title: Visualization of the actin cytoskeleton: different F-actin-binding probes tell different stories.
abstract: The actin cytoskeleton is necessary for cell viability and plays crucial roles in cell motility, endocytosis, growth, and cytokinesis. Hence visualization of dynamic changes in F-actin distribution in vivo is of central importance in cell biology. This has been accomplished by the development of fluorescent protein fusions to actin itself or to various actin-binding proteins, actin cross-linking proteins, and their respective actin-binding domains (ABDs). Although these protein fusions have been shown to bind to F-actin in vivo, we show that the fluorescent protein used for visualization changes the subset of F-actin labeled by an F-actin ABD probe. Further, different amino acid linkers between the fluorescent protein and ABD induced a similar change in localization. Although different linkers and fluorescent proteins can alter the subset of actin bound by a particular ABD, in most cases, the fusion protein did not label all of a cell's F-actin all of the time. Even LimEΔcoil and GFP-actin, which have been used extensively for cytoskeletal visualization, were highly variable in the subsets of actin that they labeled. Lifeact, conversely, clearly labeled cortical F-actin as well as F-actin in the anterior pseudopods of motile cells and in macropinocytotic cups. We conclude that Lifeact most accurately labels F-actin and is the best currently available probe for visualization of dynamic changes in F-actin networks.
authors.affiliation: Department of Molecular and Cell Biology, University of Connecticut, Storrs, Connecticut, 06269.
authors.forename: Michael G
authors.initials: MG
authors.lastname: Lemieux
authors.forename: Dani
authors.initials: D
authors.lastname: Janzen
authors.forename: Rander
authors.initials: R
authors.lastname: Hwang
authors.forename: Jeannette
authors.initials: J
authors.lastname: Roldan
authors.forename: Irene
authors.initials: I
authors.lastname: Jarchum
authors.forename: David A
authors.initials: DA
authors.lastname: Knecht
mesh_terms: D008841:Actin Cytoskeleton / Q000378:metabolism*; D000199:Actins / Q000378:metabolism*; D000818:Animals; D002460:Cell Line; D016764:Cell Polarity; D002633:Chemotaxis; D004023:Dictyostelium / Q000166:cytology; D064448:Filamins / Q000737:chemistry / Q000378:metabolism; D049452:Green Fluorescent Proteins / Q000378:metabolism; D051379:Mice; D015335:Molecular Probes / Q000378:metabolism*; D055503:Protein Multimerization; D017434:Protein Structure, Tertiary
chemical_list: D000199:Actins; D064448:Filamins; D015335:Molecular Probes; D049452:Green Fluorescent Proteins
keywords: F-actin; actin-binding protein; cytoskeleton; fluorescent protein