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Supporting InformationRosenbloom et al. 10.1073/pnas.1320044111SI Materials and MethodsSite-Directed Mutagenesis. Site-directed mutagenesis of Dronpa atV157 was performed using the Quikchange Mutagenesis kit andprotocol from Agilent Technologies. Primers were designedaccording to the Quikchange Mutagenesis protocol specifica-tions. Full-length vector constructs were replicated and muta-genized by PCR with PfuUltra DNA polymerase from Agilent.Template plasmid was removed by digestion with DpnI.
Protein Expression and in Vivo Biotinylation. Dronpa, rsKame, andPAmCherry1 cloned into pJY00 with an N-terminal 6XHis anda C-terminal avidin tag. Dronpa and rsKame are transformed intoAvB101 Escherichia coli cells stably transformed with an iso-propyl β-D-1-thiogalactopyranoside (IPTG) inducible BirACmthat will biotinylate avidin tags on proteins. Cells were grown toan OD600 of 0.7 at 37 °C, with agitation then induced with 1 mMIPTG and 50 μM added biotin. Proteins were induced for 12 h,then collected by centrifugation. The cell pellet was washed inlysis buffer [50 mM Na2HPO4 (pH 7.4), 300 mM NaCl, 10 mMimidazole, and 5% (vol/vol) glycerol] and then either stored at−80 °C or immediately purified.
Protein Purification. Affinity chromatography using nickel coatedSepharose beads (His-trap HP) from GE was used to purify His-tagged Dronpa, rsKame, and PAmCherry1. Purification was doneby fast protein liquid chromatography on an FPLC instrument(ÄKTAFPLC; GE Healthcare Life Scienes). Cell pellets wereresuspended in lysis buffer and lysed via sonication at 18 mW(5 × 20-s pulses with 1-min intervals). Cell lysate was loaded andwashed with lysis buffer at a rate of 0.5 mL/min. All column boundproteins were eluted off using a gradient elutionwith elution buffer[50 mM Na2HPO4 (pH 7.4), 300 mM NaCl, 300 mM imidazole,and 5% (vol/vol) glycerol]. Samples of the cell lysate, direct loadonto the column, and flow-throughs were collected for purityanalyses. The fractions containing fluorescent protein were di-alyzed with dialysis buffer [50 mM Na2HPO4 (pH 7.4), 300 mMNaCl, and 5% (vol/vol) glycerol] to remove imidazole by centri-fugation in size exclusion columns (15-kDa filters). Purified pro-tein was snap frozen in liquid nitrogen and stored at −80 °C.Protein purity and biotinylation was confirmed by SDS/PAGE andstaining for total protein with Coomassie Blue, and Western blotanalysis, probing for histidine with aHRP-conjugated αHis and forbiotinylation with an HRP-conjugated streptavidin. Protein sam-ples were quantified using a BCA Assay (Pierce).
BulkPhoto-SwitchingandSpectroscopy.Fluorescent proteins Dronpaor rsKame were transformed into Avb101 E. coli cells and ex-pressed. Cells were grown to OD 0.7 and induced with 1 mMIPTG for 2 h. For bulk photo-switching measurements, cells wereplated on poly-D-lysine–coated coverslips. Cells were illuminatedcontinuously with 488 nm (0.32 W/mm2 or 0.54 W/mm2). Cellswere also illuminated with 1-s pulses of 405 nm (2.83 W/mm2).The fluorescence was allowed to decay fully between pulses. Foreach sample, three fields of view were imaged, and 30 oscillationswere measured.For excitation and emission spectra, purified protein samples
were diluted to 1 μM in a 10-mm quartz cuvette. For the excitationspectra, the cuvette was loaded onto the fluorimeter (Quanta-Master 30; Photon Technology International), and an emissionwavelength of 525 nm was chosen. The emission spectra of thesame sample were taken by bombarding the sample with an exci-tation wavelength of 488 nm, and the emission spectrum was col-
lected from495 nm to 625 nm. For the absorbance spectra, purifiedprotein samples were loaded in a 10-mmquartz cuvette and loadedonto the Varian Cary 50 Bio UV/Visible spectrophotometer.Protein concentrations were determined by the absorbance of
the sampleat278nm.Thepublished e andquantumyieldofDronpawere used as the standards for calculations. The molar extinctioncoefficients, quantum yields, and brightness of theDronpamutantswere determined. The brightness is compared with EGFP withEGFP set at 100.
Cloning of Mammalian Constructs. Human BCS1L was purchasedfrom Open Biosystems. pBCS1L-Lk-PA-FP was built by insert-ing BSC1L1-160 into the pCMV Kan/Neo. The 25-amino-acidlinker (Lk) DLNMSRAQASNSAVDGTAGPGSIAT was addedto the N-terminal of a photoactivatable fluorescent protein (PA-FP) by PCR and inserted into pBCS1L1-160. To build pPA-FP-Lk-BclXl201-233, the Lk and BclXl201-233 were added to the C terminusof the PA-FPs by PCR and then cloned into pCMV Kan/Neo.prsKame-Lk-Drp1 was built by inserting rsKame into pcDNA5/
TO. Human DRP1 was a generous gift from Jodi Nunnari, Uni-versity of California, Davis. A 14-amino-acid flexible linker(AAAGGGSGGGSGGG) was added to the N terminus ofDRP1 by PCR then cloned into the prsKame-5/TO at the C ter-minus of rsKame, creating prsKame-Lk-Drp1.PAmCherry1 was a generous gift from the Liphardt laboratory,
University of California, Berkeley. A Kozak consensus sequenceGACACC was added directly N-terminal to the ATG start codonin all constructs to promote translation.
Cell Culture and Transfection. Two different cell types were used:murine EpH4 (mammary gland epithelial), and human T-RexHeLa (cervical cancer) cells. EpH4 cells were originally isolatedby C. Roskelley. T-Rex HeLa cells were purchased from Invi-trogen. All cell reagents were purchased from University of Cal-ifornia, San Francisco Cell Culture Facility. The EpH4 cells werecultured in DMEM with 2% (vol/vol) FBS with added 5 mg/L ofinsulin and50mg/Lgentamycin.T-RexHeLacellswere cultured inmodified Eagle’s medium with 10% (vol/vol) tetracycline-freeFBS, penicillin/streptomycin, and 5 mg/L blasticidin to maintainthe stably transfected tetracycline repressor gene. Cells weregrown at 37 °C with 5% CO2.Mammalian cells were transfected with one of two lipid-based
commercial transfection reagents. EpH4 cells were transfectedwith Lipofectamine 2000 (Invitrogen). Cells were transfected for4–8 h and then incubated for ∼12 h at 37 °C and 5% CO2 beforeimaging. T-Rex HeLa cells were transfected with Xfect (Clon-tech). Cells were transfected in media with serum. Cells weretransfected for 48 h and then incubated for ∼12 h at 37 °C and5% CO2 before imaging.
Outer and Inner Mitochondrial Membranes-Cell Preparation. EpH4and HeLa cells were transfected with BCS1L1-160-Lk-rsKameand/or PAmCherry1-Lk-BclXl201-233. Twelve hours before fixa-tion, cells were fed 100 nm or 40 nm Au fiducial markers coatedin FBS (60,000 particles/mL). Cells were fixed with 1% formalin(Sigma Aldrich) in 1× PHEM (60 mM Pipes, 25 mM Hepes,10 mM EGTA, and 2.0 mM MgCl2, pH 7.0) for 10 min. Fixedcells were imaged in 1× PHEM. Mitochondrial localization wasconfirmed with either MitoTracker Red CMXRos or Mito-Tracker Green.
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Immunoblotting.T-Rex HeLa cells were transfected with rsKame-Lk-Drp1 and induced for 3.5 h with 0.75 μM doxycycline. Cellswere lysed with M-PER (mammalian protein extraction reagent;Thermo Scientific), and the cytoplasmic fraction was collected bycentrifugation. Total protein within the fractions was quantifiedby A280, and equal amounts were probed for human Drp1 (anti-Drp1; Santa Cruz Biotechnology) and rsKame (anti-DronpaGreen;Amalgaam). Band densities were quantified with pixel densityanalysis in ImageJ.
In Vitro Single Molecule Fluorescence Characterization. BiotinylatedDronpa molecules were purified and immobilized on a strepta-vidin-coated coverslip in a physiological buffer [150 mM Tris (pH7.5) and 50 mM NaCl]. Protein concentration was lowered suchthatmolecules were spatially well separated enough to be detectedindividually. Slides were prepared for single-molecule imaging invitro according to ref. 1. Single molecules were simultaneouslyactivated and excited by 405 nm and by 488 nm of varying powerdensities on our custom total internal reflection microscope (1).The chosen laser power is within the range of typical laser powerappropriate for photoactivated localization microscopy (PALM)imaging (Movie S1). Dronpa and DronpaV157L samples wereexposed to 488-nm illumination (0.5–5.9 W/mm2) for 3 s beforephotoactivation with 405-nm laser (0.0–7.3 mW/mm2) and simul-taneously illuminated with 488 nm and 405 nm until all proteinswere photobleached. PAmCherry1 samples were simultaneouslyilluminatedwith 561 nm (22W/mm2) and 405 nm (0.0–3.2W/mm2).Data were analyzed using our custom MATLAB-based analysissoftware package (1). The Hoshen-Kopelman clustering algorithmwas applied to identify the PA(photo-switchable)-FP moleculesthat photo-switched multiple times. Then the photo-switching ki-netics of each single molecule was analyzed.
PALM Instrumentation. 2D PALM images were taken underpseudototal internal reflection (TIRF) illumination conditionswhereby the wave field, traveling almost along the surface of thecoverslip, can effectively excite thicker axial depths (<1 μm) whilesuppressing the majority of out-of-focus fluorescence. The PALMmicroscope was custom built on an Olympus IX71 microscopeframe. An objective of N.A. 1.45 (UAPO 150xO/TIRFM SP) wasused to illuminate a 55 μm × 55 μm sample area as well as todetect single-molecule fluorescence. Z drift was automaticallycorrected using a feedback looped piezo-stage (CRIFF; ASI).
Fluorescence images were collected with an EMCCD camera(DV887ECS-BV; Andor) at 20 Hz. The FWHM of the final PSFof the imaging system was 2.8 EMCCD pixel (301 nm).
Data Analysis.Analysis of PALM data was done by our MATLAB-based custom analysis software. The position of the single fluo-rescence molecule was first inferred by the bright local maximumpixels—at least six times brighter than the background noise andthe brightest among the neighboring 7 × 7 pixels. The subpixelposition of the single PA-FP molecule is calculated by fitting the7 × 7 image to a 2D Gaussian PSF model. XY drift for allidentified events was corrected by tracking the drift of the 100-nmor 40-nm Au fiducial marker through the PALM movie data. Thereconstruction of PALM images, two-color images overlay, and allother necessary analysis were also done using the MATLAB-basedcustom software package (1).
Simulation of the Measurement of Dynamin Related-1 Helical Dimensionwith PALM. First, the coordinates (x-y: in-plane; z: axial) of dynaminrelated-1 (Dnm1) monomers were determined from the structuralparameters of Dnm1 helix measured using cryo-EM for two con-formations: Nonconstricted, outer diameter (129 nm), number ofsubunits per turn (24); Constricted, outer diameter (67 nm), num-ber of subunits per turn (12.4) (2). The Dnm1 helix is composed oftwo parallel strands with a Dnm1 dimer occupying a site on eachstrand. Therefore, there are four Dnm1 monomers per subunit.The helical pitch (28.8 nm) does not change between the twoconformations. To account for an imperfect labeling, a subset ofthe Dnm1 monomers was randomly chosen for a given labelingefficiency. Then the coordinates of the selected Dnm1 were pro-jected onto the y = 0 plane, whereas the x-z coordinates wererandomized according to a 2D Gaussian distribution (mean: 0; SD:20 nm) to simulate the measured localization uncertainty ofrsKame. The width and the length of the helix were measured bythe difference between the maximum and minimum coordinates inx-direction and z-direction, respectively. We repeated this for 1,000independent and random samplings of the rsKame-labeled Dnm1for a given label efficiency to obtain the mean and SD of the widthand length measurement. The labeling efficiency was varied be-tween 0.05 and 1. Simulation was done for both one-turn and two-turn helix.
1. Lee SH, Shin JY, Lee A, Bustamante C (2012) Counting single photoactivatablefluorescent molecules by photoactivated localization microscopy (PALM). Proc NatlAcad Sci USA 109(43):17436–17441.
2. Mears JA, et al. (2011) Conformational changes in Dnm1 support a contractilemechanism for mitochondrial fission. Nat Struct Mol Biol 18(1):20–26.
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Fig. S1. Quantification of the photo-physical rates of Dronpa. Probability distributions describing three measured variables [the number of times singlemolecules photo-switch (NBlink), the time spent in the fluorescent state (TON), and the time spent in the dark state (TOFF)] used to determine the photo-kineticrates. Dronpa was simultaneously illuminated with 488-nm laser power (0.2, 0.5, and 5.9 W/mm2) and 405-nm laser (0.0–7.3 mW/mm2). (A) For all conditions,NBlink is fit well by a geometric distribution. (B) TON is fit well by a single exponential decay. (C) TOFF is best fit by a triple exponential decay.
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Fig. S2. Bulk spectroscopic and photo-switching properties of Dronpa and rsKame. (A) Excitation and emission spectra recorded from purified proteinswitched to the ON state by exposure to strong 405-nm laser power. (B) Fluorescent properties of Dronpa and rsKame. Brightness is compared with EGFP as100. The reduced brightness of rsKame compared with Dronpa is due to a reduced adsorption at the maximal excitation wavelength and is consistent witha longer dwell in dark or OFF state of the molecule. (C–F) E. coli cells expressing rsKame or Dronpa were continuously excited with 488 nm. (C and D) Pulsed0.5-s bursts of illumination spaced 5 s apart with strong 405-nm laser power (2.8 W/mm2) were used to photoactivate the fluorescent proteins. Fluorescence wasrecorded continuously for 30 cycles. (E) The decay rate was determined by quantifying the time from peak to valley of each cycle post 405-nm pulse. (F) Thegrowth rate was determined by quantifying the time from valley to peak of each cycle upon single pulse of 405-nm light. (Scale bar, 500 nm.)
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Fig. S3. Comparison of Dronpa and rsKame photo-switching kinetics at the single-molecule level. Single-molecule samples were excited with 5.9 W/mm2
(dashed line), 0.5 W/mm2 (solid line), or 0.2 W/mm2 (dash-dot line) 488-nm laser power and photoactivated over a range of 405-nm laser power densities (0.0–7.3 mW/mm2). (A) <NBlink> is the mean number of times single molecules underwent the photo-switching cycle. (B) <TON> is the mean time single moleculesspent actively fluorescing. (C) <TOFF> is the mean time single molecules spent on the dark state. (D) Even in the absence of 405-nm laser, both Dronpa andrsKame recover from the dark state by 488-nm laser alone. This 488 nm-induced activation is proportional to the 488-nm laser power. However, rsKame isapproximately twice less prone to this phenomenon than Dronpa. (E and F) Super resolution images of outer mitochondrial membrane (OMM) labeled withDronpa (E) or rsKame (F) in EpH4 cells. Arrowheads indicate missed localizations, and the arrow indicates an area of poor localization due to overlappingphotoactivations. (G) rsKame and Dronpa have similar single-molecule photon budgets of 320 photons per molecule on average; (H) however, the lowerphotoactivation by 488-nm illumination of rsKame leads to a lower background of five photons per event, compared with 30 for Dronpa. Photon budgets andbackgrounds were estimated from the fields of view of E and F. (I) Numerous examples of rsKame-labeled inner mitochondrial membrane (IMM) in HeLa cellsdisplaying linear and transverse fluorescence patterns. (Scale bars, 2 μm for E and F and 200 nm for E and F Insets and I.)
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Fig. S4. Comparison of mGeosM and rsKame at the single-molecule and ensemble levels. HeLa cells expressing mGeosM and rsKame targeted to the OMMwere first illuminated by 5.9 W/mm2 of 488 nm only. Then, 160 mW/mm2 of 405 nm were added to photoactivate all of the remaining molecules. (A) mGeosMexhibits a strong initial fluorescence (i) followed by rapid photobleaching (ii); upon simultaneous illumination by 405 nm and 488 nm, fluorescence recovers tohalf of its initial value (iii). (B) rsKame also exhibits initial fluorescence (i) followed by rapid photobleaching (ii); upon simultaneous illumination by 405 nm and488 nm, fluorescence increases to 1.5–3 times its initial value (iii). (C) The number of single molecule events detected in the 488 nm-only illumination portiondecreases faster for mGeosM than for rsKame. The single-molecule photon budgets (D), background fluorescence (E), and localization uncertainties (F)measured for the single-molecule events detected are similar for mGeosM and rsKame. Example of OMMs labeled with mGeosM (G) or rsKame (H). (Scale bar,200 nm.)
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Fig. S5. Photo-physical properties of rsKame and PAmCherry1 within the two-color PALM method. (A) The photoactivation rate of PAmCherry is linearlyproportional to the 405-nm laser power (R2 = 0.95). Photoactivation rates (ka) were obtained from single-molecule data taken under simultaneous illuminationwith 561-nm excitation laser power of 22.0 W/mm2 and 405-nm activation laser powers ranging from 0.0 to 3.2 W/mm2. (B) For two-color PALM imaging,samples were initially simultaneously illuminated by weak 488-nm and 405-nm laser powers to collect rsKame single-molecule data until all molecules werephotobleached. To then collect PAmCherry1 single molecule data, samples were simultaneously illuminated by strong 561-nm and 405-nm laser powers until allmolecules were photobleached. The individual localization uncertainties of all (C) rsKame (n = 8,394) or (D) PAmCherry1 (n = 6,628) fluorescent molecules werecalculated for all molecules within the analyzed fields of view.
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Fig. S6. Fluorescence labeling efficiency of Drp1 and its effect on measuring the size of Drp1 complex with PALM. (A) The cytoplasmic fractions of un-transfected or rsKame-Lk-Drp1transfected HeLa cells were immunoblotted for Drp1. The ratio of rsKame-Lk-Drp1 to Drp1, based on band density quantifi-cation, is 0.62 to 0.38. The fractions were also immunoblotted with anti-Drp1 for rsKame-Lk-Drp1 with anti-Dronpa to confirm the identity of rsKame-Lk-Drp1.This indicates that more than 62% of the Drp1 population is labeled with rsKame. Additionally, allowing for a 20% inactive population of rsKame, typical forfluorescent proteins, at least 50% of Drp1 is estimated to be detectable by PALM (1). Measurement of diameter (Left) and length (Right) of (B) one-turn and (C)two-turn Dnm1 helix was simulated for various labeling efficiency, with accounting for the 20-nm localization uncertainty of rsKame (SI Materials andMethods). Although the measurement of diameter or length of Dnm1 helix depends on the localization uncertainty of the fluorescent marker fused to Dnm1and also on the population of labeled Dnm1 subunits, their mean size change between constricted and nonconstricted conformations rather accurately androbustly reflects the actual conformational change (dotted line in B and C).
1. Ulbrich MH, Isacoff EY (2007) Subunit counting in membrane-bound proteins. Nat Methods 4(4):319–321.
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Fig. S7. Data filtering by cluster analysis. Cluster analysis was used to filter out cytoplasmic monomeric and small oligomers of rsKame fused Drp1 not involvedin mitochondrial fission, characterized by large clusters of rsKame-Lk-Drp1. (A) Unfiltered raw image. (B) The clusters with fewer than eight localized rsKamemolecules were filtered out. A nearest neighbor distance of 44 nm was used as the clustering criteria. (C) Overlay with PAmCherry1-Lk-BclXl201-233 labeledOMM images. (D) The filtered super resolution images demonstrated superior ability to observe Drp1 clusters at fission sites as opposed to their diffractionlimited counterparts. (Scale bars, 500 nm.)
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Fig. S8. Drp1 helical rings can be observed at various stages of mitochondrial fission. Four different Drp1 fission states were observed in cells transfected withPAmCherry1-Lk-BclXl201-233 and rsKame-Lk-Drp1. (A–D) Initial described Drp1 clusters on unconstricted or slightly constricted mitochondria. (E–H) Drp1 clustersfound on constricted mitochondria are labeled Constricted. (I–L) When found on mitochondrial membranes that had almost completed fission, Drp1 clustersare labeled Scissioned. (M–P) Terminal described the residual Drp1 clusters at the termini of mitochondria. (Q) Intermediate and Initial Drp1 helical ring andouter mitochondrial membrane uncorrected diameters are proportional to each other. (R) The outer mitochondrial membrane Intermediate and Initial statediameters exist in two distinct populations. The uncorrected Intermediate state diameters (mean = 102 ± 42, median = 190) are significantly reduced comparedwith the Initial state (mean = 198 ± 43, median = 95). (Scale bars, 500 nm for A–F, 200 nm for G–P.)
Movie S1. Live imaging movie of Dronpa, rsKame, and mEos2. EpH4 cells were transfected with Dronpa, rsKame, or mEos2 targeted to the outer mito-chondrial membrane. Dronpa- and rsKame-labeled cells were illuminated with 488-nm light only, whereas mEos2-labeled cells were illuminated simultaneouslyby 488 nm and 405 nm. Single-molecule fluorescence events are more clearly distinguishable for rsKame than Dronpa owing to the slower 488 nm-inducedphoto-switching of rsKame. The video shows a small fraction of the whole cell’s mitochondrial population under illumination.
Movie S1
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Movie S2. Comparison of mGeosM and rsKame. HeLa cells expressing mGeosM and rsKame targeted to the OMM were first illuminated by 5.9 W/mm2 of488 nm only. Both fluorescent proteins exhibit initial fluorescence that quickly photobleaches. At 4:10, 160 mW/mm2 of 405 nm were added to photoactivateall of the remaining molecules, and samples were imaged until fully photobleached.
Movie S2
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