Analysis of the thyroglobulin internalization process using in vitro reconstituted thyroid follicles: evidence for a coated vesicle-dependent endocytic pathway.

We have designed a new experimental system based on in vitro reconstituted thyroid follicles (RTF) to study the relative implication of macropinocytosis and micropinocytosis processes in the internalization of thyroglobulin (Tg). Thyrocytes cultured in the presence of TSH reorganize in histiotypic and functional follicles. Tg, which accumulates into the newly formed intrafollicular lumen (IL), was pulse labeled with [125I]iodide. Basal or TSH-activated Tg internalization, i.e. transfer from IL to cells, was assessed by measuring [125I]Tg in the cells and the IL; the IL fraction was collected after selective opening of lumina by a short treatment of RTF in a calcium-free medium. We used the ratio between cellular and IL labeled Tg contents as an endocytic index. TSH caused a very rapid increase in the cellular uptake of labeled Tg; the endocytic index increased by a factor of 4-8. The TSH effect was maximum after 15-20 min. TSH had no effect when the chase-incubation was performed at 4 C, but exhibited the same stimulatory action in terms of both time course and amplitude of action at 20 and 37 C. The macropinocytosis-related cellular structures, the pseudopods, were never observed in RTF maintained at 20 C; they were rare at 37 C and only found after 30 min of TSH treatment. At 20 as well as 37 C, the action of TSH on Tg endocytosis was concentration dependent in the range of 0.05-10 mU/ml. A fraction of Tg internalized by thyrocytes was found in coated vesicles. The labeled Tg content of purified coated vesicles varied with the temperature of the chase-incubation and was increased in TSH-treated RTF. Taken together, these data show that endocytosis of Tg by thyroid follicular cells in resting or moderately activated states does not proceed via the pseudopod formation-dependent mechanism, also termed macropinocytosis. Tg internalization would be related to what is referred as micropinocytosis and would involve a coated vesicle-dependent endocytic pathway.