Nanobodies as novel tools to monitor the mitochondrial fission factor Drp1

Nanobody library construction

Alpaca immunization with recombinant Drp1 as well as Nb library generation was performed as described before (Rothbauer et al, 2006). Animal immunization was authorized by the government of Upper Bavaria (Permit number 55.2-1-54-2532.0-80-14). Briefly, an alpaca (V. pacos) was immunized with recombinant human Drp1 (Drp1). An initial priming dose of 0.48 mg Drp1 was followed by booster injections of 0.24 mg each in the 3^rd, 4^th, 7^th, and 12^th wk. Serum samples of 20 ml were taken in the 9^th wk of immunization for analysis of seroconversion by ELISA. 13 wk after the first dose, 100 ml blood was drawn, and the lymphocytes isolated by Ficoll gradient centrifugation using the Lymphocyte Separation Medium (PAA Laboratories GmbH). Total RNA was isolated using TRIzol (Life Technologies) followed by reverse transcription of mRNA into cDNA using a First-Strand cDNA Synthesis Kit (GE Healthcare). To isolate the Nb repertoire, three nested PCR reactions were performed with the following primers: (i) CALL001 and CALL002, (ii) forward primer set FR1-1, FR1-2, FR1-3, FR1-4 and reverse primer CALL002, and (iii) forward primer FR1-ext1 and FR1-ext2 and reverse primer set FR4-1, FR4-2, FR4-3, FR4-4, FR4-5, and FR4-6 to introduce SfiI and NotI restriction sites. The sequences for all primers (synthesized by Integrated DNA Technologies) used in this study are listed in Table S6. The amplificated comprising the Nb repertoire was then subcloned into the pHEN4 phagemid vector (Arbabi Ghahroudi et al, 1997) using the Sfil/NotI restriction sites thereby generating the Drp1-Nb library.

Nanobody screening

To select for Drp1-specific Nbs, E. coli, TG1 cells containing the Drp1-Nb library in pHEN4 were infected with the M13K07 helper phage. Next, the hereby generated Nb-presenting phages were isolated from culture supernatant by PEG precipitation and 1 × 10¹¹ phages were used for subsequent biopanning. In each selection round, extensive blocking was carried out with 5% milk or BSA in PBST (PBS, 0.05% Tween 20, pH 7.4) (Pardon et al, 2014). To deplete non-specific binders, phages were first added to Nunc Immuno MaxiSorp tubes (Thermo Fisher Scientific) coated with 10 μg/ml GFP before transferring them to tubes coated with 10 μg/ml Drp1 or 10 μg/ml GFP as non-related antigen for 2 h at RT. Next, unbound phages were removed by washing with stringency increasing each round. Bound phages were eluted with 100 mM triethylamine (pH 12.0) and immediately neutralized with 1 M Tris–HCl pH 7.4. After each round of panning, log phase E. coli TG1 cells were infected with the eluted phages and grown on selection plates to rescue enriched phages. Drp1-specific phage enrichment was monitored by determining the CFUs after each round. After two rounds of panning, 260 individual clones were randomly selected and screened by phage ELISA using immobilized Drp1 or GFP as control. HRP-labelled anti-M13 monoclonal antibody (GE Healthcare) and 1-Step Ultra TMB solution (Thermo Fisher Scientific) were used for detection of bound phages. The reaction was stopped with 100 μl of 1 M H₂SO₄ and the signal was detected on a plate reader (Pherastar) at 450 nm. Phage ELISA-positive clones were defined by a twofold signal above the control.

Expression plasmids

For bacterial Nb expression, Nb encoding cDNA were cloned into the pHEN6C vector as previously described (Rothbauer et al, 2008). For mammalian expression of bivalent Nbs, cDNAs comprising the two coding sequences of the respective Nbs connected by a flexible Gly-Ser linker [(G₄S)₄] and a C-terminal sortase tag (L-P-E-T-G) followed by a His₆-tag were generated and inserted into pCDNA3.4 expression vector variant comprising N-terminal signal peptide for secretion (Wagner et al, 2021) as following: For bivD7, each Nb sequence was amplified separately by PCR using bivD7-1 and bivD7-2 or bivD7-3 and downEcoRI-rev primer pairs. Next, both Nbs were fused by overlap-extension PCR using the primers bivNshort_for and downEcoRI-rev. For bivD63 cloning, the primer pairs bivD63forN and bivNtermGS-rev as well as bivD63forC and downEcoRI-rev were used followed by bivD63_fusion and downEcoRI-rev for overlap-extension PCR. Finally, the resulting sequence was inserted into pCDNA3.4 via restriction digestion with Esp3I and EcoRI. To generate unmodified Cbs, Nb sequences were cloned into pTagRFP by BglII and HindIII restriction digest as previously described (Panza et al, 2015). For turnover-accelerated Cbs, the Nb sequence was inserted via PstI and BspEI into the previously described pEGFP-Ubi-R-3xGS-tagRFP plasmid (Keller et al, 2018). The coding sequence of Drp1 was amplified from pAcGFP-Drp1 plasmid (Jenner et al, 2022) with Drp1_GTPaseFor and Drp1_C_term_rev and cloned into pEGFP-N1 backbone. For deletion constructs for domain mapping, all sequences were amplified from AcGFP-Drp1 and cloned into pEGFP-N1 with Drp1-GTPaseFor forward primer and the respective reverse primer: Drp1-VD-rev (GTPase-MD-VD), Drp1-MD-rev (GTPase-MD), Drp1-GTPase-rev (GTPase). The correct sequences of all constructs were confirmed by Sanger sequencing. All expression constructs used in this study are listed in Table S7.

Protein expression and purification

Monovalent Drp1-Nbs were expressed and purified as previously described (Rothbauer et al, 2008; Wagner et al, 2021). Bivalent Drp1-Nbs were produced using the ExpiCHO system (Thermo Fisher Scientific) according to the manufacturer’s instructions (Wagner et al, 2021; Fagbadebo et al, 2022). Purity of produced Nbs was assessed using SDS–PAGE. Thus, protein samples were denatured (10 min, 95°C) with 2x SDS-sample buffer (60 mM Tris–HCl, pH 6.8; 2% [wt/vol] SDS; 5% [vol/vol] 2-mercaptoethanol; 10% [vol/vol] glycerol, 0.02% bromphenol blue) and gels were stained with InstantBlue Coomassie (Abcam). Protein concentrations were determined using Bradford and spectrophotometer measurements. Drp1 was purified as described in (Jenner et al, 2022). Briefly, DRP1 (isoform 1) was expressed from a pTYB2 vector in E. coli BL21-CodonPlus (DE3)-RIPL. Bacteria were grown at 37°C in LB medium containing 100 μg/ml ampicillin and 35 μg/ml chloramphenicol to an OD_{600 nm} of 0.6. Protein expression was induced using 1 mM isopropyl 1-thio-β-d-galactopyranoside (IPTG) at 14°C for 18 h. Cell pellets were resuspended in 20 mM HEPES/KOH pH 7.4, 500 mM NaCl and 1 mM MgCl₂ containing 1 mM PMSF, 1 μg/ml DNAse I (Roche Diagnostics), and protease inhibitor (Complete; EDTA-free Protease Inhibitor Mixture; Roche Applied Science), homogenized by mechanical rupture and cell debris was removed by centrifugation. The supernatant was purified using chitin resin affinity purification (New England Biolabs, Inc.). Drp1 was eluted by cleavage from the affinity resin using 30 mM DTT at pH 8 for 48 h at 4°C and further purified using anion exchange chromatography (HiTrap Q FF, GE Healthcare) at pH 8. Pure elution fractions were pooled and dialyzed against 20 mM HEPES/KOH pH 7.4, 500 mM NaCl and 1 mM MgCl₂. Purity was assessed using SDS–PAGE as described and protein concentration was determined using Bradford and spectrophotometer measurements. Purified protein was stored with 20% (vol/vol) glycerol.

Affinity measurements by biolayer interferometry

Binding kinetics analysis of Drp1-Nbs was performed using the Octet RED96e system (Sartorius) according to the manufacturer’s recommendations. In brief, streptavidin-coated biosensor tips (SA, Sartorius) were incubated for 40 s with 1.7–10 μg/ml biotinylated Drp1-Nbs diluted in Octet buffer (HEPES, 0.1% BSA). In the association step, a four-step twofold dilution series of Drp1 starting from 125 nM (D7), 62.5 nM (D63) or 120 nM (bivD7 and bivD63) were applied for 300 s followed by dissociation in Octet buffer for 720 s. Each concentration was normalized to a reference applying Octet buffer only for association. Data were analysed with the Octet Data Analysis HT 12.0 software using the 1:1 ligand model and global fitting.

Drp1 GTPase activity measurements

GTPase activities for Drp1 were measured using a continuous, regenerative assay described by (Ingerman & Nunnari, 2005). In brief, Drp1 (100 nM final) was incubated with 0, 10, 100 or 1,000 nM D7, D63, or GFP Nb as negative control. Upon addition of 1 mM DTT, 1 mM PEPK, 600 μM NADH, ≥20 U/ml PK/LDH (all Sigma-Aldrich) and 1 mM GTP, GDP (both Jena Bioscience), or no nucleotide, the reactions were started with the final buffer containing 20 mM Hepes pH 7.4, 150 mM KCl, 2 mM MgCl₂. Upon thermal equilibration of the microtiter plates, NADH absorbances were measured for 120 min using a Safire Tecan-F129013 microplate reader (Tecan) operating at 340 nm and 37°C. Using BSA without nucleotide or GDP for rapid NADH depletion, absorbances were converted into NADH concentrations. GTPase rates were calculated using linear fits between 30 and 120 min of the traces.

Cell culture and transfections

U2OS and HEK293 cell lines were obtained from ATCC (CRL3216, HTB-96); the HeLa Kyoto cell line (Cellosaurus no. CVCL_1922) was acquired from S. Narumiya (Kyoto University, Japan). Drp1 KO HeLa cells were generated in and provided by the laboratory of Thomas Langer (Max Plack Institute, Cologne, Germany). For mycoplasma testing, the mycoplasma kit Venor GeM Classic (Minerva Biolabs) and Taq polymerase (Minerva Biolabs) were applied. No additional authentication was performed as this study does not contain any cell-line-specific experiments. Culturing of cell lines was carried out by standard protocols. In brief, cells were grown in DMEM high glucose, pyruvate, GlutaMax (Thermo Fisher Scientific) with 10% (vol/vol) FCS (Thermo Fisher Scientific) and 1% (vol/vol) penicillin/streptomycin (Thermo Fisher Scientific). For routine passaging of cells, 0.05% trypsin–EDTA (Thermo Fisher Scientific) was used. Cells were cultivated at 37°C and 5% CO₂ in a humidified incubator. U2OS cells were transiently transfected with Lipotectamine 2000 (Thermo Fisher Scientific) according to the manufacturer’s recommendations. HEK293 and HeLa cells were transfected with Polyethyleneimine (PEI, Sigma-Aldrich) as previously described (Braun et al, 2016).

Nanobody immobilization on NHS-sepharose matrix

1.2 mg Drp1-Nbs per 1 ml NHS-Sepharose (Cytiva) were immobilized according to the manufacturer’s instructions.

Sortase labelling of nanobodies

For sortase coupling, 50 μM Nb, 250 μM sortase peptide (H-Gly-Gly-Gly-propyl-azide, synthesized by AG Maurer, University of Tübingen) dissolved in sortase buffer (50 mM Tris, pH 7.5, and 150 mM NaCl) and 10 μM sortase were mixed in coupling buffer (sortase buffer with 10 mM CaCl₂) and incubated for 4 h at 4°C. Uncoupled Nb and Sortase were removed by immobilized metal affinity chromatography, and the excess peptide was depleted via Amicon Ultra Centrifugal Filters (Merck Millipore). To label the azide-coupled nanobody with a fluorophore, SPACC (strain-promoted azide–alkyne cycloaddition) click chemistry reaction was applied. For this, fivefold molar excess of dibenzocyclooctyne-AlexaFluor647 (DBCO-AF647) (Jena Bioscience) was added to the azide-coupled Nb followed by incubation for 2 h at room temperature and subsequently purified by dialysis and hydrophobic interaction chromatography.

Mammalian cell lysis and protein extraction

For intracellular immunoprecipitation (IC-IP), 2.5–3 × 10⁶ HEK293 cells were seeded in a 100 mm culture dish (P100). The next day, cells were transfected with the chromobody-encoding plasmid. After 24 h, cells were harvested at 90% confluency. This workflow was also followed for the domain mapping constructs. For immunoprecipitations (IPs) using nanotraps, T175 flasks or P100 dishes of HEK293, U2OS or HeLa cells were harvested at ∼90% confluency. HEK293 cells were detached by light pipetting and U2OS or HeLa cells by trypsinization. Subsequently, the cells were washed with 1 ml PBS, and the pellets were stored at −80°C until lysis. For lysis, T175 flask pellets were resuspended in 600 μl, P100 pellets in 200 μl lysis buffer (50 mM Tris–HCl, pH7.5; 150 mM NaCl; 1 mM EDTA, pH 8; 0.5% TritonX-100; 1 μg/μl DNaseI; 2.5 mM MgCl₂; 2 mM PMSF; 1x Protease inhibitor Mix M [Serva]). The lysate was passed through 20G, 23G, and 27G needle gauges multiple times with intermediate vortexing and 10 min incubation steps on ice. Subsequently, the lysate was centrifuged for 10 min at 18,000g at 4°C. The supernatant was transferred to a fresh tube and diluted 1.5-fold with dilution buffer (50 mM Tris–HCl pH 7.5; 150 mM NaCl; 1 mM EDTA, pH 8; 2 mM PMSF).

Immunoprecipitation

Soluble protein extracts were generated from HEK293, HeLa and U2OS cells as described above. For analysis of the input (IN), 20 μl of the lysate was mixed with an equal amount of 4x SDS-Sample buffer. For IP, 80 μl slurry of each nanotrap was equilibrated in IP buffer (50 mM Tris–HCl pH 7.5; 150 mM NaCl; 1 mM EDTA, pH 8) and added to equal amounts of lysate. As positive (domain mapping) or negative control (precipitation of endogenous Drp1), 80 μl GFP-Trap slurry (ChromoTek) was used. For IC-IP, 80 μl of RFP-Trap slurry (ChromoTek) was used. Soluble protein extracts were incubated with the nanotraps overnight at 4°C on an end-over end rotor. For positive control (precipitation of endogenous Drp1), 4 μl anti-Drp1 antibody was added to the lysate and after overnight incubation at 4°C 80 μl equilibrated Protein A/G sepharose slurry were added followed by additional 3–4 h incubation at 4°C. Beads were harvested by centrifugation (2,000g, 2 min, 4°C) and supernatant (non-bound sample) was mixed 1:1 with 4x SDS-sample buffer. The beads were washed three times with 500 μl wash buffer (IP buffer with 2 mM PMSF, for MS samples in addition to 1x protease inhibitor mix and 0.05% Tween20). With the third wash, beads were transferred into a fresh tube. The beads were boiled in 60 μl 2x SDS-sample buffer for 10 min at 95°C and the supernatant was transferred into a fresh tube (bound sample).

For immunoblotting of IP samples, 1% of input and non-bound and 33% of the bound sample were loaded on an SDS-Page and blotted on nitrocellulose membrane as previously described (Fagbadebo et al, 2022). Immunoblots were probed with the following antibodies: anti-Drp1, anti-TagRFP, anti-GAPDH (Fig S14A–C). All antibodies used in this study are listed in Table S8. For immunoblot scanning, a Typhoon-Trio laser scanner (GE Healthcare) was used with excitation 633 nm and emission filter 670 nm BP 30 settings.

• Download figure
• Open in new tab
• Download powerpoint

Figure S14. Full-size Western Blots images shown in this study.

(A) Full size of Western blots depicted in Fig 2B (domain mapping) stained with an anti-GFP antibody. (B) Full size of Western blots depicted in Figs 3A and S3. Top half stained with anti-Drp1 antibody; bottom half stained with anti-GAPDH antibody. (C) Full size Western blots depicted in Fig 6A. Top half stained with anti-Drp1 antibody; bottom half stained with anti-TagRFP antibody. Molecular weights in kD are shown on the left of each blot.

Chromobody imaging

wt HeLa cells (8,000 cells/well) or Drp1 KO HeLa cells (11,000 cells/well) were seeded in μclear 96 well plates (Greiner Bio-One). The next day, cells were transfected with the respective plasmids. 24 h post transfection, cells were stained with 400 nM MitoTracker green (Life Technologies) and 4 μg/ml Hoechst33258 for 30 min. Afterwards, medium was replaced with live-cell visualization medium DMEMgfp-2 (Evrogen) supplemented with 10% FBS, 2 mM L-glutamine with or without 20 μM CCCP. Images were taken 10, 70, and 130 min after compound addition with ImageExpress Micro Confocal High Content screening system (Molecular devices) at 40x magnification. During imaging, cells were kept at 37°C and 5% CO₂. Raw microscopy images were adjusted in brightness and contrast using MetaExpress software (64 bit, 6.2.3.733 or higher, Molecular devices).

Immunofluorescence in fixed cells

Cells were seeded in μclear 96 well plates (Greiner Bio-One) in the same densities as described for Cb imaging. The next day, cells were transfected with GFP-Drp1 plasmid or left untransfected. 24 h post-transfection, cells were washed twice with PBS and subsequently fixed with 3.7% PFA for 10 min at RT. After three times washing with PBS, cells were incubated with TBST for 10 min at RT. After additional washes, cells were blocked with 5% BSA in TBST for 1 h. Afterwards, the cells were incubated with 100–1,000 nM Nb and anti-Drp1 antibody (1:100–1:300) in 5% BSA/TBST overnight at 4°C. Then, unbound Nb and antibody were washed away and the cells incubated with secondary antibody (anti-rabbit labelled with AF647; 1:1,000) and Cy5-conjugated goat anti-alpaca antibody (1:500) for 1 h at RT in 2.5% BSA/TBST. Nuclei staining with 4’, 6-diamidino-2-phenylindole (DAPI; Sigma-Aldrich) was performed concurrently. After additional washing with TBST and PBS, images were acquired using an ImageExpress Micro Confocal High Content screening system (Molecular devices) at 40x magnification. Raw microscopy images were adjusted in brightness and contrast using MetaExpress software (64 bit, 6.2.3.733 or higher, Molecular devices).

Liquid chromatography-MS sample preparation and measurement

Immunoprecipitation of Drp1 using the D7 and D63 nanotraps were compared with the GFP-Trap (negative control) in three technical replicates. Immunoprecipitation was performed as described above and proteins were subsequently separated by SDS–PAGE (4–12% NuPAGE tris gel [Invitrogen]) for 7 min at 200 V and stained with Coomassie blue. The protein gel pieces were subjected to tryptic digestion as described previously (Shevchenko et al, 2006). Purified peptide samples were measured on a Q Exactive HF mass spectrometer (Thermo Fisher Scientific) online-coupled to an Easy-nLC 1200 UHPLC (Thermo Fisher Scientific). Peptides were separated using a 20-cm-long, 75–μm–inner diameter analytical HPLC column (ID PicoTip fused silica emitter; New Objective) packed in-house with ReproSil-Pur C18-AQ 1.9-μm silica beads (Dr Maisch GmbH) and eluted using a 60 min segmented gradient from to 10–90% of solvents A (0.1% formic acid) and B (80% acetonitrile in 0.1% formic acid) at a constant flow rate of 200 nl/min. The column temperature was maintained at 40°C using an integrated column oven. Peptides were ionized by nanospray ionization and a capillary temperature of 275°C. The mass spectrometer was operated in the positive ion mode. Full MS scans were acquired in a range of 300–1,750 m/z at resolution of 60,000. Seven most intense multiple-charged ions were selected for HCD fragmentation with a dynamic exclusion period of 30 s and tandem MS (MS/MS) spectra were acquired at resolution of 15,000.

Mass spectrometry data analysis

The acquired raw files were processed by MaxQuant software (version 2.0.3.0.) (Cox & Mann, 2008) and searched against the Uniprot Homo sapiens database (105.079 entries, downloaded 2022/08/01), V. pacos derived nanotraps and commonly observed protein contaminants. For MS and MS/MS, the peptide mass tolerance was set at 4.5 ppm and 20 ppm, respectively. Only two missed cleavages were allowed for the tryptic digestion. Carbamidomethylation (C) was used as fixed modification, whereas oxidation (M) and acetylation (protein N-term) were defined as variable modifications. False discovery rate was set to 1% at both peptide and protein levels. For label-free quantification, a minimum ratio count of two was requested. Intensity-based absolute quantification (iBAQ) was enabled. Downstream analysis of the “proteinGroups.txt” output table was performed in Perseus (version 1.6.15.0). Contaminants, reversed proteins and proteins only identified by site were filtered out. Only proteins that were quantified in two of three replicates of either the bait or control pulldown were retained in the dataset. Missing values were imputed (width 0.3, downshift 1.8). To determine significantly enriched proteins, a multi-volcano analysis (Hawaiian plot) was performed. The s₀ and FDR parameters of the multi-volcano analysis were: for class A (higher confidence, s₀ = 0.1, FDR = 0.01) and class B (lower confidence, s₀ = 0.1, FDR = 0.05). Additional graphical visualization was performed in the R environment (version 4.1.1) and in GraphPad (version 8.0.1.).

Cell cultivation, seeding, and staining for super resolution microscopy

U2OS wt or U2OS mEGFP-Drp1 cells were cultivated in DMEM (low glucose) supplemented 10% (vol/vol) FBS and 1% (vol/vol) penicillin/streptomycin (Invitrogen) at 37°C and 5% (vol/vol) CO₂. For microscopy, the cells were seeded in 35 mm μ-Dish 1.5H glass bottom imaging chambers (for confocal imaging) or on 35 mm 1.5H glass coverslips in in a six-well plate (for SMLM) at a density of 3 × 10⁵ cells per dish/well and grown for 18 h. If required, mitochondria were stained using 200 nM MitoTracker Green FM (Invitrogen) for 20 min. Cells were fixed in 4% (vol/vol) PFA in DMEM (pre-warmed to 37°C) for 10 min at RT and washed twice with PBS. To quench unreacted fixative, the cells were incubated in 50 mM NH₄Cl for 15 min followed by permeabilization in 0.25% (vol/vol) Triton X-100 for 8 min. The cells were washed with PBS three times for 5 min and incubated with 1% (wt/vol) BSA in PBS for 1 h to block unspecific binding sites. DRP1 was labelled using 250 nM bivD7 coupled to AlexaFluor 647 (Invitrogen) in 1% (wt/vol) BSA in PBS over night at 4°C protected from light. The cells were washed three times with PBS and stored at 4°C in the dark until imaging.

Confocal fluorescence microscopy

U2OS mEGFP-Drp1 cells were prepared as described above. Confocal fluorescence microscopy images were acquired on an inverted Infinity Line confocal dual-color STED 775 QUAD laser scanning microscope (Abberior Instruments) equipped with a UPLXAPO 60x/1.42 Oil objective (Olympus). mEGFP was excited at 488 nm and AF647 was excited at 640 nm wavelength. Images were acquired with a pixel size of 65 nm and a pixel dwell-time of 5 µs. The fluorescence emission signal was collected on avalanche photodiode detectors. Raw microscopy images were adjusted in brightness and contrast using Fiji/ImageJ (Schindelin et al, 2012). Colocalization of endogenous mEGFP-Drp1 and bivD7_AF647 was assessed by calculating the Pearson’s correlation coefficient of the respective emission signals using Just Another Co-localization Plugin (JACoP) (Bolte & Cordelières, 2006) in Fiji/ImageJ (Schindelin et al, 2012). For this, the images were cropped to remove unspecific bivD7 signal outside of the cell and in the nucleus. The experiments are representative for n = 3 individual replicates with n = 11 cells in total.

Single-molecule localization microscopy

U2OS WT cells were seeded and stained as described above and mounted on custom-made imaging chambers or single concave depression microscopy slides covered with 100–300 μl imaging buffer (50 mM Tris–HCl pH 8, 10 mM NaCl, 10% [wt/vol] glucose, 35 mM cysteamine [MEA], 0.5 mg/ml glucose oxidase [Sigma-Aldrich] and 40 μg/ml catalase [Sigma-Aldrich]) and sealed using Twinsil speed dental glue (Picodent). The samples were imaged on a home-built wide-field/TIRF microscope or on a SR GSD (3D) inverted DMI6000 B super-resolution wide-field/TIRF microscope (Leica Microsystems) equipped with a 100x, 1.47 N.A. GSD Objective, 405, 488, 532, and 642 nm excitation lasers and an Andor iXon Ultra 897 EMCCD camera. Image series were acquired with an exposure time between 30 and 50 ms and an EM gain of 300. AF647 was excited using 640 nm wavelength and the 405 nm activation laser intensity was manually adjusted to keep a constant number of localizations per frame. Typically, 70,000–100,000 frames were recorded. Analysis was performed using the LAS X Softeware (Leica) and ThunderStorm (Ovesny et al, 2014). Images were rendered using a Gaussian with a width according to the localization precision. Image analysis was performed using Fiji/ImageJ (Schindelin et al, 2012). Images are representative for n = 6 individual experiments.

Image analysis and statistics

Image analysis was performed with MetaExpress software (64 bit, 6.2.3.733 or higher, Molecular devices) for n > 130 cells per construct. Using the Custom editor (version 2.5.13.3 or higher) or the MetaExpress software, we developed an algorithm to determine the average signal intensity per transfected cell. Based on the assumption that cells usually are transfected with both constructs, the GFP-NLS signal was used as a marker for transfection and cell area. Cell average intensity of the TagRFP signal was determined for all GFP-positive cells. Using the DAPI signal in GFP-NLS negative cells, the TagRFP signal intensity was measured for background determination. The average background signal was determined (n = 2125 cells) and subtracted from all measurements. A workflow is shown in Fig S15A and B.

• Download figure
• Open in new tab
• Download powerpoint

Figure S15. Workflow of the automated image segmentation used for Cb signal quantification.

For quantitative image analysis, cells were co-transfected with GFP-NLS (green) and chromobody (Cb, red), and nuclei were stained using DAPI (blue). (A) Workflow for background signal determination. Area of GFP-NLS-expressing cells and nuclei were determined individually (second row). Then, nuclei of GFP-NLS-expressing cells were excluded (third row). Next, the DAPI signal was used to create a cell shape and the RFP intensity was measured in this area (forth row). (B) Workflow for Cb signal intensity determination. The GFP signal was used to determine the cell shape and the RFP signal was measured in this area.

Mean and SD were calculated for each condition. For comparison of unmodified and turnover-accelerated Drp1-Cbs, significance was calculated using Kruskal Wallis test with Dunn’s multiple comparisons test. For comparison of turnover-accelerated Cbs in wt HeLa and Drp1 KO HeLa cells, significance was calculated using two-way ANOVA with Sidak’s multiple comparisons test. Tests were performed using GraphPad Prism software (Version 8 or higher). All graphs were prepared using GraphPad Prism.