Free and polymerized tubulin in cultured bone cells and Chinese hamster ovary cells: the influence of cold and hormones

Abstract

Free and polymerized tubulin were measured in [rat] bone cells and Chinese hamster ovary (CHO) cells cultured on plastic substrata. Polymerized tubuin was stabilized in a microtubule-stabilizing medium (MSM) containing 50% glycerol and separated from free tubulin by centrifugation. Tubulin content was assayed in both fractions by the colchicine-binding assay. The measured degree of polymerization in both bone cells and CHO cells varied with stabilization conditions. The degree of polymerization in attached cells increased up to 73% during the first 20 min after addition of MSM at 24.degree. C, and remained constant thereafter. Stabilization at 0.degree. C resulted in a decrease down to 62% in the degree of polymerization during the first 20 min after addition of the MSM, which again remained constant thereafter. Confluent bone cells maintained at 0.degree. C for 1 h before stabilization contained significantly less polymerized tubulin than control cells kept at 37.degree. C using stabilization both at 0.degree. C and at 24.degree. C. Changes in bone cell morphology induced by incubation of cells with prostaglandin E1 or E2, parathyroid hormone and dibutyryl cAMP were not associated with a change in the degree of tubulin polymerization. This was confirmed morphologically by immunofluorescence using affinity-purified tubulin antibodies: microtubules in hormone-treated cells were not noticeably reorganized when compared to mircotubule organization in control cells. They were, however, squeezed closer together in cellular pseudopods due to the altered cell shape. This altered cell shape appears to be correlated with disorganization of the microfilament system, since microfilaments, detected using affinity-purified actin antibodies, did alter drastically their appearance and distribution after hormone addition.