Basic Characterization of Plant Actin Depolymerizing Factors: A Simplied, Streamlined Guide

Actin depolymerizing factors (ADFs) are small monomeric actin-binding proteins that alter the oligomeric state of cellular actin. Members of the ADF family can bind both the G-actin and F-actin in plants, and their functions are regulated by cellular pH, ionic strength and availability of other binding partners. Actin depolymerization activity is reportedly essential for plant viability. By binding to the ADP-bound form of actin, ADFs severe actin laments and thereby provide more barbed lament ends for polymerization. They also increase the rate of dissociation of F-actin monomer by changing the helical twist of the actin lament. These two activities together make ADF the major regulator of actin dynamics in plant cell. Therefore, it is essential to measure the binding and depolymerization activity of the plant ADFs. Here, we present a simplied, streamlined step-by-step protocol to quickly measure these important functions of the ADF proteins in vitro.


Introduction
Actin depolymerizing factors (ADFs) represent a large family of small monomeric proteins and their enzymatic activity includes actin binding and subsequent depolymerization or severing of F-actin or actin bundles. Different isoforms of ADF may show different levels of enzymatic activity and such difference may dictate their diverse physiological roles. It is therefore useful to envision a relatively quick and streamlined protocol to screen the functionality of large ADF protein families in plants or alternatively a broader biochemical characterization of ADF homologues across the kingdom. In this protocol, we describe the basic principles, materials and detailed procedures for assessment of the actin binding and depolymerizing activity of the recombinant ADF proteins in vitro. We also describe a quick protocol for single actin lament visualization and live documentation of ADF functionality with respect to lament organization and depolymerization. These assays together constitute a basic yet comprehensive protocol for characterization of ADF proteins.

Reagents (A) Kits used
For cloning of the genes: Champion pET200 directional TOPO Expression kit Champion pET200 directional TOPO Expression kit (Thermo Fisher Scienti c, USA) was used for gateway-based cloning of the cDNAs from donor plasmid containing the cDNAs encoding ADFs. The kit includes linearized topoisomerase I-activated Champion pET expression vector, carrying an N-terminal His-tag and buffer/salt solution. A proofreading enzyme such as Biorad iProof high delity DNA polymerase (Biorad Inc, USA) is used for PCR to generate a blunt end PCR product with minimum error.
For protein puri cation: QIAGEN N-NTA spin columns The Ni-NTA resin from QIAGEN, USA is a standard a nity puri cation protocol for one-step puri cation of His-tagged proteins. Depending on the extent of overexpression, the puri ed proteins may attain 60-90% homogeneity. Ni-NTA spin columns provide a convenient microspin set-up for simultaneous processing of multiple samples. 10 mM imidazole -0.68 g imidazole (MW 68.08 g/mol) 100 mM NaH2PO4 -13.80 g NaH2PO4·H2O (MW 137.99 g/mol) 100 mM Tris-Cl -12.10 g Tris base (MW 121.1 g/mol) Adjust pH to 6.3 using 1N HCl and lter sterilize (0.2 or 0.45 µm).

SDS-PAGE and western blot buffers
Construction of recombinant plant expression vectors 1. CLone ADF cDNAs in pET200 prokaryotic expression vector carrying a N-terminal His-tag. Alternatively, the CDNAs can be cloned in any bacterial expression vector carrying a C-terminal or N-terminal a nity tag suitable for puri cation using standard cloning procedure.
2. Transform the ADF expression constructs into E.coli BL21 (DE3) cells under Ampicillin selection and con rm positive clones by PCR ampli cation.
2. Add IPTG (100 mL from 1M stock) at a nal concentration of 1mM and grow for an additional 4 hours under same conditions to induce the recombinant protein expression.

Harvest the cells by centrifugation (5000 rpm) at 4 °C for 15 minutes and wash in sonication buffer.
Sonicate the cells in 20 mM Tris-Cl, pH 8.0 supplemented with PMSF and β-ME at the pulse of 30 seconds each of 35% amplitude until the lysate is clear. To avoid foaming and protein degradation, sonication over ice in glass vials is recommended for small proteins. Addition of protease inhibitor cocktail can also increase protein recovery.
4. Separate the soluble fraction from membrane fraction by centrifugation (12,000 rpm) at 4 °C for 15 minutes. Collect the supernatant and pellet separately. 4. Con rm the expression of protein with the mouse monoclonal anti-His antibody in a 1:2000 dilution and a HRP-conjugated anti-mouse secondary antibody in a 1:10000 dilution by standard western blotting procedures (we used an ECL chemiluminescence kit (Pierce, USA) following manufacturer's instruction).
6. Majority of the proteins are expressed in the membrane fraction (as observed from the western blots in our experiments). It may not be the case for all ADFs, but if more protein is observed in the membrane fraction, native puri cation protocol should be followed as per manufacturer's instruction. The buffer compositions for both native and denaturing conditions are described in the reagent section.
7. Solubilize the membrane fraction in 8 M Urea, 100 mM NaH 2 PO 4 and 100 mM Tris-Cl (pH 8.0) and purify with Ni-NTA resin columns (QIAGEN, USA). Improved protein elution is obtained in acidic condition and addition of 200-250 mM imidazole. About 70-80% protein homogeneity can be obtained.
9. Check the concentration of protein using Bradford Assay. Brie y, 10µL of each standard or unknown sample is added to 390µL of the Coomassie Plus Reagent (Pierce, USA), mixed and incubated for 10 minutes at RT. Measure the absorbance at or near 595 nm with water as blank. Prepare a standard curve by plotting the average blank-corrected 595 nm measurement for each BSA standard vs its concentration in µg/mL. This standard curve is used to determine the protein concentration of each unknown sample.

Actin preparation
Human platelet G-actin (85% beta and 15% gamma iso-forms) can be obtained from Cytoskeleton Inc, USA. Non-muscle actin has an approximate molecular weight of 43 kDa. The protein is provided as a lyophilized white powder, which is stable for 6 months when stored desiccated to <10% humidity at 4 °C. It will then be in the following buffer: Actin polymerization 1. Add 25µL sterile distilled water to a 250 µg supplied lyophilized aliquot to make a 10 mg/mL solution.
Store the actin in 5 mM Tris-HCl pH 8.0, 0.2 mM CaCl 2 , 0.2 mM ATP, 5% sucrose, and 1% dextran. Dilute High and low speed co-sedimentation with actin 1.Incubate the ADF proteins with bundled/polymerized actin in binding buffer at RT for 2-3 h, and then centrifuge at ~40,000 g at 4 ºC for 1 h (low-speed) or 100,000g for 20 min (high-speed).
2. Remover the supernatant carefully so as to not disturb the pellet. Dissolve the pellet in actin binding buffer and precipitate with acetone.
3. Suspend the pellet in Laemmli sample buffer and run in a 12% SDS-PAGE.
4. Measure the protein quantity in-gel by assessing band density using imageJ and imagesoft lite software (NIH, LICOR). · Puri cation may yield less protein due to the high membrane localization tendency of ADF proteins. This issue can be resolved by adding 10 mM Imidazole in the bacterial growth medium, which showed a better puri cation pro le in our experiments.

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Do not freeze-thaw the pyrene-labeled actin to avoid/minimize variation between replicates.

Time Taken
Time required to complete the entire protocol is around 2 weeks contingent upon the success of each step.

Anticipated Results
Plant ADFs express in a low amount in bacterial cells, and high expression often results in aggregation of cells. For a very active ADF protein, actin binding should start at low concentrations of input proteins as we found in some of the isoforms we used. The gel-based quantitation of actin sedimentation is, however, not always accurate and it is important to support the results with uorescence-tagged actin.
Once the basic information of the activity pattern of all ADF members is obtained, it is suggested to test them across larger pH range and in presence or absence of inhibitors and co-factors. Depolymerization assay should always accompany co-sedimentation. Lastly, for an active ADF protein, polymerized actin laments (F-actin; 2-8 μm length) should start depolymerizing immediately after addition of protein; both severing and depolymerization from pointed end can be observed very de nitively under live imaging.