Detergent-based isolation of marginal bands of microtubules from nucleated erythrocytes

We have developed a new, detergent-based method for the isolation of marginal bands (MBs) of microtubules from non-mammalian vertebrate erythrocytes. The critical step in MB isolation is selective removal of the "membrane skeleton" network (MS), within which the MB is enclosed. To test potential MS solubilizing agents systematically, we prepared dogfish (Mustelus canis) erythrocyte cytoskeletons in the presence of protease inhibitors and stored them at -20 degrees C in medium containing 50% glycerol and 10 microM taxol to stabilize the MB. Using this as a standard starting material, we found that low concentrations of sodium dodecyl sulfate (SDS) (0.025-0.1%) in the presence of Triton X-100 (0.1-0.4%) released both MBs and nuclei intact from cytoskeletons. Either detergent alone was ineffective. MB release from cytoskeletons was blocked by excess Triton X-100 and slowed by glycerol, and this was useful for stopping the release reaction during quantitative time-course studies. Most MBs (greater than 90%) were liberated from cytoskeletons in 5 to 30 min, depending upon detergent concentrations and other conditions, and they were sufficiently stable for mass isolation by differential centrifugation. Added standard proteins were not proteolyzed during MB release, nor was release blocked by protease inhibitors, indicating that endogenous proteases were not involved. As observed in thin sections and negatively stained whole mounts (transmission electron microscopy) and in critical-point dried preparations (scanning electron microscopy), the isolated MBs consisted principally of bundled microtubules, with some additional adhering material. SDS-polyacrylamide gel electrophoresis showed the isolated MBs to be composed primarily of four tubulin region polypeptides, with the same stoichiometry as in the whole cytoskeleton. As determined by immunofluorescence microscopy, isolated MBs bound antibody to both chicken brain and erythrocyte tau, in addition to anti-tubulin. Thus, proteins of the tau family may be involved in bundling of MB microtubules. Unlike previous MB isolation methods, that employed here is applicable to erythrocytes of diverse species, including the marine toad (Bufo marinus) and the chicken (Gallus domestica), both of which should be of value for comparative studies.