Severing enzymes amplify microtubule arrays through lattice GTP-tubulin incorporation.

Protein expression and purification

Drosophila melanogaster full-length spastin was purified by affinity chromatography and ion exchange as previously described ( 62 ) . Caenorhabditis elegans MBP-tagged katanin Mei1/Mei2 ( 12 ) was purified on amylose resin. The affinity tag was removed by Tobacco Etch Virus protease and the protein was further purified on an ion exchange MonoS column (GE Healthcare) as previously described ( 63 ). Peak fractions were concentrated, buffer exchanged into 20 mM Hepes 7.0, 300 mM KCl, 10 mM MgCl 2 and 1 mM TCEP and flash frozen in small aliquots in liquid nitrogen. Homo sapiens EB1-GFP was expressed and purified as previously described ( 64 ).

Human a1AßIII tubulin with an engineered FLAG-tag at the β-tubulin

C-terminus was expressed using baculovirus and purified as described previously ( 38 ). Transmission electron microscopy of microtubule severing reactions Taxol-stabilized GDP microtubules were prepared by polymerizing 10 μl of 100 μM glycerol-free porcine tubulin (Cytoskeleton Inc., Denver, CO) in 80 mM K-PIPES pH 6.8, 1 mM MgCl 2 , 1 mM EGTA, 10 % DMSO, 1 mM GTP for 1 hour in 37 °C water bath. Taxol was added to 20 μM final concentration and the reaction was incubated on the bench top for 1–2 hours. Microtubules were loaded on 60 % glycerol cushion (BRB80, 60 % (v/v) glycerol and 20 μM taxol) at 37 °C using a pipette tip with the tip cut off. Non-polymerized tubulin was removed by centrifugation in a TLA100 rotor at 35,000 rpm for 15 min at 37 °C. The pellet was gently re-suspended to 2.5 μM of tubulin in BRB80, supplemented with 20 μM taxol and 1mM GTP at 37 °C using a pipette tip with the tip cut off. For GDP microtubules, all polymerization and severing reactions were performed at 37 °C. 20 μl of 100 μM glycerol-free porcine tubulin (Cytoskeleton Inc.) was polymerized in 10 % DMSO, 1 mM GTP and 10 mM MgCl 2 for 1 hour at 37 °C water bath. The microtubules were passed through 60 % glycerol cushion (BRB80, 60 % (v/v) glycerol and 1mM GTP) using a TLA100 rotor at 53,000xg for 15 min to remove non-polymerized tubulin. The pellet was washed twice using 50 μl buffer (BRB80, 10 % DMSO, 1 mM GTP) and gently re-suspended to 30 μM in the same buffer using a pipette tip with the tip cut off. GMPCPP microtubules were prepared by polymerizing 20 μl of 100 μM glycerol-free porcine tubulin (Cytoskeleton Inc.) in 1mM GMPCPP in BRB80 (80 mM PIPES-KOH pH6.8, 1mM MgCl 2 , 1mM EGTA, 1mM DTT) on ice for 5 min and then in a water bath at 37 °C for 1 hour. Non-polymerized tubulin was removed by centrifugation in a TLA100 rotor at 126,000 × g for 5 min at 37 °C. The pellet was washed twice with 50 μl of BRB80 at 37 °C and re-suspended in 50 μl of ice-cold BRB80. The reaction was kept on ice for 30 min and periodically mixed up and down to fully depolymerize microtubules. GMPCPP was added to 1 mM, the polymerization reaction was kept on ice for 10 min and then transferred to 37 °C for 2–4 hrs or overnight. Non-polymerized tubulin was removed by centrifugation and washed as described above. The microtubule pellet was gently re-suspended to 2.5 μM of tubulin in BRB80 using a pipette tip with the tip cut off. We found that performing severing reactions in the tube followed by pipetting onto electron microscopy grids resulted in microtubule breakage. We therefore first carried out severing reactions on the electron microscopy grid. Briefly, 2μl microtubule solution (at 1 to 3 μM) in BRB80 (80mM PIPES pH 6.8, 1 mM MgCl2, 1 mM EGTA) was applied to a glow-discharged Cu grid, followed by pipetting of 2μl ATP solution (10mM ATP in BRB80 supplemented with 20mM taxol, for taxol stabilized microtubules) and 2μl spastin (at 100 nM). The reaction was allowed to proceed on grid for one minute or as specified, after which the liquid was wicked off with calcium-free filter paper, and the grid was stained with 0.75% (w/v) uranyl formate, and air-dried. Images were collected on a FEI Morgagni 286 electron microscope operated at 80kV and equipped with AMT lens-coupled 1k x 1k CCD camera. For the solution severing reaction time courses, 20 μl of GMPCPP or taxol-stabilized microtubules in BRB80 buffer at 2.5 μM or 1 μM were applied to parafilm followed by addition of 20 μl of 50 nM spastin or 200 nM katanin in 20 mM HEPES pH 7.5, 300 mM KCl, 10 mM MgCl 2 , 1mM TCEP and 1mM ATP to a final concentration of 25 nM spastin and 100 nM for katanin. For the solution severing reaction time courses of non-stabilized GDP microtubules, 20 μl of 30 μM GDP microtubules in the presence of 10% DMSO were incubated with 2 μl of 20 nM katanin. Buffer without severing enzymes was added to microtubules as a negative control. The severing reactions were incubated for 30 sec, 2 or 5 mins and carbon- coated grids (Carbon Film only on 400 mesh, Ted Pella, Inc.) were dipped in the reactions. Excess liquid was blotted using filter paper. Grids were washed three times with 40 μl BRB80, stained with 0.75 % (w/v) uranyl formate and air-dried. Images were collected on a T12 Technai electron microscope (FEI) equipped with a 2k x2k Gatan US1000 CCD camera. Images were collected at nominal magnifications of 550x, 13,000x or 30,000x corresponding to pixel sizes of 84 Å/pix, 3.55 Å/pix, or 1.54 Å/pix, respectively. TIRF based assays of tubulin incorporation into stabilized microtubules damaged by spastin and katanin Double-cycled, GMPCPP-stabilized microtubules ( 65 ) were polymerized from 2 mg/ml porcine brain tubulin (Cytoskeleton). First polymerization was 1 h, the second polymerization step was at least 4 h to obtain long microtubules. Then microtubules were centrifuged, resuspended in warm BRB80 (80 mM K-PIPES pH 6.8, 1 mM MgCl 2 , 1 mM EGTA) and stored at 37°C or RT before use. The same results were obtained regardless of whether the storage temperature was 37°C or RT. Taxol-stabilized microtubules ( 62 ) were polymerized from 5 mg/ml porcine brain tubulin containing 1% biotinylated and 20% HiLyte647-labeled tubulin (Cytoskeleton) in BRB80 with 10% DMSO, 0.5 mM GTP and 10 mM MgCl 2 . After 1 h incubation at 37°C, 20 μ M Taxol was added and the mixture was further incubated ON. Microtubules were then centrifuged through a 60% glycerol cushion for 12 min at 109,000 g, 35°C. The microtubule pellet was washed with warm BRB80 supplemented with 14.3 mM 2-mercaptoethanol, 20 μ M Taxol and resuspended gently in the same buffer. Chambers for TIRF microscopy were assembled as previously described ( 62 ). Double-cycled GMPCPP microtubules containing 1% biotinylated tubulin and 20% HiLyte647-labeled tubulin (or unlabeled tubulin for the DIC assays) assembled as above were immobilized in the chamber with 2 mg/ml Neutravidin (Thermo Fisher Scientific) and imaged by TIRF or DIC microscopy in severing buffer (BRB80 buffer with 2 mg/ml casein, 14.3 mM 2-mercaptoethanol, 2.5% glycerol, 50 mM KCl, 2.5 mM MgCl 2 , 1 mM ATP, 1% Pluronic F127 (Life Technologies) and oxygen scavengers). To introduce and detect nanoscale damage in microtubules ( Fig. 2 ), immobilized microtubules were then incubated with 10 nM spastin or 2 nM katanin in severing buffer for 35 s or 90 s respectively. Microtubules in control experiments were incubated without severing enzyme. The enzyme mixture was then replaced with 1 μM HiLyte488-labeled tubulin (Cytoskeleton), 1 mM ADP, 0.5 mM GTP, 1% F127 Pluronic, 2.5 mg/ml casein in BRB80 and left to incubate for 5 min. The tubulin containing solution was then washed out with 45 μl of BRB80 supplemented with oxygen scavengers, 1.5 mg/ml casein, 10 mM 2-mercaptoethanol and 1% F127 Pluronic. Microtubules and HiLyte488-labeled tubulin were imaged by TIRF. Multiple fields of view were imaged. The same assay was performed for taxol-stabilized microtubules, but in this case the repair step was performed with 0.1 μM soluble tubulin to prevent microtubule nucleation in the presence of taxol. For time course experiments, the same protocol was used except that microtubules were incubated with 2 nM spastin ( fig. S3 ) or 2 nM katanin ( fig. S4 ) for 35–120 sec. Control microtubules were incubated without severing enzyme for 120 sec. 1 μM HiLyte488-labeled tubulin was used for the repair step. For repair with 1 μM recombinant human tubulin ( fig. S6 ), nanodamaged microtubules were incubated for 5 min with recombinant tubulin. Unincorporated tubulin was washed away and tubulin incorporated into microtubules was detected by anti-FLAG M2 antibodies (Sigma Aldrich, diluted 1:500) and goat anti-mouse antibodies conjugated with Alexa Fluor 488 (Invitrogen, diluted 1:1000). All assays were performed at room temperature. Details regarding image acquisition and analysis are described in the subsection below. Image acquisition and analysis of tubulin incorporation in GMPCPP-and taxol-stabilized microtubules by TIRF microscopy Images were acquired using a Nikon Ti-E microscope equipped with a 100× 1.49 NA oil objective and a TI-TIRF adapter (Nikon). The 488 excitation laser (Coherent Inc.) was set at 20 mW and the 647 nm laser (Coherent Inc) was set to 2 mW before being coupled into the Ti-TIRF optical fiber (Nikon). Two-color simultaneous imaging was performed using a TuCAM (Andor) device that splits the emission on to two separate EMCCD cameras (Andor iXon 897). The excitation and emission were split by a quad band dichroic (Semrock) and the emission was further split by an FF640 filter (Semrock) and further filtered with a FF01–550/88 (Semrock) for the 488 channel and a FF01–642/LP (Semrock) for the 640 channel. The Tucam imaging system introduces an extra 2X magnification yielding a final pixel size of 77 nm. The images from the two cameras were aligned by first imaging a grid of spots (Nanogrid MiralomaTech) on each camera and using the GridAligner plug-in for ImageJ. DIC illumination was provided by a SOLA-SE-II (Lumencor) coupled to the microscope by a liquid light guide. A standard set of polarizer and analyzer (Nikon 100 X-II High NA/Oil) prisms were used and the image was captured on a CoolSNAP (Photometrics) camera. The final pixel size for DIC images was 65 nm. Raw DIC images were processed using an FFT bandpass filter. DIC images were scaled and transformed to overlay with fluorescent images by imaging fluorescent microtubules in both channels for image registration. The entire imaging setup was controlled by Micro-Manager ( 66 ). For data shown in figs. S3 and S4 , images were analyzed using scripts in ImageJ and MATLAB. First, the offset between 640 and 488 channels was corrected with the GridAligner plugin. Then microtubules were selected with 7 px-wide line selection and line scans were generated. These line scans were imported into a MATLAB script that identified the peaks in the 488 channel and recorded the number, intensity and full-width-at-half-maximum (FWHM) of the repair sites. The FWHM for a diffraction-limited spot was obtained using 100 nm TetraSpeck beads (Thermo Fischer Scientific). Data were exported to PRISM software for graphing. Transmission electron microscopy of microtubules repaired with recombinant tubulin GMPCPP microtubules at 1 μM concentration in 1x BRB80 were applied to parafilm in a humidity chamber and incubated with 20 nM spastin in microtubule-severing buffer (20 mM HEPES pH 7.5, 300 mM KCl, 10 mM MgCl 2 , 1 mM TCEP and 0.5 mM ATP). Buffer containing 0.5 mM ATPγS instead of ATP was used as a control. Severing was allowed to proceed for 30 seconds followed by addition of 0.6 μM soluble FLAG-tagged single-isoform recombinant neuronal human α1AβIII tubulin to repair the microtubule lattice in the presence of 1 mM GTP and 5mM ADP to inactivate the enzyme. The repair reaction was carried out for 5 min. Microtubules were then stabilized by the addition of 5 volumes of 0.2% glutaraldehyde in 1xBRB80 (80mM PIPES, 1mM MgCl 2 , 1mM EGTA). After 3 minutes, crosslinking was quenched by the addition of Tris-HCl pH 7.5 to 20 mM final concentration and crosslinked microtubules were transferred into a 10 ml centrifuge tube (Beckman Coulter). The microtubule severing and healing procedure was repeated three more times, reactions were pooled into the same centrifuge tube and microtubules were then spun down in a MLA-80 rotor at 100,000 × g for 15 min at 30 °C. The microtubule pellet was gently washed with 200 μl of 1x BRB80 at 37 °C twice and re-suspended in 50 μl of warm 1x BRB80. 5 μl of 6.7 μM of monoclonal mouse-raised anti-FLAG M2 antibody (Sigma Aldrich) and 5 μl of 11.45 μM of goat anti-mouse antibody conjugated to 4 nm spherical gold nanoparticles, C11–4-TGAMG-50, (Nanopartz) were added to microtubules to label repaired sites. Antibody labeling was allowed to proceed for 5 min and the reaction was mixed with 10 volumes of 30 % glycerol in 1x BRB80. Microtubules in 30% glycerol were loaded on 1x BRB80 cushion containing 40% glycerol and spun down onto glow-discharged carbon-coated grids (Carbon Film only on 400 mesh, Ted Pella, Inc.) at 4,200 x g for 20 min at 30 °C. Excess liquid was blotted using filter paper. Grids were washed three times with 30 μl of BRB80, stained with 0.75 % (w/v) uranyl formate and air-dried. Images were collected on a T12 Technai electron microscope (FEI) equipped with a 2k x 2k Gatan US1000 CCD camera. Images were collected at nominal magnifications of 6,800x and 18,500x corresponding to pixel sizes of 6.8 Å/pix, 2.5 Å/pix, respectively. Images in fig. S7F were collected on a TF20 electron microscope (FEI) equipped with a K2 camera (Gatan). Images were collected at 50,000x magnification and 9,600x magnifications corresponding to pixel sizes of 0.73 Å/pix and 3.65 Å/pix, respectively. Live imaging of severing and tubulin incorporation into nanodamaged GMPCPP- microtubules and GMPCPP-capped GDP-microtubules To observe microtubule severing and tubulin incorporation at damage sites simultaneously ( Fig. 3 and figs. S5A – C ), GMPCPP-stabilized double-cycled microtubules labeled with 1% biotin and 20% HiLyte647-tubulin were immobilized in imaging chambers. Image acquisition was started using 100 ms continuous exposure in the 647 and 488 channels simultaneously and the chamber was perfused with severing buffer containing 0.5 mM GTP, 20 nM spastin and 0, 0.1 or 2 μM HiLyte488-labeled tubulin. Severing rates were calculated by manual counting of severing events (microtubule breaks) as a function of time. Tubulin incorporation sites were readily visible in the 488 channel. To observe the live incorporation of single tubulin dimers into microtubules damaged by spastin ( Figs. 3F , G ), double-cycled GMPCPP microtubules composed of 20% HiLyte647- labeled and 1% biotinylated tubulin were immobilized in imaging chambers as above. The chamber was then perfused with severing buffer and images of microtubules were acquired. Microtubules were then incubated for 30 sec with 20 nM spastin in severing buffer. Image acquisition was started during the spastin incubation step and a solution containing fluorescently labeled tubulin (50 nM Alexa488-labeled tubulin (PurSolutions LLC, USA) in BRB80 with 2 mg/ml casein, 14.3 mM 2-mercaptoethanol, 50 mM KCl, 2.5 mM MgCl2, 1 mM ADP, 0.5 mM GTP, 1% Pluronic F127 and oxygen scavengers) was flushed in. Images were acquired for 5 min at 10 Hz in the 488 nm channel. After tubulin perfusion, the 640 laser was turned off to prevent photobleaching or microtubule photodamage. Images of fluorescent tubulin molecules landing on the microtubule were analyzed using a 7×7 pixel box and the intensity of tubulin molecules incorporated into the microtubule was calibrated against the intensity of single tubulin dimers obtained by immobilizing 0.5 nM Alexa488 tubulin on glass with an anti-ß tubulin antibody (SAP.4G5, Sigma Aldrich) and imaging under the same conditions. For imaging of non-stabilized microtubules GDP microtubules with a GMPCPP cap, sea urchin axonemes purified as described ( 67 ) were non-specifically adhered to the coverslip and 15 μM tubulin containing 20% HiLyte647 tubulin, and 1mM GTP added to start microtubule growth from the axonemes. After the desired microtubule length was achieved (10–20 μm), the solution was exchanged quickly to introduce HiLyte488 tubulin (20%) and 0.5 mM GMPCPP. After the growth of the GMPCPP cap, tubulin and nucleotide were washed out and spastin (5 nM) was introduced in the chamber with 1 mM ATP in the absence or presence of soluble tubulin at 2 μM (500 nM HiLyte488 tubulin + 1.5 μM unlabeled tubulin) or 5 μM (500 nM HiLyte488 tubulin + 4.5 μM unlabeled tubulin) and 0.5 mM GTP. Polymerization and imaging were performed at 30°C. Microtubule dynamics measurements and EB1 recognition of lattice-incorporated GTP- tubulin TIRF microscopy chambers were prepared as described above. 10% HiLyte647-labeled microtubules were polymerized at 30°C at 10 μM tubulin. 25 nM spastin or katanin and 10 μM porcine brain tubulin containing 10% HyLite647-labeled tubulin was perfused into the chamber in severing assay buffer (50 mM KCl, 1% F127 Pluronic, 0.2 mg/ml casein, 6.2 mM 2-mercaptoethanol, 1.5% glycerol, 0.1% methylcellulose 4000cP and oxygen scavengers in 1xBRB80) with 1 mM GTP and 1 mM ATP) together with 50 nM EB1-GFP. Images were acquired in the 647 and 488 channels simultaneously at 2 Hz. Microtubule rescues are defined as transition of microtubules from shrinkage to growth. Rescue frequency was calculated as the number of rescues divided by the time spent depolymerizing. Catastrophes are defined as the transition of microtubules from growth to shrinkage. Catastrophe frequency was calculated as the number of catastrophes divided by the time spent in the polymerization state. The EB1 puncta and the microtubule rescue site were considered co-localized when the distance between the EB1 spot and the end of the depolymerizing microtubule was less than two pixels. The cutoff for an EB1 puncta was defined as having a mean intensity in a 5×5 pixel box that is at least 3 standard deviations above the mean background EB1 lattice intensity. Background EB1 lattice intensity was determined from control chambers without severing enzymes. Background EB1 lattice intensity was the same in the absence of severing enzymes or the presence of severing enzymes but in the absence of ATP. For statistical significance calculation, rescue site analysis was also performed using synthetic data generated by shifting the position of the EB1 spots by 7 pixels on the microtubule (alternatively, both towards the plus and the minus ends). For the GTP-tubulin and EB1-GFP co-localization experiments shown in Fig. 6E , microtubule extensions were grown in the absence of fluorescent tubulin for 8 minutes at 30°C at 12 μM porcine brain tubulin (Cytoskeleton) in severing assay buffer. 20 nM spastin, 50nM EB1-GFP and 12μM porcine brain tubulin containing 10% HyLite647-labeled tubulin was perfused into the chamber in severing assay buffer. Image acquisition was started during perfusion in the 640 and 488 channels simultaneously at 5 Hz. The offset between the 640 and 488 channels was corrected using a nanogrid (Nanogrid Miraloma Tech) and the GridAligner plug-in in ImageJ.

Laser ablation of microtubules with spastin or katanin generated GMPCPP-islands

GMPCPP-stabilized unmodified microtubule seeds were immobilized on glass. To pre-grow microtubules, 16μM tubulin containing 12.5% HiLyte647-labeled tubulin with 1mM GTP was perfused into the chamber and incubated for 10 minutes at 30°C. Microtubules were then capped using 6μM tubulin with 10% HiLyte 647 and 0.5mM GMPCPP. The chamber was washed after 2 minutes with severing assay buffer without GTP and then incubated with 4nM spastin, 6μM tubulin containing 25% HiLyte-labeled 488 tubulin in the presence of 200μM GMPCPP in severing assay buffer (50 mM KCl, 1% F127 Pluronic, 0.2 mg/ml casein, 6.2 mM 2-mercaptoethanol, 2.5% glycerol, 0.1% methylcellulose 4000cP and oxygen scavengers in 1xBRB80) with or without 1mM ATP for 3 minutes. The chamber was washed with buffer containing severing assay buffer. Microtubules were ablated with a 405nm laser at 40% power using the iLas laser illuminator (BioVision). Images in the 488 and 647 channels were acquired sequentially with 100 ms exposure. For the rescue frequency measurements, 15% HiLyte647-labeled tubulin at 7μM in severing assay buffer containing 1mM GTP was perfused into the chamber. For the katanin experiments, the chamber was washed after microtubule capping with severing assay buffer without GTP and then incubated with 20nM katanin and 8μM tubulin containing 25% HiLyte-labeled 488 tubulin in the presence of 200μM GMPCPP in severing assay buffer with or without ATP for 45 sec. Microtubule depolymerization rates through the GMPCPP islands were determined by dividing the length of the island by the time it takes to depolymerize through it.

Laser ablation of dynamic microtubules with enzyme-generated GTP islands

TIRF microscopy chambers were prepared as described above. HiLyte647-labeled microtubule extensions were polymerized for 8 minutes at 30°C at 12 μM porcine brain tubulin (Cytoskeleton) containing 20% HiLyte647-labeled tubulin in severing assay buffer. 25 nM spastin or katanin, 50nM EB1-GFP and 12μM porcine brain tubulin containing 20% HyLite647-labeled tubulin was perfused into the chamber in severing assay buffer with ATP or ATPγS. Microtubules were ablated using a DeltaVision OMX™ with the 405nm laser at 100% power for 1 sec or with a 405nm laser at 40% power using an iLas laser illuminator (BioVision). Images were acquired in the 647 and 488 channels at 5 Hz on the DeltaVision OMX and 2.9Hz on the iLas system.. Live imaging of tubulin incorporation and severing into dynamic microtubules Chambers for TIRF microscopy were prepared as described above. GMPCPP-stabilized, unmodified microtubules containing 2% biotinylated tubulin were immobilized with 0.1 mg/ml NeutrAvidin (Thermo Fisher Scientific). Microtubule extensions were polymerized for 12 minutes at 30°C at 10 or 12 μM porcine brain tubulin (Cytoskeleton) containing 10% HiLyte647 tubulin in severing assay buffer (50 mM KCl, 1% F127 Pluronic, 1 mM ATP, 1 mM GTP, 0.2 mg/ml casein, 6.2 mM 2-mercaptoethanol, 1.5% glycerol, 0.1% methylcellulose 4000cP and oxygen scavengers in 1xBRB80). Then, 25 nM katanin or spastin with 12 μM porcine brain tubulin containing 10% HyLite488-labeled tubulin was perfused into the chamber in severing assay buffer. Images were acquired with 488 and 640 lasers simultaneously at 2 Hz at 100 ms exposure. The incorporation of the HiLyte488 tubulin was immediately visible upon perfusion only at microtubule tips in the control and along the microtubules and the dynamic tips in the enzyme and ATP conditions. Total polymer mass was obtained by measuring the background corrected total integrated fluorescence in both the 488 and 640 channels. The laser ablation controls were performed at the same enzyme and tubulin concentrations but with 1mM ATPγS. Microtubules were ablated with a 405nm laser at 40% power using an iLas laser illuminator (BioVision) for the katanin experiments and the Deltavision OMX for spastin.

Quantification and Data Analysis

N numbers and statistical tests are reported for all experiments in figure legends. All experiments were performed multiple times and only representative images are shown. ImageJ was used for image analysis. Prism (Graphpad) was used for graphi006Eg and statistical analysis.

Supplementary Material MovieS1 MovieS2 MovieS3 MovieS4 MovieS5 MovieS6 MovieS7 Supplemental Materials