Visualization of the Actin Microfilaments and Microtubules in Response to ATP-depletion

Two of the three main components of the cellular cytoskeleton are microfilaments, composed of actin, and microtubules, composed of tubulin. Changes in cell shape, movement, and division are all dependent upon polymerization and depolymerization of these dynamic cytoskeletal proteins. Actin and tubulin can be visualized in mouse fibroblast cells using the technique of immunofluorescent microscopy. Prior research has shown that upon depletion of ATP (adenosine triphosphate) through the application of energy metabolism inhibitors, changes can be observed in the cytoskeleton; stress fiber structures composed of actin are disrupted, while an opposite effect of stabilization occurs in microtubules. When microtubules are stabilized, post-translational modifications often accumulate as a result. These changes suggest that ATP may control microtubule depolymerization within mouse fibroblast cells. Because of the differences in these 2 cytoskeletal protein filament systems, imaging both microtubules and actin filaments simultaneously involves special fixation techniques. My project involves using a methanol fixation technique along with antibodies to both actin and tubulin to determine the temporal and spatial changes in both microtubules and actin filaments as ATP becomes depleted in cells. Thus far, I have learned sterile technique to grow mouse fibroblast cells and have performed immunofluorescent microscopy to see the normal distribution of microtubules in cells with normal levels of ATP. Next, I’ll look at both cytoskeletal filaments under ATP-depleting conditions in order to see whether stress fiber disassembly occurs in regions where microtubules are more stable.