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 bone cells and Chinese hamster ovary (CHO) cells cultured on plastic substrata. Polymerized tubulin was stabilized in a microtubule- stabilizing medium (MSM) containing 50 percent glycerol and separated from free tubulin by centrifugation. Tubulin content was assayed in both fractions by the colchicines- binding assay. The measured degree of polymerization in both bone cells and CHO cells varied with stabilixation conditions. The degree of polymerization in both bone cells and CHO cells varied with stabilization conditions. The degree of polymerization in both bone cells and CHO cells varied with stabilization conditions. The degree of polymerization in attached cells was found to increase up to 73 percent during the first 20 min after addition of the MSM at 24 degrees C, and remained constant thereafter. Stabilization of 0 degrees C resulted in a decrease down to 62 percent in the degree of constant thereafter. Stabilization at 0 degrees C resulted in a decrease down to 62 percent in the degree of polymerization during the first 20 min after addition of the MSM at 24 degrees C, and remained constant thereafter. Confluent bone cells maintained at 0 degrees C for 1 h before stabilization contained significantly less polymerized tubulin than control cells kept at 37 degrees C using stabilization both at 0 degrees C and at 24 degrees C. Changes in bone cell morphology induced by incubation of cells with prostaglandin E(1) or E(2), parthyroid hormone, and dibutyryl cyclic AMP 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 microtubule 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.