title: Optimized two-color super resolution imaging of Drp1 during mitochondrial fission with a slow-switching Dronpa variant.
abstract: We studied the single-molecule photo-switching properties of Dronpa, a green photo-switchable fluorescent protein and a popular marker for photoactivated localization microscopy. We found the excitation light photoactivates as well as deactivates Dronpa single molecules, hindering temporal separation and limiting super resolution. To resolve this limitation, we have developed a slow-switching Dronpa variant, rsKame, featuring a V157L amino acid substitution proximal to the chromophore. The increased steric hindrance generated by the substitution reduced the excitation light-induced photoactivation from the dark to fluorescent state. To demonstrate applicability, we paired rsKame with PAmCherry1 in a two-color photoactivated localization microscopy imaging method to observe the inner and outer mitochondrial membrane structures and selectively labeled dynamin related protein 1 (Drp1), responsible for membrane scission during mitochondrial fission. We determined the diameter and length of Drp1 helical rings encircling mitochondria during fission and showed that, whereas their lengths along mitochondria were not significantly changed, their diameters decreased significantly. These results suggest support for the twistase model of Drp1 constriction, with potential loss of subunits at the helical ends.
authors.affiliation: Department of Molecular and Cell Biology, California Institute for Quantitative Biosciences, Jason L. Choy Laboratory of Single Molecule Biophysics.
authors.forename: Alyssa B
authors.initials: AB
authors.lastname: Rosenbloom
authors.affiliation: California Institute for Quantitative Biosciences, Jason L. Choy Laboratory of Single Molecule Biophysics, Howard Hughes Medical Institute, and.
authors.forename: Sang-Hyuk
authors.initials: SH
authors.lastname: Lee
authors.affiliation: Department of Molecular and Cell Biology.
authors.forename: Milton
authors.initials: M
authors.lastname: To
authors.affiliation: Jason L. Choy Laboratory of Single Molecule Biophysics, Department of Physics, University of California, Berkeley, CA 94720.
authors.forename: Antony
authors.initials: A
authors.lastname: Lee
authors.affiliation: California Institute for Quantitative Biosciences, Jason L. Choy Laboratory of Single Molecule Biophysics, Howard Hughes Medical Institute, and.
authors.forename: Jae Yen
authors.initials: JY
authors.lastname: Shin
authors.affiliation: Department of Molecular and Cell Biology, California Institute for Quantitative Biosciences, Jason L. Choy Laboratory of Single Molecule Biophysics, Howard Hughes Medical Institute, and Department of Physics, University of California, Berkeley, CA 94720 carlos@alice.berkeley.edu.
authors.forename: Carlos
authors.initials: C
authors.lastname: Bustamante
mesh_terms: D003116:Color; D034281:Dynamins / Q000737:chemistry / Q000378:metabolism; D006367:HeLa Cells; D006801:Humans; D021621:Imaging, Three-Dimensional; D008164:Luminescent Proteins / Q000378:metabolism*; D008853:Microscopy; D063154:Mitochondrial Dynamics; D051336:Mitochondrial Membranes / Q000378:metabolism; D050505:Mutant Proteins / Q000378:metabolism*; D017433:Protein Structure, Secondary
chemical_list: D008164:Luminescent Proteins; D050505:Mutant Proteins; D034281:Dynamins
keywords: PALM; photo-physics; suborganelle structures