title: Live-cell imaging of neurofilament transport in cultured neurons.
abstract: Neurofilaments, which are the intermediate filaments of nerve cells, are space-filling cytoskeletal polymers that contribute to the growth of axonal caliber. In addition to their structural role, neurofilaments are cargos of axonal transport that move along microtubule tracks in a rapid, intermittent, and bidirectional manner. Though they measure just 10nm in diameter, which is well below the diffraction limit of optical microscopes, these polymers can reach 100 μm or more in length and are often packed densely, just tens of nanometers apart. These properties of neurofilaments present unique challenges for studies on their movement. In this article, we describe several live-cell fluorescence imaging strategies that we have developed to image neurofilament transport in axons of cultured neurons on short and long timescales. Together, these methods form a powerful set of complementary tools with which to study the axonal transport of these unique intracellular cargos.
authors.affiliation: Department of Neuroscience, The Ohio State University, Columbus, OH, USA.
authors.forename: Atsuko
authors.initials: A
authors.lastname: Uchida
authors.affiliation: Department of Neuroscience, The Ohio State University, Columbus, OH, USA.
authors.forename: Paula C
authors.initials: PC
authors.lastname: Monsma
authors.affiliation: Department of Neuroscience, The Ohio State University, Columbus, OH, USA.
authors.forename: J Daniel
authors.initials: JD
authors.lastname: Fenn
authors.affiliation: Department of Neuroscience, The Ohio State University, Columbus, OH, USA.
authors.forename: Anthony
authors.initials: A
authors.lastname: Brown
mesh_terms: D000818:Animals; D001370:Axonal Transport / Q000502:physiology*; D001369:Axons / Q000378:metabolism*; D018929:Cell Culture Techniques; D002478:Cells, Cultured; D002540:Cerebral Cortex / Q000166:cytology; D003599:Cytoskeleton; D005727:Ganglia, Spinal / Q000166:cytology; D007382:Intermediate Filaments / Q000378:metabolism*; D007734:Kymography / Q000379:methods; D051379:Mice; D008856:Microscopy, Fluorescence; D008870:Microtubules / Q000378:metabolism*; D051381:Rats; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D012984:Software; D013194:Staining and Labeling; D017783:Superior Cervical Ganglion / Q000166:cytology; D014162:Transfection
chemical_list: D011993:Recombinant Fusion Proteins
keywords: Axonal transport; Electroporation; Fluorescence microscopy; Fluorescent fusion protein; Kymograph analysis; Lipofection; Live-cell imaging; Magnetofection; Microinjection; Neurofilament; Photoactivation; Photobleaching; Primary cell culture; Time-lapse imaging; Transfection