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Genes were cloned into the defined vectors by application accepted enzymatic brake abstract and articulation with T4 DNA ligase. To accomplish constructs breadth abbreviate tags (e.g. HA or Flag epitope tag) or arresting sequences were added to the protein, the tag was included in the primers acclimated to PCR-amplify the gene. PCR articles were digested with brake enzymes and ligated into cut vectors (e.g. pcDNA3 and pDisplay). In all cases, the CMV apostle was acclimated for announcement in beastly cells. The abiogenetic constructs cloned and acclimated for this abstraction as able-bodied as the arrangement advice for primers are abbreviated in Supplementary Table 1.
UnaG cloned into pGEX-2T was attentive provided by Dr. Miyawaki (RIKEN, Japan)23. To aftermath UnaG protein, the plasmid was adapted into Escherichia coli ache BL21 (DE3) cells. Proteins were bidding by consecration of 0.25 mM isopropyl β-d-1-thiogalactopyranoside at 18 °C for 18 h. The beef were harvested and lysed in 25 mM sodium phosphate pH 7.8, 400 mM sodium chloride, 5 mM dithiothreitol, 1 mM phenylmethylsulfonyl fluoride at 4 °C. UnaG was antiseptic application GST affection chromatography, and the GST tag was removed with thrombin protease over pH 8.0 brief at 4 °C. The protein was antiseptic added by ion barter (HiTrap Q HP, GE Healthcare) and size-exclusion chromatography on a Superdex 200 cavalcade (GE Healthcare). Reconstitution was agitated by bond antiseptic ApoUnaG and BR with 1:2 stoichiometry. Afterwards reconstitution application BR, chargeless BR and DMSO were alone application a HiTrap desalting cavalcade (GE healthcare) equilibrated with a PBS buffer.
The assimilation and fluorescence spectra were acquired by application a UV/VIS assimilation spectrometer (Lambda25, Perkin Elmer) and a fluorescence spectrometer (Quantamaster, Photon Technology International). To achromatize the holoUnaG, a sample cuvette was placed in advanced of a dejected laser (TECBL-20GC-488, World Star Tech), admitting the cuvette was placed in a aphotic allowance afterwards abacus BR during fluorescence recovery.
Pictures for fluorescence discharge of antiseptic UnaG showed in Fig. 1c were taken with a camera on cellular buzz (V20, LG). An discharge clarify (HQ525/50m, Chroma) was absorbed in advanced of the camera to address alone the fluorescence.
The photo-oxidation articles were extracted by application altered solubility of the compounds amid aqueous and amoebic solvents. We added agnate aggregate of chloroform (528730, Sigma Aldrich) into albino holoUnaG, and again acerb vortexed the alloyed sample. The UnaG proteins were denatured and aggregated as a white pellet beneath this condition, breadth the amoebic band was gradually angry into chicken blush advertence that the UV-absorbing articles were attenuated in the amoebic layer. We acclaim took the amoebic band and evaporated the bread-and-butter at 60 °C with exhaustion pump. The consistent chicken pellet was primarily attenuated in dimethyl sulfoxide (D8418, Sigma Aldrich), and added adulterated for bristles times with deionized baptize afore analysis.
LC break was performed by a Nexera X2 ultra aerial achievement aqueous chromatography (UHPLC) arrangement (Shimadzu Accurate Instruments) with an ACE Excel 2 C18 cavalcade (50 × 2.1 mm i.d., 2 µm, ACE). The adaptable phases were (A) 0.1% formic acerbic in 95/5 water/acetonitrile and (B) 0.1% formic acerbic in acetonitrile. The bread-and-butter affairs (gradient) consisted of captivation bread-and-butter (A/B 90:10) for 1 min, the linearly converting to bread-and-butter (A/B 60:40) for 7 min, the linearly converting to bread-and-butter (A/B 15:85) for 2 min and captivation for 10 min (Supplementary Fig. 4a). The breeze amount was 200 µL min−1 and the bang aggregate was 10 µL. The cavalcade temperature was set to 40 °C. For LC-UV/vis detection, the LC arrangement was accompanying to a photodiode arrangement detector (DAD, SPD-M30A, Shimadzu Accurate Instruments). For LC/MS analysis, the LC arrangement was interfaced to a Thermo Finnigan LTQ XL beeline ion allurement accumulation spectrometer (Thermo Accurate Inc.) via Ion Max electrospray ionization (ESI) source. For ESI, the voltage was set to 4.0 kV and capillary temperature was 250 °C. Full accumulation spectra were recorded with the accumulation ambit of 100–2000 Da. For HRMS and HRMS/MS analyses, a Q Exactive orbitrap accumulation spectrometer (Thermo Accurate Inc.) was utilized. For collision-induced break (CID) HRMS/MS, forerunner abreast amplitude and normalized blow activity were set to 2 Da and 35%, respectively. The resolutions of full-MS browse and data-dependent MS/MS (ddMS2) modes for HRMS and HRMS/MS were set to 70,000 and 17,500, respectively.
Cos7 beef (Korean Collection for Type Cultures (KCTC)) were able on a coverslip-bottomed 8-well alcove (155409, LAB-TEK) in Dulbecco’s adapted Eagle’s average (DMEM, SH30022.01, Hyclone) supplemented with 10% v/v fetal bovine serum (97068-085, VWR Life Science) and 1% v/v antibiotic–antimycotic (15240-062, Gibco). The beef were transfected at ~70% of confluency by application an electroporator (MPK5000, Invitrogen) with ~500 ng mL−1 of plasmids. Mediums provided from the aggregation were acclimated during transfection. Afterwards 12–24 h post-transfection, we anchored the beef with 3% paraformaldehyde (50-980-495, Electron Microscopy Sciences) with 0.2% glutaldehyde (16020, Electron Microscopy Sciences) for 10 min at allowance temperature for all ambition structures except the ER, which was incubated in the fixation band-aid for 30 min at 37 °C to bottle the aerial structure. Afterwards fixation, we done the sample with PBS absorber rigorously.
For the live-cell checkered imaging, MitoTracker Red (1 µM, M7512, Invitrogen), ER Tracker Red (10 µM, E34250, Invitrogen) and LysoTracker Red (1 µM, L7528, Invitrogen) were activated to the animate beef for 1 min at 37 °C, and the beef were done three times with DMEM afore imaging. HaloTag staining with bootleg Alexa Flour 647 labeled HaloTag ligand (Promega) was agitated by incubating the animate beef in 3 µM of HaloTag ligand absolute advance average for 15 min at 37 °C.
For the immunostaning of the vimentin filaments, primary (anti-vimentin, MA5-11883, Thermo Fisher Scientific) and accessory antibodies (Alexa Flour 488 labeled anti-mouseIgG, A-10680, Thermo Fisher Scientific; bootleg Atto 488 labeled anti-mouseIgG) were adulterated to 2 µg mL−1 absorption in PBS, and activated to the anchored beef for 1 h at allowance temperature sequentially. Atto 488-NHS ester (41698, Sigma Aldrich) was affiliated to the bald anti-mouse IgG via bang acknowledgment with 3:1 stoichiometry, and antiseptic by application a admeasurement exclusion cavalcade (Amicon Ultra, 10 k, UFC5010, Sigma Aldrich) to exclude the unreacted reagents.
To investigate the cellular toxicity of UnaG beneath SML imaging conditions, the corpuscle activity was abstinent in two altered ways: (1) a beaming live/dead appraisal (L3224, Thermo Fisher Scientific), (2) bright-field imaging with aerial deepening for cellular dynamics and morphology. Additionally, a beaming ROS indicator (CellROX Deep Red, C10422, Thermo Fisher Scientific) was acclimated to assay any accessible ROS accident during SML imaging, by staining the beef with 5 µM of CellROX Deep Red for 30 min at 37 °C.
For pull-down assay, Cos7 beef were transfected with able plasmids (UnaG-APtag BirA, UnaG-mCherry, pSNAP-FtnA and UnaG-FtnA) at ~500 ng mL−1 absorption and argent on the corpuscle ability bowl with 150 mm diameter. The beef grew up for 2 canicule in a biotin-containing average to aftermath biotinylated UnaG. At >70% confluency, beef were done with algid PBS and calm by application TrypLE (12605, Thermo Accurate Inc.). Calm beef were disconnected into 6 aliquots and centrifuged at 3000 g for 5 min at 4 °C. Afterwards auctioning the supernatants, the consistent corpuscle pallets were stored at −20 °C for added analysis.
Lysis of the beef was agitated out in a lysis absorber (50 mM HEPES pH 7.4, 150 mM NaCl, 1 mM EDTA, 10% v/v glycerol and 1% v/v Triton X-100) supplemented with 1% v/v protease inhibitor cocktail (P8340, Sigma-Aldrich) and 1% v/v phosphatase inhibitor cocktail (P5726, Sigma-Aldrich). Beef were acclaim abeyant and incubated at 4 °C for 30 min, with added bond accomplish for every 10 min. Afterwards incubation, the corpuscle suspensions were centrifuged at 15,000 g for 10 min at 4 °C, and consistent supernatants were added filtered in a size-exclusion cavalcade (Amicon Ultra, 10 k, UFC5010, Sigma Aldrich).
To attach the corpuscle extracts on the coverslip, we congenital a breeze alcove with PEG-coated coverslips and double-holed accelerate glasses. The coverslips and accelerate glasses were sonicated in alconox, deionized water, acetone and 1 M KOH sequentially for 20 min each. Cleaned glasses were incubated in aminosilane acknowledgment absorber (200 mL of MeOH 10 mL of acerb acid 2 mL of aminosilane) for 20 min, with 1-min sonication in the average of incubation. PEG blanket was agitated by incubating the glasses in PEG acknowledgment band-aid (320 µL of 100 mM sodium bicarbonate 80 mg of mPEG 1 mg of biotin-PEG) for 3 h. Able glasses were sandwiched by double-sided band and closed by epoxy. Afterwards architecture a breeze chamber, 1.25 mg mL−1 of streptavidin and the 1/100 adulterated corpuscle lysates were incubated for 10 min sequentially, with accurate abrasion accomplish with PBS. Biotinylated UnaG from UnaG-APtag was absorbed to the apparent via biotin–avidin alternation for 20 min in lysis buffer. For UnaG-mCherry, added 1/500 adulterated biotinylated anti-RFP antibiotic (ab34771, Abcam) was incubated for 1 h in lysis buffer, afterward UnaG-mCherry evolution for 20 min in lysis buffer.
FtnA clusters were anchored by applying 1/100 adulterated corpuscle lysate on to the poly-l-lysine coated coverslip for 30 min. For SnapTag-FtnA, the anchored lysate was blocked with 10% BSA in PBS for 30 min and advised with 0.1 µM of SNAP-Surface Alexa Flour 647 (S9136S, New England Biolabs) in staining absorber (10% BSA, 0.1 v/v Triton X-100 in PBS) for 6 h at allowance temperature.
Fixed beef were beheld in imaging absorber absolute Tris pH 8 (10 mM, TRI01, LPS Solution), NaCl (50 mM, 7548–4405, Daejung), glucose (10% w/v, G7021, Sigma Aldrich), GLOX (560 µg mL−1, G2133, Sigma Aldrich), CAT (400 µg mL−1, C100, Sigma Aldrich) and BR (600–1,000 nM, B4126, Sigma Aldrich). To anticipate the arrival of oxygen from the air, the top of the sample alcove was deeply closed with coverslip application biologically non-toxic grease during measurement. For the live-cell imaging, the Cos7 beef were able on a coverslip (18 mm diameter, 0117580, Deckgläser) in the aforementioned action as declared above, and were beheld afterwards 12–24 h post-transfection. Able coverslip was primarily army on a alluring alcove (Chamlide CMB, Animate Corpuscle Instrument), which acts as an able backlog of the absorber solution. A DMEM-based imaging absorber was alien to accumulate the beef animate for a continued time, which independent DMEM (no phenol red, aerial glucose, 21063-029, Gibco), HEPES pH 8 (75 mM, H4034, Sigma Aldrich), glucose (2% w/v), GLOX (560 µg mL−1), CAT (400 µg mL−1) and BR (300–1000 nM). To about-face off Alexa Flour 647 for the checkered imaging, 10 mM of β-mercaptoethylamine was supplemented to the absorber additionally.
A home-built imaging bureaucracy with accepted Epi axle was acclimated to access all imaging results, except for confocal z assemblage images in Supplementary Fig. 10b that were acquired by application a bartering spinning deejay confocal arrangement (DragonFly, Andor). Three laser diode ablaze sources (488 nm: 150 mW, OBIS, Coherent; 561 nm: 150 mW, OBIS, Coherent; 647 nm: 120 mW, OBIS, Coherent) were co-aligned and accompanying with a single-mode optical cilia (3.4 µm diameter, PM-488-FC, Thorlabs) for axle cleanup. The cilia achievement was 25 times broadcast with broadcast lenses to enlarge the field-of-view, and delivered into a microscope anatomy (Eclipse Ti-E, Nikon) through a lens (f = 400 mm, AC508-200-A-ML, Thorlabs) to focus the laser at the centermost of the aback breach of cold lens (Plan Apo TIRF, 100×, NA1.49, oil, Nikon) to accomplish Epi illumination. A dichroic beam-splitter (ZT405/488/561/647rpc, Chroma) was alien to bear the laser lights to cold lens, breadth the fluorescence anesthetized through the dichroic beam-splitter and was added filtered by an discharge clarify (ZET405/488/561/647m-TRF, Chroma). A absolute focus arrangement kept the imaging even connected during all measurements.
For single-color imaging, filtered fluorescence was anon recorded with a accurate CMOS camera (sCMOS, Prime95b, Photometrics) after any added eyes except the tube lens. The camera was set to 12-bit acute apprehension approach after any pre- and post-processing, with 110 nm of pixel size. In adjustment to access exact photon numbers per anatomy of holoUnaG, the able photoelectrons per A/D calculation (gain, G) of camera was abstinent by application the relation, G = counts/variance(counts), giving a amount of 0.44 commensurable to manufacturer’s advice (G = 0.65). For checkered imaging, the filtered fluorescence was delivered to a multichannel imaging arrangement (QV2, Photometrics) to activity the single-molecule signals from altered dyes to altered regions of the EMCCD camera (iXon Ultra 888, Andor) and almanac the assorted images at the aforementioned time. We acclimated a attenuated band-pass clarify for the red dyes (ET605/30m, Chroma) to abbreviate the fluorescence arising of UnaG, and abstinent band-pass filters for blooming (ET525/50m, Chroma) and far-red (ET700/75m, Chroma) dyes. The EMCCD camera recorded all the channels accompanying with 130 nm of pixel size, 30 MHz of pixel readout acceleration and 30–300 of EM gain.
To angel the animate samples, temperature of the sample holder and the cold lens were kept at 37 °C by application a temperature ambassador (Chamlide TC, Animate Corpuscle Instrument), if needed.
An accessible antecedent imaging software, µManager66, was acclimated to access widefield and raw single-molecule images. Another advisedly broadcast software, ThunderSTORM67, analyzed the raw abstracts and reconstructed the SML images. For the live-cell SML imaging with 1-s time resolution, the raw images were recorded in overactivated altitude and candy with HAWK software that enables artifact-free high-density localizations68, afore analyzed with ThunderSTORM. The SML images consisted of 5- or 10-nm pixels, and rendered by normalized Gaussian. The checkered images recorded in altered regions of the camera were accumbent application arrangement images of broadband beaming chaplet (TetraSpek, T14792, Invitrogen) that appeared in both channels and a home-built MatLab cipher that adapted the signals from red and far-red regions to agnate blooming arena by application second-order polynomial function.
Labeling advantage of vimentin fiber was analyzed by the afterward steps. First, we manually traced thin, alone filaments and straightened the anecdotal images application ImageJ. Then, a custom MatLab cipher adapted a straightened angel into a bifold angel and affected the atom of covered breadth of the average three pixels.
To assay the abyss of labeling from FtnA array images, we aboriginal analyzed the photon numbers from distinct UnaG and Alexa Fluor 647 molecules beneath switching condition. Then, the fluorescence intensities from >100 FtnA clusters labeled with anniversary fluorophore were calm at the aforementioned axle intensity, camera settings including the acknowledgment time and absorber conditions. Since FtnA is non-existent in beastly beef and forms homo-oligomer consisting of 24 monomers69, 24-times of the single-molecule acuteness was acclimated as the abstract admiration amount anatomy the FtnA cluster, and the abyss of labeling was affected by the allotment of abstinent array acuteness to the abstract estimation.
Further advice on analysis architecture is accessible in the Nature Analysis Reporting Summary affiliated to this article.
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