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

1999; Elsevier BV; Volume: 823; Issue: 1-2 Linguagem: Inglês

10.1016/s0006-8993(99)01147-6

1872-6240

N Devineni, Laurie S. Minamide, Mengda Niu, Daniel Safer, Rama Shanker Verma, James R. Bamburg, V T Nachmias,

Cell Image Analysis Techniques

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 (Tβ4). It is not clear whether Tβ4 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: Tβ4, Tβ10, 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 Tβ4 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 Tβ4 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 Tβ4. Combining the contributions of Tβ4, 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) Tβ4 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.