title: Live cell imaging of cytoplasmic dynein movement in transfected embryonic rat neurons.
abstract: Live cell imaging of the movement of various membrane-bounded organelle cargos has enhanced our understanding of their function. Eukaryotic cells utilize microtubules and two classes of microtubule-based motor proteins, cytoplasmic dynein and members of the kinesin family, to deliver a variety of membrane-bounded organelles and other cargos to their appropriate locations. In order to better understand the functions and regulation of cytoplasmic dynein, we developed a method to study its location and motility in living cells. The technique takes advantage of the long thin axons of cultured hippocampal neurons. We use calcium phosphate to transfect fluorescent-tagged dynein intermediate chain (IC) subunits (DYNC1I) into cultured neurons. When the ICs are expressed at low levels, they are effective probes for the location of the cytoplasmic dynein complex in axons when living cells are imaged with fluorescence microscopy. The fluorescent subunit probes can be used to identify specific cargos of dynein complexes with different IC isoforms as well as the kinetic properties of cytoplasmic dynein.
authors.affiliation: Department of Cell Biology, University of Virginia, Charlottesville, VA, USA.
authors.forename: Mitchell W
authors.initials: MW
authors.lastname: Ross
authors.affiliation: Department of Cell Biology, University of Virginia, Charlottesville, VA, USA.
authors.forename: David J
authors.initials: DJ
authors.lastname: Mitchell
authors.affiliation: Department of Cell Biology, University of Virginia, Charlottesville, VA, USA.
authors.forename: John C
authors.initials: JC
authors.lastname: Cain
authors.affiliation: Department of Cell Biology, University of Virginia, Charlottesville, VA, USA.
authors.forename: Kiev R
authors.initials: KR
authors.lastname: Blasier
authors.affiliation: Department of Cell Biology, University of Virginia, Charlottesville, VA, USA.
authors.forename: K Kevin
authors.initials: KK
authors.lastname: Pfister
mesh_terms: D000818:Animals; D001370:Axonal Transport / Q000502:physiology*; D001369:Axons / Q000378:metabolism*; D002130:Calcium Phosphates; D002478:Cells, Cultured; D056445:Cytoplasmic Dyneins / Q000378:metabolism*; D004622:Embryo, Mammalian / Q000378:metabolism; D049452:Green Fluorescent Proteins / Q000235:genetics; D006624:Hippocampus / Q000166:cytology; D007734:Kymography / Q000379:methods*; D008164:Luminescent Proteins / Q000235:genetics; D008856:Microscopy, Fluorescence; D008870:Microtubules / Q000378:metabolism; D051381:Rats; D014162:Transfection; D000097573:Red Fluorescent Protein
chemical_list: D002130:Calcium Phosphates; D056445:Cytoplasmic Dyneins; D049452:Green Fluorescent Proteins; D008164:Luminescent Proteins; D000097573:Red Fluorescent Protein; C020243:calcium phosphate
keywords: Axonal transport; Cytoplasmic dynein; Fluorescent protein; Intermediate chain; Live cell microscopy; Motor protein; Neurons; Transfection