title: Imaging protein dynamics in live mitotic cells.
abstract: To ensure that genetic material is accurately segregated during mitosis, eukaryotic cells assemble a mitotic spindle, a dynamic structure composed of microtubules and associated regulatory, structural and motor proteins. Although much has been learned in the past decades from direct observations of live cells expressing fluorescently tagged spindle proteins, a complete understanding of spindle assembly requires a detailed analysis of the dynamic behavior of component parts. Proteins tagged with conventional fluorophores, however, make such an analysis difficult because all of the molecules are uniformly fluorescent. To alleviate this problem, we have tagged proteins with a photoactivatable variant of GFP (PA-GFP), thereby allowing one to follow the behavior of a subset of tagged molecules in the cell. Here, we describe methods to tag and express proteins with PA-GFP, locally photoactivate the recombinant protein and record the dynamic behavior of the photoactivated molecules in live cells. We provide examples of photoactivable proteins in mammalian and yeast cells to illustrate the power of this approach to examine the dynamics of spindle formation and function in diverse cells.
authors.affiliation: Department of Biology, University of Massachusetts, Amherst, MA 01003, USA.
authors.forename: Nick P
authors.initials: NP
authors.lastname: Ferenz
authors.forename: Nan
authors.initials: N
authors.lastname: Ma
authors.forename: Wei-Lih
authors.initials: WL
authors.lastname: Lee
authors.forename: Patricia
authors.initials: P
authors.lastname: Wadsworth
mesh_terms: D000818:Animals; D049452:Green Fluorescent Proteins / Q000235:genetics / Q000378:metabolism; D006801:Humans; D008027:Light; D057054:Molecular Imaging; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D008941:Spindle Apparatus / Q000235:genetics / Q000378:metabolism
chemical_list: D011994:Recombinant Proteins; D049452:Green Fluorescent Proteins