title: Formins filter modified actin subunits during processive elongation.
abstract: Fission yeast cells reject actin subunits tagged with a fluorescent protein from the cytokinetic contractile ring, so cytokinesis fails and the cells die when the native actin gene is replaced by GFP-actin. The lack of a fluorescent actin probe has prevented a detailed study of actin filament dynamics in contractile rings, and left open questions regarding the mechanism of cytokinesis. To incorporate fluorescent actin into the contractile ring to study its dynamics, we introduced the coding sequence for a tetracysteine motif (FLNCCPGCCMEP) at 10 locations in the fission yeast actin gene and expressed the mutant proteins from the native actin locus in diploid cells with wild-type actin on the other chromosome. We labeled these tagged actins inside live cells with the FlAsH reagent. Cells incorporated some of these labeled actins into actin patches at sites of endocytosis, where Arp2/3 complex nucleates all of the actin filaments. However, the cells did not incorporate any of the FlAsH-actins into the contractile ring. Therefore, formin Cdc12p rejects actin subunits with a tag of ~2 kDa, illustrating the stringent structural requirements for this formin to promote the elongation of actin filament barbed ends as it moves processively along the end of a growing filament.
authors: Department of Molecular Cellular and Developmental Biology, Yale University, P.O. Box 208103, New Haven, CT 06520-8103, USA.
authors: Qian
authors: Q
authors: Chen
authors: Shalini
authors: S
authors: Nag
authors: Thomas D
authors: TD
authors: Pollard
mesh_terms: D008841:Actin Cytoskeleton / Q000378:metabolism / Q000648:ultrastructure*; D051376:Actin-Related Protein 2-3 Complex / Q000235:genetics / Q000378:metabolism; D000199:Actins / Q000378:metabolism*; D001665:Binding Sites; D048749:Cytokinesis; D003598:Cytoskeletal Proteins / Q000235:genetics / Q000378:metabolism; D005786:Gene Expression Regulation; D008856:Microscopy, Fluorescence; D050505:Mutant Proteins / Q000235:genetics / Q000378:metabolism / Q000648:ultrastructure; D017434:Protein Structure, Tertiary; D012568:Schizosaccharomyces / Q000235:genetics* / Q000378:metabolism*; D029702:Schizosaccharomyces pombe Proteins / Q000235:genetics / Q000378:metabolism
chemical_list: D051376:Actin-Related Protein 2-3 Complex; D000199:Actins; C531994:Cdc12 protein, S pombe; D003598:Cytoskeletal Proteins; D050505:Mutant Proteins; D029702:Schizosaccharomyces pombe Proteins