A quantitative analysis of G-actin binding proteins and the G-actin pool in developing chick brain

Abstract

The large G-actin pool in individual actively motile cells has been shown to be maintained primarily by the actin sequestering protein thymosin beta four (Tβ 4). It is not clear whether Tβ 4 or an isoform also plays a primary role in neural tissue containing highly motile axonal growth cones. To address this question we have made a definitive analysis of the relative contributions of all the known G-actin sequestering proteins: Tβ 4, Tβ 10, profilin, and phosphorylated (inactive) and unphosphorylated (potentially active) forms of both ADF and cofilin, in relation to the G-actin pool in developing chick brain at embryonic days 13 and 17. From our measurements we estimate the intracellular concentration of G-actin as 30–37 μM and of Tβ 4 as 50–60 μM in an `average' brain cell in embryonic chick brain. No other beta thymosin isoforms were detected in these brain extracts. The ratio of soluble, unphosphorylated ADF to Tβ 4 is only 1:7 at 13 embryonic days, but increases to 1:4 at 17 days. Profilin and cofilin concentrations are an order of magnitude lower than Tβ 4. Combining the contributions of Tβ 4, unphosphorylated ADF and unphosphorylated cofilin, we estimate a mean G-actin critical concentration of ∼0.45 μM and ∼0.2 μM, respectively, in day 13 and day 17 embryonic brain extracts, suggesting a significant developmental decrease. We conclude that (a) Tβ 4 is the major actin sequestering protein in embryonic chick brain and the only beta thymosin isoform present; (b) ADF may play a significant developmental role, as its concentration changes significantly with age; (c) the known G-actin binding proteins can adequately account for the G-actin pool in embryonic chick brain.