Tissues and spermatogenic cells
Human testis tissue was obtained surgically from patients undergoing a testicular sperm extraction procedure (courtesy of Professor Dr. med. Wolfgang Schulze, Department of Andrology, University Hospital Hamburg-Eppendorf, Germany). Informed consent and Ethic Committee Approval was obtained (OB/X/2000), and the ethical principles for research involving human tissues as stated in the 52nd World Medical Association Declaration of Helsinki were strictly observed. For protein extraction, tissue samples were submerged immediately in a cryoprotectant and snap-frozen in liquid nitrogen.
All animal housing and operation practices were in compliance with German Animal Welfare laws, and the Guiding Principles in the Care und Use of Laboratory Animals (DHEW Publication, NIH, 80-23) were observed in all cases. For protein extraction, male Wistar rats were obtained from the UKE animal house. Animals were sacrificed at days 10, 15, 20, 22, 26, 28, 30, 45, and 60 (n = 10 per age group) by decapitation (5- and 10-days-old animals) or CO2 inhalation (all others). Control tissues were taken from 60-days-old animals. Tissues were snap-frozen in liquid nitrogen immediately after removal. For the preparation of germ cells, adult male Sprague Dawley-rats (n = 3) were obtained from Charles River Inc. Animals were killed by CO2-asphyxiation, the testes removed, transferred to 32°C PBS solution and decapsulated.
Polyclonal rabbit antisera against synthetic peptides were generated as described [20
]. 14- and 15-mer linear oligopeptides from PFN2 and PFN3, i.e. CAYSMAKYLRDSGF and CEVGVLTGPDRHTFL, respectively, were used as antigen (Bioscience, Göttingen, Germany; Pineda-Antibodies Service, Berlin, Germany). Other antibodies used included a rabbit polyclonal anti-PFN1 (Novus Biologicals, Littleton, USA), rabbit anti-PFN4 peptide antiserum [20
], monoclonal anti-α-tubulin DM1A (Sigma, Munich, Germany), monoclonal anti- actin JLA20 (Calbiochem, Schwalbach, Germany), rabbit anti-VASP [22
], goat anti-GST (GE Healthcare, Freiburg, Germany) and monoclonal anti-c-myc (BD Biosciences, Heidelberg, Germany). Peroxidase-conjugated secondary antibodies were AffiniPure Fc-fragment goat anti-rabbit IgG and AffiniPure rabbit anti-mouse IgG (H+L), respectively (both from Jackson ImmunoResearch, Newmarket, UK). Antibodies used for immunocytochemistry are indicated below.
Tissue samples were homogenized three times for 10 s with the UltraTurrax (Art Labortechnik, Mühlheim) in 10 mM Tris, 1 mM EDTA, 0.5 mM DTT, 50 mM NaCl, 0.4% NP-40, 0.2% NaDOC, 0.04% SDS, Protease-inhibitor-cocktail (complete mini EDTA-free, Roche), pH 7.8 (100 μl solubilisation buffer per 30 mg tissue). After 30 min of solubilisation at 4°C on a rotating wheel, debris was removed by centrifugation (30 min, 13000 rpm, 4°C) and the supernatants collected. Protein concentrations were estimated employing the BioRad Protein-Assay according to the suggestions of the supplier (BioRad, München, Germany).
In vitro translation and co-immunoprecipitation
C-myc-tagged profilins and HA-tagged β-actin were synthesized from pGBKT-7 and pGADT-7 plasmid constructs using the TNT®-T7 Quick Coupled Transcription, Translation System (Promega, Mannheim, Germany) according to manufacturer's instruction. 25 μl of β-actin in vitro translation reaction was co-incubated with 25 μl of profilin in vitro translation reaction at room temperature for 2 h. After incubation, 3 μl of anti c-myc-antibody (BD Biosciences) and 200 μl protein A-agarose (Roche Diagnostics, Penzberg, Germany) in PBS were added and the mixtures incubated overnight at 4°C. Protein A beads were then washed three times (10 s, 4°C, 7000 rpm) with 500 μl co-immunoprecipitation buffer containing 50 mM Tris (pH 8.0), 100 mM NaCl, 1 mM EDTA, 0.5% NP-40. Proteins were eluted at 95°C for 5 min in sample buffer (40 mM Tris, pH 6.8, 2% SDS, 100 mM DTT, 1 mM EDTA, 8% glycerol). After removal of protein A beads by centrifugation, 25 μl of protein supernatant were separated on a 4–12% gradient SDS-PAGE (see below), and profilins and actin visualized employing c-myc monoclonal antibody (BD Biosciences) and anti-HA-high affinity monoclonal antibody (clone 3F10, Sigma).
Western blot analysis
Western blot analysis of in vitro translated proteins and protein extracts was carried out by standard procedures. Briefly, approximately 80 μg proteins per lane were separated on 4–12% NuPage®Novex Bis-Tris gradient gels (Invitrogen, Karlsruhe, Germany) and transferred to polyvinylidene difluoride membranes (Amersham) in a discontinuous buffer system using a semi-dry blotter. Immunodetection was carried out by blocking for 1 h in 1% Western-blocking reagent (Boehringer Mannheim, Germany) or in 5% ECL-blocking agent when using the ECL-Plus-system (GE Healthcare, Freiburg, Germany), followed by incubation with the first antibody over night at 4°C. Antibody dilutions were 1:1000 for anti-PFN1, 1:10000 for anti-PFN2, 1:700 for anti-PFN3 and 1:500 for anti-PFN4. Antibody binding was detected either by Cy5-conjugated AffiniPure goat anti-rabbit IgG or a peroxidase-conjugated AffiniPure Goat anti-rabbit IgG (both from Jackson Immuno Research, Newmarket, UK).
Profilin expression in E. coli and PLP affinity chromatography
2xYT with kanamycin was inoculated 1:50 from a fresh ON culture of BL21(DE3)pLys cells harbouring the mouse PFN3 or -4 gene under control of the bacteriophage T7-promoter in the vector pET28a(+). At an OD600 of 0.5, protein expression was initiated by adding IPTG to a final concentration of 1 mM. After induction, the bacteria were grown for 4 h at 42°C. Then, the cells were pelleted by centrifugation (15 min at 6000 rpm, 4°C), resuspended in 25 ml of lysis buffer (50 mM Tris-HCl, 10 mM NaCl, 10 mM EDTA, 1.5% TritonX-100, 1:1000 Trasylol, 1 μM Pepstatin A, 50 μM Pefabloc SC) and incubated for 20 min on ice. After adding lysozyme, the solution was frozen overnight at -80°C. Next day, the solution was thawed at 37°C and then sonicated on ice 10 times 30 s at 80 W probe energy with 30-s intervals. The lysate was centrifuged at 4°C for 50 min at 14000 rpm. The supernatant was loaded onto a poly-L-proline column, washed and equilibrated with washing buffer (20 mM Tris-HCl, 150 mM NaCl). The column was washed with 5–10 column volumes of washing buffer and then with washing buffer including 2 M urea, to remove unbound protein. To analyze the binding affinity, profilins were then eluted with 4 M and 8 M elution buffer (4 M/8 M Urea in washing buffer). Fractions were collected and checked by standard SDS-PAGE.
Yeast Two-Hybrid Interaction
Yeast strains AH109 and Y187 and pGBKT7 and pGADT7 plasmids were from the Matchmaker Two-Hybrid System
3 (Clontech), providing HIS3, ADE2 and lacZ reporters and allowing high stringency assays. For bait and prey construction from human and mouse testes cDNAs, oligodeoxynucleotide primers as given in table were employed in RT-PCR amplification and amplicons subcloned into the multiple cloning site of pGBKT7 and pGADT7 vectors. The coding region of the human VASP cDNA was likewise subcloned into pGADT7 and pGBKT7. The yeast strains were transformed with the constructs ([48
]; Quick and Easy Transformation Protocol
) and colonies grown according to the Yeast Protocols Handbook
(Clontech, Heidelberg, 2001). Plasmid selection was maintained by growing cells in minimal medium (0.67% yeast nitrogen base, 2% glucose) supplemented with lysine, histidine, adenine and tryptophan (for pGADT7 selection) or leucine (for pGBKT7 selection). Mating tests were performed under conditions of increasing stringency according to the manufacturer's suggestions. Diploid colonies were replica-plated on minimal medium with high stringency and grown at 30°C for 4–8 days. Colonies were isolated and tested for the expression of the lacZ reporter using the β-Galactosidase Assay Kit
(Pierce). Prey plasmids were isolated, transformed into Escherichia coli
, and inserts verified by sequence analysis (MWG). Interactions were verified by plate growth assays on minimal mediums in the absence of histidine or adenine or both.
Actin polymerization assay
Muscle actin was purified from rabbit skeletal muscle as described [49
] and labelled with pyrene according to Kouyama and Mihashi [29
]. Recombinant profilins were expressed as glutathion-S transferase (GST) fusion proteins in Escherichia coli
ER2566 (New England Biolabs, Heidelberg, Germany) and purified by glutathione sepharose affinity chromatography according to the manufacturer's instructions (GE Healthcare). Eluted profilins were dialysed against 20 mM Tris-Cl, pH 7.4, 0.2 mM CaCl2
, 1 mM dithiothreitol and stored on ice. To determine their influence on actin polymerization, 5 μM α-actin (5% pyrene-labelled) in G-buffer (2 mM Tris-HCl, pH 7.5, 0.2 mM ATP, 0.1 mM CaCl2
, 0.5 mM DTT) was incubated for 10 min at 20°C with or without 15 μM recombinant GST-fused profilins. Polymerization was initiated by the addition of MgCl2
and KCl (final concentration 1 mM and 50 mM, respectively). Fluorescence was monitored for 2 h at 366 nm excitation and 407 nm emission using a LS50B fluorimeter (Perkin Elmer, Langen, Germany).
Protein-lipid overlay assay
PIP Strips™ membranes (Molecular Probes, Eugene, USA) were employed following the manufacturer's instructions. Briefly, after blocking with 3% bovine serum albumin (BSA) in TBS-T (10 mM Tris-Cl, ph 8.0, 150 mM NaCl, 0.1% (v/v) Tween 20) the lipid-containing membranes were incubated with 0.5 μg/ml GST fused profilins for 2.5 h. Membranes were washed three times with TBS-T + 3% BSA and the bound proteins detected by anti-GST antibody in conjunction with HRP-labelled secondary antibody and enhanced chemiluminescence.
Spermatogenic cells were collected from mechanically dissociated seminiferous tubular fragments (identified with a dissecting stereomicroscope as corresponding to stages I-XIV of rat spermatogenesis according to their transillumination pattern). Cells were placed in a drop of 3.7% paraformaldehyde (electron microscopy grade) in 0.1 M sucrose in phosphate buffer, pH 7.4, on microscope slides coated with Vectabond (Vector Laboratories, Burlingame, CA). This fixation procedure results in the preservation of the Golgi-acrosome-acroplaxome-manchette-nuclear relationship in spermatids, a condition that facilitates structure identification and access of antigenic probes. After 15-min fixation at room temperatures a coverglass was placed on top of the preparation. The glass coverslip was removed and the microscope slide- containing fixed spermatogenic cells was used for immunocytochemistry (see below). Cells were immunoreacted with affinity purified PFN3 and PFN4 (working dilution: 1:200) and α-tubulin monoclonal antibody (working dilution 1:100; Sigma-Aldrich, St. Louis, MO), followed by anti-rabbit IgG-conjugated with fluorescein isothiocyanate or anti-mouse IgG conjugated with rhodamine (working dilution 1:200; Jackson Immunoresearch Laboratories, West Grove, PA), respectively. Phalloidin-Texas Red-X was used to detect F-actin according to the manufacturer's protocol (Molecular Probes, Eugene, OR). Specimens were mounted with Vectashield (Vector Laboratories) and examined in a Zeiss Universal phase-contrast/fluorescence microscope equipped with episcopic illumination. Images were recorded using a Magnafire digital CCD camera (Optronics, Goleta CA).
Generation of 3-dimensional models
Homology models for both PFN3 and PFN4 from mouse and man were generated using the SWISS-MODEL server [50
], based on the closest sequence homologues found from the PDB for each protein. For PFN3
, the model is based on bovine profilin 1 (PDB entry 1PNE
], and for PFN4, the template was Acanthamoeba profilin II (PDB entry 2ACG
]. The structures were superimposed with each other and profilins 1 and 2 using the SSM method [52
] in Coot [53
]. Protein structure figures were generated using Pymol, Dino, and POV-Ray.