Tubulin is a heterodimer composed of one alpha- and one beta-tubulin molecules. There are multiple genes for slightly different forms, or isotypes, of both alpha- and beta-tubulin. These have different tissue- and developmental stage- expression patterns, and all are subject to numerous posttranlational modifications (PTM). Blood cells express a particular isotype of beta-tubulin restricted to these cells, and an isotype of alpha tubulin that is shared by other types of cells as well as blood cells. We have pursued a mass-spectrometry-based study of these tubulins, in order to look for changes during development or following exposure to MT-targeted chemotherapy drugs. We have used readily available chicken red blood cells for this study, but the tubulin isotypes are the same as those expressed in human platelets and white blood cells. We have found that the alpha tubulin contains a PTM that has not been reported previously. This PTM appears to be restricted to blood cells, since we do not find it in tubulin from brain or epithelial cells. We do not yet understand the biological significance of this PTM, its chemical or enzymatic origin, nor how it changes during blood cell differentiation or following exposure to chemotherapy drugs. These are all subjects of ongoing research. Natural products have historically been the source of most of the microtubule (MT)-targeting small molecules whose properties have allowed them to become useful drugs. That remains true of most but not all of the compounds that we have used in this study. These include the clinically established MT-active drugs colchicine, combretastatin, vincristine, taxol, and others. Almost all such agents were developed first in pre-clinical research that included in vitro studies of the effect of the compounds on polymerization of tubulin to microtubules as well as the effect of such compounds on cell behavior, especially examining the ability of the compounds to disrupt mitosis through effects on the MT arrays that comprise the mitotic spindle. The in vitro studies with purified tubulin are a critical way in which compounds are evaluated, and indeed this is often used as a screening method to discover new active compounds as well as to quantitatively evaluate established ones. To further this, we have reviewed approaches to quantitating MT assembly by optical methods, with the goal of maximizing the information that may be obtained and minimizing the errors that the system can produce. We used both optical density-based and fluorescence-based approaches, and evaluated sources of error as well as underappreciated sources of extra information on the polymerization process.
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