Synergy between Cyclase-associated protein and Cofilin accelerates actin filament depolymerization by two orders of magnitude.

Purification and labeling of rabbit muscle actin

Rabbit skeletal muscle actin was purified from acetone powder 40 generated from frozen ground hind leg muscle tissue of young rabbits (PelFreez, Rogers, AR). Lyophilized acetone powder stored at −80 °C was mechanically sheared in a coffee grinder, resuspended in G-buffer (5 mM Tris-HCl pH 7.5, 0.5 mM Dithiothreitol (DTT), 0.2 mM ATP, 0.1 mM CaCl 2 ), and then cleared by centrifugation for 20 min at 50,000 × g . Actin was polymerized by the addition of 2 mM MgCl 2 and 50 mM NaCl and incubated overnight at 4 °C. F-actin was pelleted by centrifugation for 150 min at 361,000 × g , and the pellet solubilized by dounce homogenization and dialyzed against G-buffer for 48 h at 4 °C. Monomeric actin was then precleared at 435,000 × g , and loaded onto a S200 (16/60) gel-filtration column (GE Healthcare, Marlborough, MA) equilibrated in G-Buffer. Fractions containing actin were stored at 4 °C. For biotinylation of actin, the F-actin pellet described above was dounced and dialyzed against G-buffer lacking DTT. Monomeric actin was then polymerized by adding an equal volume of 2× labeling buffer (50 mM imidazole pH 7.5, 200 mM KCl, 0.3 mM ATP, 4 mM MgCl 2 ). After 5 min, the actin was mixed with a 5-fold molar excess of NHS-XX-Biotin (Merck KGaA, Darmstadt, Germany) and incubated in the dark for 15 h at 4 °C. Labeled F-actin was pelleted as above, and the pellet was rinsed briefly with G-buffer, then by homogenized with a dounce, and depolymerized by dialysis against G-buffer for 48 h at 4 °C. Biotinylated monomeric actin was purified further on an S200 (16/60) gel-filtration column as above. Aliquots of biotin-conjugated actin were snap frozen in liquid nitrogen and stored at −80 °C. To fluorescently label actin, G-actin was polymerized by dialyzing overnight against modified F-buffer (20 mM PIPES pH 6.9, 0.2 mM CaCl 2, 0.2 mM ATP, 100 mM KCl) 24 . F-actin was incubated for 2 h at room temperature with Alexa-488 NHS ester dye (Life Technologies) at a final molar concentration five times in excess of actin concentration. F-actin was then pelleted by centrifugation at 450,000 × g for 40 min at room temperature. The pellet was resuspended in G-buffer, and homogenized with a dounce, and incubated on ice for 2 h to depolymerize filaments. Actin was then re-polymerized on ice for 1 h after adding KCl and MgCl 2 (final concentration of 100 and 1 mM respectively). F-actin was pelleted by centrifugation for 40 min at 450,000 × g at 4 °C. The pellet was homogenized with a dounce and dialyzed overnight at 4 °C against 1 l of G-buffer. Next, the solution was centrifuged at 450,000 × g for 40 min at 4 °C. The supernatant was collected, and the concentration and labeling efficiency was determined by measuring the absorbance at 280 and 495 nm. Molar extinction coefficients used were as follows: ε 280 actin = 45,840 M −1 cm −1 , ε 495 Alexa-488 = 71,000 M −1 cm −1 and ε 280 AF488 = 7810 M −1 cm −1 . Purification and biotinylation of SNAP tagged CapZ SNAP-CapZ 41 was expressed in E. coli BL21 DE3 (Stratagene, La Jolla, CA) by growing cells to log phase at 37 °C in TB medium, then inducing expression using 1 mM IPTG at 18 °C overnight. Cells were harvested by centrifugation and pellets were stored at −80 °C. Frozen pellets were resuspended in lysis buffer (20 mM NaPO 4 pH 7.8, 300 mM NaCl, 1 mM DTT, 15 mM imidazole, 1 mM PMSF) supplemented with a protease inhibitor cocktail (0.5 µM each of pepstatin A, antipain, leupeptin, aprotinin, and chymostatin). Cells were lysed by sonication with a tip sonicator while keeping the tubes on ice. The lysate was cleared by centrifugation at 150,000 × g for 30 min at 4 °C. The supernatant was then flowed through a HisTrap column connected to a Fast Protein Liquid Chromatography (FPLC) system. The column with the bound protein was first extensively washed with the washing buffer (20 mM NaPO 4 pH 7.8, 300 mM NaCl, 1 mM DTT and 15 mM imidazole) to remove nonspecifically bound proteins. SNAP-CapZ was then eluted with a linear zero to 250 mM imidazole gradient in 20 mM NaPO 4 pH7.8, 300 mM NaCl, and 1 mM DTT. The eluted protein was concentrated and labeled with Benzylguanine-Biotin (New England Biolabs) according to the manufacturer’s instructions. Free biotin was removed using size-exclusion chromatography by loading the labeled protein on a Superose 6 gel-filtration column (GE Healthcare, Pittsburgh, PA) eluted with 20 mM HEPES pH 7.5, 150 mM KCl, 0.5 mM DTT. Fractions containing the protein were combined and concentration was determined by measuring the absorbance ( ε 280 = 102,165 M −1 cm −1 ). Purified protein was aliquoted, snap frozen in liquid N 2 and stored at ‐80 °C. Purification and labeling of ADF/Cofilin Wild-type yeast Cofilin (Cof1) was purified as follows 42 . The protein was expressed fused to a glutathione-S-transferase (GST) tag with a thrombin cleavage site and expressed in E. coli BL21 DE3. Cells were grown to log phase at 37 °C in TB medium, then induced with 1 mM IPTG at 18 °C overnight. Cells were harvested by centrifugation and pellets were stored at −80 °C. Frozen pellets were resuspended in lysis buffer (20 mM NaPO 4 pH 7.8, 300 mM NaCl, 1 mM DTT, 1 mM PMSF + protease inhibitors as described above). Cells were lysed by sonication with a tip sonicator while keeping the tubes on ice. The lysate was cleared by centrifugation at 150,000 × g for 30 min at 4 °C. The supernatant was then incubated with glutathione-agarose beads for 1 h on a rotator at 4 °C. The beads were first washed thoroughly with washing buffer (50 mM Tris pH 7.5, 150 mM NaCl and 1 mM DTT) to remove unbound protein and then incubated with thrombin (0.05 mg/ml) to cleave Cofilin from bead-bound GST. The cleaved protein was recovered by centrifugation. The supernatant containing the protein was concentrated and loaded on to a Superose 12 gel-filtration column (GE Healthcare, Pittsburgh, PA) pre-equilibrated with 10 mM Tris pH 7.5, 50 mM NaCl and 1 mM DTT. The fractions containing Cofilin were pooled, concentrated, snap frozen in liquid N 2 and stored at −80 °C. To prepare fluorescently labeled Cofilin, Cof1(T46C/C62A) 18 was purified as described above and dialyzed overnight against 10 mM Tris-HCl pH 7.5, 50 mM NaCl, and 0.2 mM Tris(2-carboxyethyl)phosphine (TCEP) at 4 o C. The dialyzed protein was then mixed with a 10-fold molar excess of Cy3-maleimide (GE Healthcare, Pittsburgh, PA) and incubated overnight in the dark at 4 °C. Free dye was removed using a PD-10 desalting column. The labelled protein was then aliquoted, snap frozen in liquid N 2 and stored at −80 °C. Human Cofilin1 and ADF were purified as follows 43 . The proteins were expressed in E. coli BL21 DE3 by growing cells to log phase at 37 °C in TB medium, then induced with 1 mM IPTG at 18 °C overnight. Cells were harvested by centrifugation and pellets were stored at −80 °C. Frozen pellets were resuspended in 20 mM Tris pH 8.0, 50 mM NaCl, 1 mM DTT, and protease inhibitors as described above. Cells were lysed by sonication with a tip sonicator while keeping the tubes on ice. The lysate was cleared by centrifugation at 150,000 × g for 30 min at 4 °C. The supernatant was loaded on a 1 ml HiTrap HP Q column (GE Healthcare, Pittsburgh, PA), and the flow-through was harvested and dialyzed against 20 mM HEPES pH 6.8, 25 mM NaCl, and 1 mM DTT. The dialyzed solution was then loaded on a 1 ml HiTrap SP FF column (GE Healthcare, Pittsburgh, PA) and eluted using a linear gradient of NaCl (20–500 mM). Fractions containing ADF/Cofilin were concentrated, dialyzed into 20 mM Tris pH 8.0, 50 mM KCl, and 1 mM DTT, snap frozen in liquid N 2 and stored at −80 °C. Purification and labeling of Srv2/CAP polypeptides His-tagged full-length S. cerevisiae Srv2, Srv2–90, N-terminal fragments N-Srv2, Srv2ΔCARP (Fig. 2a ) 23 , 44 and mouse N-CAP1 22 were expressed in E. coli BL21 DE3 by growing cells to log phase at 37 °C in TB medium. Cells were induced with 1 mM IPTG at 18 °C overnight. Cells were harvested by centrifugation and pellets were stored at −80 °C. Frozen pellets were resuspended in 50 mM NaPO 4 pH 8.0, 1 mM PMSF, 1 mM DTT, 20 mM imidazole, 300 mM NaCl and protease inhibitors as described above. Cells were lysed by sonication with a tip sonicator while keeping the tubes on ice. The lysate was cleared by centrifugation at 150,000 × g for 30 min at 4 °C. The lysate was loaded on a 1 ml HisTrap HP column (GE Healthcare, Pittsburgh, PA) and nonspecifically bound proteins were removed by washing the column with 20 mM NaPO 4 pH 8.0, 50 mM imidazole, 300 mM NaCl and 1 mM DTT. The bound protein was then eluted using a linear gradient of 50–250 mM imidazole in the same buffer. Fractions containing the protein were concentrated and dialyzed into 10 mM imidazole pH 8.0, 150 mM NaCl and 1 mM DTT. The protein was then aliquoted, snap frozen in liquid N 2 and stored at −80 °C. For fluorescent labeling of Srv2∆CARP, the same procedure as above was followed with the exception that 1 mM DTT in the elution buffer was replaced with 0.2 mM Tris(2-carboxyethyl)phosphine (TCEP) 45 . The eluted fractions were concentrated and incubated with at least fivefold molar excess of Cy5‐maleimide dye (GE Healthcare, Pittsburgh, PA) for 30 min at 25 °C and additionally for 14 h at 4 °C. The excess dye was then quenched by addition of 5 mM DTT. Free dye was then separated from labeled protein using a PD‐10 column with 10 mM imidazole pH 8, 50 mM KCl, 1 mM DTT and 5% glycerol. Labeled protein was concentrated. The concentration and labeling efficiency was determined by measuring the absorbance at 280 and 649 nm. Molar extinction coefficients used were as follows: ε 280 Srv2∆CARP = 38,390 (Mmonomers) −1 cm −1 , ε 649 Cy5 = 250,000 M −1 cm −1 and ε 280 Cy5 = 12,500 M −1 cm −1 . The protein was then aliquoted, snap frozen in liquid N 2 and stored at −80 °C. Since Srv2/CAP self-assembles into hexamers, Srv2/CAP concentrations given in this paper refer to the concentration of Srv2/CAP hexamers.

Purification of Twinfilin S. cerevisiae Twinfilin

Twf1 was expressed as a GST-fusion protein 13 in E. coli BL21 DE3 by growing cells to log phase at 37 °C in TB medium. Cells were induced with 0.4 mM IPTG at 18 °C overnight. Cells were harvested by centrifugation and pellets were stored at −80 °C. Frozen pellets were resuspended in 10 ml of PBS supplemented freshly with 0.5 mM DTT, 1 mM PMSF + protease inhibitors as described above. Cells were incubated with lysozyme (0.5 mg/ml) on ice for 15 min and then sonicated. The cell lysate was clarified by centrifugation at 12,500 × g for 20 min and incubated at 4 °C (rotating) for at least 2 h with 0.5 ml glutathione-agarose beads (Sigma-Aldrich, St. Louis, MO). Beads were washed three times in PBS supplemented with 1 M NaCl and then washed two times in PBS. Twinfilin was cleaved from GST by incubation with PreScission Protease (GE Healthcare, Marlborough, MA) overnight at 4 °C. Beads were pelleted, and the supernatant containing the protein was concentrated and then purified further by size-exclusion chromatography on a Superose 12 column (GE Healthcare) equilibrated in 20 mM HEPES pH 7.5, 1 mM EDTA, 50 mM KCl and 0.5 mM DTT. Peak fractions were pooled, aliquoted, snap frozen in liquid N 2 and stored at −80 °C.

Purification of Profilin Human

Profilin-1 was expressed in E. coli BL21 DE3 by growing cells to log phase at 37 °C in TB medium, then inducing expression using 1 mM IPTG at 37 °C for 3 h. Cells were harvested by centrifugation and pellets were stored at −80 °C. Cell pellets were resuspended in lysis buffer (50 mM Tris-HCl, pH 8.0, 1 mM EDTA, 0.2% Triton X-100, lysozyme + protease inhibitors as described above), kept on ice for 30 min, and then further lysed by sonication. Lysates were cleared for 25 min at 272,000 × g at 4 °C, and the supernatant was collected and loaded on a HiTrap Q column (Buffer: 20 mM Tris-HCl pH 8.0) followed by a Superdex 75 column equilibrated in 20 mM Tris-HCl, pH 8.0, 50 mM NaCl. Peak fractions were pooled, snap frozen in aliquots, and stored at −80 °C.

Microfluidics-assisted TIRF microscopy

Actin filament depolymerization was monitored by microfluidics-assisted Total Internal Reflection Microscopy (mf-TIRF) 12 , 24 , 46 . Coverslips were first cleaned by sonication in detergent for 60 min, followed by successive sonications in 1 M KOH and 1 M HCl for 20 min each and in ethanol for 60 min. Coverslips were then washed extensively with H 2 O and dried in an N 2 stream. The cleaned coverslips were coated with a 80% ethanol solution adjusted to pH 2.0 with HCl containing 2 mg/ml mPEG-silane, MW 2,000 and 2 μg/ml Biotin-PEG-silane, MW 3,400 (Laysan Bio Inc., Arab, AL) and incubated overnight at 70 °C. A 40 µm high PDMS mold with three inlets and one outlet was mechanically clamped onto a PEG-Silane coated coverslip. The chamber was then connected to Maesflow microfluidic flow-control system (Fluigent, France), rinsed with TIRF buffer (10 mM imidazole pH 7.4, 50 mM KCl, 1 mM MgCl 2 , 1 mM EGTA, 0.2 mM ATP, 10 mM DTT, 15 mM glucose, 20 μg/ml catalase, 100 μg/ml glucose oxidase) and incubated with 1% BSA and 10 µg/ml streptavidin in TIRF buffer for 5 min. CapZ was then anchored on the surface by flowing in 1 nM biotin-SNAP-CapZ for 5 min. Preformed actin filaments (10% Alexa-488 labeled) were then introduced and captured by anchored CapZ at their barbed ends with their distal pointed ends free in solution. All experiments were carried out at room temperature in TIRF buffer. Each experiment was repeated at least three times, and yielded similar results. Data from a single replicate are presented in the figures. Actin filaments were first aged to ADP-F-Actin under continuous flow for 15 min and then exposed to specific biochemical conditions (e.g. Cofilin and/or Srv2 in TIRF buffer). Single- and multi-wavelength time-lapse TIRF imaging were performed using a Nikon-Ti200 inverted microscope equipped with a 150 mW Ar-Laser (Mellot Griot, Carlsbad, CA), a ×60 TIRF-objective with a numerical aperture of 1.49 (Nikon Instruments Inc., New York, NY) and an EMCCD camera (Andor Ixon, Belfast, Northern Ireland). One pixel was equivalent to 143 × 143 nm. During measurements, optimal focus was maintained by the Perfect Focus system (Nikon Instruments Inc.). Images were corrected for background fluorescence using the Fiji 47 rolling ball background subtraction algorithm (ball radius 5 pixels), analyzed using the Fiji kymograph plugin, and the kymograph slopes were measured to determine the depolymerization rates of individual filaments. One actin subunit was taken to contribute 2.7 nm to the filament length.

Single-molecule imaging and analysis

Flow cells were formed by placing two PEG-silane biotin coated coverslips (as described above) orthogonally on top of each other with lines of silicone grease as a spacer. The flow cell was then rinsed with TIRF buffer and incubated with 1% BSA and 10 µg/ml streptavidin in TIRF buffer for 5 min. Actin filaments were anchored along their lengths were then elongated by introducing a solution containing 1 μM G-actin (10% Alexa-488 labeled and 0.5% biotinylated) and 4 μM Profilin. The flow cell was then rinsed with TIRF buffer to remove free monomers. These filaments were then exposed to a mixture of Cy5-Srv2ΔCARP and yeast Cof1 in TIRF buffer. The flow cell was excited by 488 nm (0.15 mW) and 633 nm (1 mW) lasers simultaneously and imaged on a micro-mirror TIRF microscope 48 by spectrally separating the emission with a 635 long-pass filter. Images were acquired continuously (65 or 100 ms integration time) using custom-written LabVIEW software (GLIMPSE; https://github.com/gelles-brandeis/Glimpse ) and captured by an EMCCD camera (Andor Ixon Ultra). One pixel was equivalent to 130 × 130 nm. For image processing, the pointed-end of an actin filament was tracked using a thresholding algorithm (Supplementary Movie 3 ) written in MATLAB. The integrated intensity in the Cy5-Srv2ΔCARP channel was determined in a 5 × 5 pixel box drawn at the location of the pointed end of the filament. The integrated intensity values were background corrected by subtracting the sum of 25 median pixel intensity values collected from the perimeter of a 19 pixel by 19 pixel square centered on the area of interest. The average intensity over time was smoothed using a moving window of 0.71 s (second-order polynomial, Savitzky-Golay filter) (Fig. 4d ; Supplementary figure 6 ). The residence time of an individual Cy5-Srv2ΔCARP molecule at the pointed end was determined from this intensity profile over time. Only residence times longer than four frames were counted as true Cy5-Srv2ΔCARP binding events (Fig. 4e ). Depolymerization rates during periods when Cy5-Srv2ΔCARP was present or absent at the pointed end were measured by a linear fit over measured filament lengths as a function of time during the bound or unbound duration (Fig. 4h ). The mean time between Srv2ΔCARP binding events at the filament ends was calculated 49 as 1 documentclass[12pt]{minimal} usepackage{amsmath} usepackage{wasysym} usepackage{amsfonts} usepackage{amssymb} usepackage{amsbsy} usepackage{mathrsfs} usepackage{upgreek} setlength{oddsidemargin}{-69pt} begin{document}$$m = fm^prime - frac{{1 - f}}{{k_{{mathrm{off}}}}},$$end{document} m = f m ′ − 1 − f k off , where m ′ is the observed mean time between consecutive Cy5-Srv2ΔCARP dwells at the actin filament pointed end, f is the fraction of Cy5-Srv2ΔCARP molecules with at least one dye as determined in the analysis of Fig. 4b , and k off is the dissociation rate constant of the Cy5-Srv2ΔCARP molecule from the filament end. The second-order association rate constant for Srv2ΔCARP binding to the filament end was then calculated as k on = 1 / ( mc ) where c was the concentration of Srv2ΔCARP in the experiment. Reporting summary Further information on research design is available in the Nature Research Reporting Summary linked to this article.

Supplementary information Supplementary Movie 1 Supplementary Movie 2 Supplementary Movie 3 Supplementary Information Peer Review File Reporting Summary Description of Additional Supplementary Files