Perturbations in microtubule mechanics from tubulin preparation

Microtubules are essential structures for cellular organization. They support neuronal processes and cilia, they are the scaffolds for the mitotic spindle, and they are the tracks for intracellular transport that actively organizes material and information within the cell. The mechanical properties of microtubules have been studied for almost 30 years, yet the results from different groups are startlingly disparate, ranging over an order of magnitude. Here we present results demonstrating the effects of purification, associated-protein content, age, and fluorescent labeling on the measured persistence length using the freely fluctuating filament method. We find that small percentages (<1%) of residual microtubule-associated proteins left over in the preparation can cause the persistence length to double, and that these proteins also affect the persistence length over time. Interestingly, we find that the fraction of labeled tubulin dimers does not affect the measured persistence length. Further, we have enhanced the analysis method established by previous groups. We have added a bootstrapping with resampling analysis to estimate the error in the variance data used to determine the persistence length. Thus, we are able to perform a weighted fit to the data to more accurately determine the persistence length.