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 slowswitching 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.photo-physics | PALM | suborganelle structures P hotoactivated localization microscopy (PALM) allows for subdiffraction optical resolution via the stochastic temporal separation of individual photoactivatable or reversibly photoswitchable fluorescent proteins (PA-FPs or PS-FPs) and their subsequent localization in space. Successful PALM imaging requires two conditions: (i) temporally separated stochastic activation of a population of PA-FPs or PS-FPs, and (ii) accurate localization of each molecule in space (1, 2).For PALM imaging to be more biologically applicable, robust two-color PALM is necessary. Several published protocols for two-color PALM exist, one featuring EosFP and Dronpa, a GFPlike photo-switchable protein (3-5). Initially dark, upon photoactivation by 405-nm light, Dronpa becomes fluorescent with an excitation maximum at 503 nm and an emission maximum at 515 nm. When excited by 488 nm, activated Dronpa emits green fluorescence ("ON" state) until it is photo-induced back into the dark state ("OFF" state) and can be reactivated multiple times before photobleaching (Fig. 1A). In addition, Dronpa was observed to spontaneously recover from the OFF to the ON state in tens of seconds even under 488-nm illumination alone, which hypothetically has been ascribed to thermal activation (4, 6, 7). As a result, large overlapping populations of Dronpa molecules can be excited simultaneously, especially in densely labeled samples, hindering single-molecule identification and localization. rsFastLime, a Dronpa variant, has the amino acid mutation V157G (8). The valine, not directly part of the chromophore, is involved in the photoactivated isomerization of the chromophore. The V157G mutation is thought to reduce the steric ...

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