important, rather than being one of multiple factors.

Moreover, often cell will adapt to an inhibitor by increasing or decreasing other kinds of activity. For example, it could be that actin polymerization inhibition prevents amoeba from eating. In this case, because any imaginable motive mechanism requires energy, starving amoeba may become unable to move, even though it was not using actin for movement. In order to refute such scenarios, where actin polymerization is not directly inducing movement, and where cytochalazine does not inhibit movement directly mechanism, supplementary experiments were performed.

DNP induces something similar to starvation, yet, in these experiments, did not abolish movements when given alone. Nor did DNP rescue movement, because the combination (DNP + cytochalazine) is virtually identical to cytochalazine alone. In both circumstances, DNP did nothing, thus showing that ATP depletion is not influencing movement. This is proof that actin polymerization is directly causing amoeba movement, and not an indirect ATP-mediated effect. Similarly, cytochalazin's effect is independent of protein synthesis, once more supporting the hypothesis that actin polymerization is important on its own.

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When you work with living cells, there are usually more pathways which can be affected, when you start treating the cell a drug. To make sure, which pathway is specifically inhibited, you need these controls, as an inhibition of any of them could theoretically have the same effect:

  • Protein synthesis (cycloheximide) as this provides new actin monomers which are important for the enlongation.
  • ATP synthesis (dinitrophenol) as ATP is necessary for the movement along actin filaments.
  • Inhibition of microtubuli (colchicine) inhibits the microtubuli found in the flagella of amoeba, which also provide a form of movement

The controls show that only the addition of Cytochalasin B to the culture inhibits the movement by inhibiting the enlongation of the actin filaments which proves the hypothesis of the group.

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