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Proc Natl Acad Sci U S A. 2014 Sep 9;111(36):13093-8. doi: 10.1073/pnas.1320044111. Epub 2014 Aug 22.

Optimized two-color super resolution imaging of Drp1 during mitochondrial fission with a slow-switching Dronpa variant.

Author information

1
Department of Molecular and Cell Biology, California Institute for Quantitative Biosciences, Jason L. Choy Laboratory of Single Molecule Biophysics.
2
California Institute for Quantitative Biosciences, Jason L. Choy Laboratory of Single Molecule Biophysics, Howard Hughes Medical Institute, and.
3
Department of Molecular and Cell Biology.
4
Jason L. Choy Laboratory of Single Molecule Biophysics, Department of Physics, University of California, Berkeley, CA 94720.
5
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.

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.

KEYWORDS:

PALM; photo-physics; suborganelle structures

PMID:
25149858
PMCID:
PMC4246937
DOI:
10.1073/pnas.1320044111
[Indexed for MEDLINE]
Free PMC Article

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