Cysteine-rich protein 1 (CRP1) regulates actin filament bundling.

Proteins and DNA constructs α-Actinin was purified from chicken gizzard as previously described [ 27 ]. Non-muscle actin (99% pure; 80% β-actin, 20% γ-actin) was polymerized following the manufacturer's protocol (Cytoskeleton, Inc., Denver, CO). The plasmid containing chicken CRP1 cloned into the EcoRI site of pBlueScript II KS (pBSIIKS, Stratagene) was generously provided by Mary C. Beckerle (Univ. of Utah) [ 15 ]. The EcoRI insert of CRP1-pBSIIKS was sub-cloned into the enhanced cyan fluorescent fusion protein vector pECFP-C1 (BD Biosciences) and the BamHI-HindIII insert sub-cloned into pProEx HTb (Invitrogen). Nucleotide sequences were confirmed by sequence analysis. His-tagged CRP1 protein was expressed in BL21 bacteria and purified using Ni-NTA resin (Qiagen) following procedures described by the manufacturer. The his tag was cleaved from CRP1 while still bound to the resin using recombinant TEV protease (Invitrogen) following the manufacturer's protocol. The untagged CRP1 protein was concentrated and buffer-exchanged (10 mM HEPES, pH 7.0, 50 mM NaCl, 1 mM EDTA) in a centrifugal filter device (Amicon). YFP-α-actinin was constructed by subcloning the α-actinin gene [ 28 ] into the HindIII restriction site of the enhanced yellow fluorescent fusion protein vector pEYFP-N1 (BD Biosciences).

F-actin bundling assays

The bundling of F-actin was determined by sedimentation assays as previously described [ 28 , 29 ]. F-actin (10.4 μM) was incubated with the indicated concentration of CRP1 or α-actinin in bundling buffer (10 mM HEPES, pH 7.0, 50 mM NaCl, 1 mM EDTA) for 30 min at room temperature and centrifuged at 10,000 × g for 30 min. The supernatant and pellet were separated and the proteins analyzed by electrophoresis. Proteins were detected by Gelcode Blue (Pierce) staining and quantified using a KODAK ImageStation 440CF. F-actin bundles were visualized by fluorescence microscopy following modification of previously described procedures [ 30 , 31 ]. Briefly, 50 μl of assay solution was incubated on a glass coverslip inside a 12-well tissue culture dish. After 30 min, the proteins were fixed by adding 3% formaldehyde in phosphate buffered saline for an additional 30 min. Coverslips were then stained and processed for microscopy as described previously[ 25 ].

Cell culture and fluorescence microscopy Rat embryonic fibroblasts

(REFs) were cultured as described previously [ 25 ]. Cells were transfected with pECFP-CRP1 and pEYFP-α-actinin using FuGENE 6 (Roche) following manufacturer's protocols. The expression curve of CFP-CRP1 was carried out by varying the ratio of FuGENE to DNA in a final volume of 100 μL serum-free media. The transfection conditions were: 6 μL FuGENE, no DNA; 3 μL FuGENE, 0.5 μg DNA; 3 μL FuGENE, 1.0 μg DNA; 6 μL FuGENE, 1.0 μg DNA; and 6 μL FuGENE, 2.0 μg DNA. Twenty-four hours after transfection, cells were prepared for fluorescence microscopy or scraped into ice-cold lysis buffer (10 mM Tris, pH 7.4, 150 mM NaCl, 1 mM EGTA, 1 mM EDTA, 2 mM Na 3 VO 4 , 1% Triton X-100, 0.5% NP-40, 30 mM sodium pyrophosphate, 50 mM NaF, 1 μg/ml leupeptin, and 1 μg/ml aprotinin) as described previously [ 25 ]. The lysates were centrifuged at 10,000 × g for 10 min at 4°C, protein from the supernatant and pellet separated by electrophoresis, and immunoblotted with anti-GFP (Santa Cruz), anti-α-actinin (Chemicon), or anti-actin (Sigma). Proteins were detected by enhanced chemiluminence (Pierce) and quantified using a KODAK ImageStation 440CF. For fluorescence microscopy, cells were fixed for 30 min at room temperature with 3% formaldehyde (Tousimis) in PBS or in Triton X-100 buffer (20 mM Tris, pH 7.4, 50 mM NaCl, 1 mM EGTA, 5 mM EDTA, 100 μM Na 3 VO 4 , 50 mM sodium pyrophosphate, 1 μg/mL leupeptin, 1 μg/mL aprotinin, and 0.5 % Triton X-100). Digital images were captured using a Zeiss axiovert 100S microscope equipped with a Photometrics CoolSNAP HQ CCD camera controlled by MetaMorph software. Co-localization studies were carried out using a Zeiss LSM 510 confocal microscope. The scatter plots and correlation coefficients were determined using Zeiss Physiology Software v3.2.

Fluorescence resonance energy transfer

(FRET) microscopy REFs were co-transfected with pECFP-CRP1 and pEYFP-α-actinin, cultured for an additional 24 hrs, fixed using 3% formaldehyde in Triton X-100 buffer, and prepared for confocal microscopy as described above. The FRET assays were carried out following the procedure described by Karpova et al. [ 23 ]. Briefly, cells were imaged with a Zeiss LSM 510 confocal microscope operated by Zeiss Physiology Software v3.2 using a 63 × 1.3 NA Zeiss oil immersion lens. The microscopy system was configured in multitracking mode to excite the CFP with a 458 nm and YFP with a 514 nm laser line. A region of interest (ROI) containing actin stress fibers was selected for photobleaching. Using the time series function, 5 images of the cell were collected followed by selective photobleaching of the YFP within the ROI with the 514 nm laser line (typically, 150 iterations at 100% laser power was sufficient), and then the collection of 5 additional images. The FRET efficiency was calculated as a percentage using the following formula E = 100 × (I postbleach – I prebleach )/I prebleach , where I is the intensity of CFP fluorescence within the ROI. As a control, ROIs were selected from non-bleached regions of the cell.

Supplementary Material Additional File 1 Immunostaining for endogenous CRP1. Fluorescence microscopy of REFs stained with antibodies recognizing the C-terminal 17 amino acids residues (PKGFGFGQGAGALVHSE) of rat CRP1. Bar = 10 μm. Click here for file Additional File 2 Immunostaining for endogenous CRP1. Fluorescence microscopy of REFs stained with antibodies recognizing the C-terminal 17 amino acids residues (PKGFGFGQGAGALVHSE) of rat CRP1. Bar = 10 μm. Click here for file