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

Alyssa B. Rosenbloom, Sang Hyuk Lee, Milton To, Antony Lee, Jae Yen Shin, Carlos Bustamante

Research output: Contribution to journalArticlepeer-review

50 Scopus citations

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 localizationmicroscopy imagingmethod to observe the inner and outermitochondrialmembrane 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.

Original languageEnglish (US)
Pages (from-to)13093-13098
Number of pages6
JournalProceedings of the National Academy of Sciences of the United States of America
Volume111
Issue number36
DOIs
StatePublished - 2014

All Science Journal Classification (ASJC) codes

  • General

Keywords

  • PALM
  • Photo-physics
  • Suborganelle structures

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