title: SPEED Microscopy and Its Application in Nucleocytoplasmic Transport.
abstract: In eukaryotic cells, the nuclear pore complexes (NPCs) selectively mediate the bidirectional trafficking of macromolecules between the cytoplasm and the nucleus. The selective barrier is formed by intrinsically disordered phenylalanine-glycine (FG) nucleoporins anchored on the wall of the submicrometer NPC, which allows for passive diffusion and facilitated translocation through the nuclear pore. Dysfunction of nucleocytoplasmic transport has been associated with many human diseases. However, due to the technical challenge of imaging the native tomography of the FG-nucleoporin barrier and its interactions with transiting molecules in the native NPC, the precise nucleocytoplasmic transport mechanism remains unresolved. To refine the transport mechanism, single-molecule fluorescence microscopy methods have been employed to obtain the transport kinetics and the spatial transport route of individual fluorescent molecules through the NPC. In this method paper, we particularly highlight a newly developed high-speed super-resolution three-dimensional microscopy approach, termed as SPEED (single-point edge-excitation subdiffraction) microscopy, and its application in characterizing nucleocytoplasmic transport.
authors.affiliation: Department of Biology, Temple University, Biology Life Sciences Building, 1900 North 12th St., Philadelphia, PA, 19122, USA.
authors.forename: Jiong
authors.initials: J
authors.lastname: Ma
authors.affiliation: Department of Biology, Temple University, Biology Life Sciences Building, 1900 North 12th St., Philadelphia, PA, 19122, USA.
authors.forename: Joseph M
authors.initials: JM
authors.lastname: Kelich
authors.affiliation: Department of Biology, Temple University, Biology Life Sciences Building, 1900 North 12th St., Philadelphia, PA, 19122, USA. weidong.yang@temple.edu.
authors.forename: Weidong
authors.initials: W
authors.lastname: Yang
mesh_terms: D002460:Cell Line; D002467:Cell Nucleus / Q000378:metabolism*; D003593:Cytoplasm / Q000378:metabolism*; D006801:Humans; D008856:Microscopy, Fluorescence; D057054:Molecular Imaging / Q000379:methods; D022022:Nuclear Pore / Q000378:metabolism; D010539:Permeability; D021381:Protein Transport; D000072760:Single Molecule Imaging / Q000379:methods
keywords: Nuclear pore complex (NPC); Nucleocytoplasmic transport; Single-molecule tracking; Single-point edge-excitation subdiffraction microscopy (SPEED); Super-resolution microscopy; Transport receptor