Thyroidal stimulation of tubulin and actin in rat brain cytoskeleton

In cultures of neonatal rat brain cells, labeled with 35S-methionine in the presence or absence of triiodothyronine (T3), the hormone promoted a significant enhancement of labeled tubulin and actin in the insoluble fraction (30,000 g pellet) of cell homogenate. To identify the specific sub-cellular fraction associated with this induction, organ cultures of 1 day rat cerebra were labelled with 35S-methionine in the presence and absence of T3 and the insoluble fraction (30,000 g pellet) was subfractionated into mitochondria, plasma membrane and cytoskeleton. Analysis of the labeled proteins by SDS-PAGE, autoradiography and densitometry revealed a T3-induced increase of 50-80% for both tubulin and actin, only in the cytoskeleton fraction without any significant effect on the other fractions. Similar results were obtained when plasma membrane or cytoskeleton were isolated directly from labeled cerebrum by conventional methods instead of fractionating from the 30,000 g pellet. Analysis of relative stimulation of labeled tubulin and actin by T3 in cytoskeleton fraction derived from primary cultures of neuronal (N) and glial (G) cells labeled with 35-methionine show that the stimulatory effect is predominantly on the N cells. Studies on the kinetics of induction of labeled tubulin and actin by T3 in the cytoskeleton fraction prepared from cerebra labeled with 35S-methionine for 2, 8 and 18 hrs revealed no significant difference at 2 hrs; at 8 hrs, an increased incorporation into both tubulin and actin was reproducibly seen in the controls relative to T3-treated samples. However by 18 hrs, this pattern reversed and an enhanced accumulation of both labeled tubulin and actin was observed under the influence of T3. The mechanism of this apparently intriguing effect of T3 on the kinetics of association of tubulin and actin with the cytoskeleton has been discussed in the light of the dual effect of the hormone on tubulin viz. enhancing its stability as well as rate of synthesis. The overall results indicate that the thyroid hormones play a major role in the cytoskeletal transport of tubulin and actin from their site of synthesis to that of assembly thus facilitating axodendritic outgrowth and morphological differentiation