title: Avoiding artefacts when counting polymerized actin in live cells with LifeAct fused to fluorescent proteins.
abstract: When tagged with a fluorescent protein, actin is not fully functional, so the LifeAct peptide fused to a fluorescent protein is widely used to localize actin filaments in live cells. However, we find that these fusion proteins have many concentration-dependent effects on actin assembly in vitro and in fission yeast cells. mEGFP-LifeAct inhibits actin assembly during endocytosis as well as assembly and constriction of the cytokinetic contractile ring. Purified mEGFP-LifeAct and LifeAct-mCherry bind actin filaments with Kd values of ∼10 μM. LifeAct-mCherry can promote actin filament nucleation and either promote or inhibit filament elongation. Both separately and together, profilin and formins suppress these effects. LifeAct-mCherry can also promote or inhibit actin filament severing by cofilin. These concentration-dependent effects mean that caution is necessary when overexpressing LifeAct fusion proteins to label actin filaments in cells. Therefore, we used low micromolar concentrations of tagged LifeAct to follow assembly and disassembly of actin filaments in cells. Careful titrations also gave an estimate of a peak of ∼190,000 actin molecules (∼500 μm) in the fission yeast contractile ring. These filaments shorten from ∼500 to ∼100 subunits as the ring constricts.
authors.affiliation: Department of Molecular, Cellular and Developmental Biology, Yale University, PO Box 208103, New Haven, Connecticut 06520-8103, USA.
authors.forename: Naomi
authors.initials: N
authors.lastname: Courtemanche
authors.affiliation: Department of Molecular, Cellular and Developmental Biology, Yale University, PO Box 208103, New Haven, Connecticut 06520-8103, USA.|Departments of Molecular Biophysics and Biochemistry, Yale University, PO Box 208103, New Haven, Connecticut 06520-8103, USA.|Department of Cell Biology, Yale University, PO Box 208103, New Haven, Connecticut 06520-8103, USA.
authors.forename: Thomas D
authors.initials: TD
authors.lastname: Pollard
authors.affiliation: Department of Molecular, Cellular and Developmental Biology, Yale University, PO Box 208103, New Haven, Connecticut 06520-8103, USA.
authors.forename: Qian
authors.initials: Q
authors.lastname: Chen
mesh_terms: D008841:Actin Cytoskeleton / Q000378:metabolism*; D051339:Actin Depolymerizing Factors / Q000378:metabolism; D000199:Actins / Q000378:metabolism*; D000818:Animals; D018797:Cell Cycle Proteins / Q000378:metabolism*; D048749:Cytokinesis / Q000502:physiology; D003598:Cytoskeletal Proteins / Q000378:metabolism*; D004705:Endocytosis / Q000502:physiology; D051304:Profilins / Q000378:metabolism; D012568:Schizosaccharomyces / Q000378:metabolism; D029702:Schizosaccharomyces pombe Proteins / Q000378:metabolism
chemical_list: D051339:Actin Depolymerizing Factors; D000199:Actins; D018797:Cell Cycle Proteins; D003598:Cytoskeletal Proteins; D051304:Profilins; D029702:Schizosaccharomyces pombe Proteins