Effects of profilin and thymosin beta4 on the critical concentration of actin demonstrated in vitro and in cell extracts with a novel direct assay.
The free actin concentration at steady state, Ac, is a variable that determines how actin regulatory proteins influence the extent of actin polymerization. We describe a novel method employing fluorescence anisotropy to directly measure Ac in any sample after the addition of a trace amount of labeled thymosin beta4 or thymosin beta4 peptide. Using this assay, we confirm earlier theoretical work on the helical polymerization of actin and confirm the effects of actin filament-stabilizing drugs and capping proteins on Ac, thereby validating the assay. We also confirm a controversial prior observation that profilin lowers the critical concentration of Mg2+-actin. A general mechanism is proposed to explain this effect, and the first quantitative dose-response curve for the effect of profilin on Ac facilitates its evaluation. This mechanism also predicts the effect of profilin on critical concentration in the presence of the limited amount of capping protein, which is the condition often found in cells, and the effect of profilin on critical concentration in cell extracts is demonstrated for the first time. Additionally, nonlinear effects of thymosin beta4 on the steady state amount of F-actin are explained by the observed changes in Ac. This assay has potential in vivo applications that complement those demonstrated in vitro.
Yarmola
Elena G
EG
Department of Medicine, University of Florida College of Medicine, Gainesville, Florida 32610, USA.
Bubb
Michael R
MR
D000199:Actins / Q000032:analysis* / Q000737:chemistry; D000255:Adenosine Triphosphate / Q000378:metabolism; D000595:Amino Acid Sequence; D000818:Animals; D002417:Cattle; D002457:Cell Extracts / Q000737:chemistry*; D055598:Chemical Phenomena; D002627:Chemistry, Physical; D003285:Contractile Proteins / Q000494:pharmacology*; D005454:Fluorescence Polarization / Q000379:methods*; D006801:Humans; D008274:Magnesium / Q000032:analysis / Q000737:chemistry; D008840:Microfilament Proteins / Q000494:pharmacology*; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000737:chemistry; D011108:Polymers / Q000737:chemistry; D051304:Profilins; D017433:Protein Structure, Secondary; D011994:Recombinant Proteins; D015203:Reproducibility of Results; D013947:Thymosin / Q000737:chemistry / Q000494:pharmacology*; D000099201:Thymosin beta(4)
D000199:Actins; D000255:Adenosine Triphosphate; D002457:Cell Extracts; D003285:Contractile Proteins; D008274:Magnesium; D008840:Microfilament Proteins; D010446:Peptide Fragments; D011108:Polymers; D051304:Profilins; D011994:Recombinant Proteins; D013947:Thymosin; D000099201:Thymosin beta(4); C495738:PFN1 protein, human