Specimens of the collembolan species Isotomurus maculatus (Arthropoda, Schäffer 1986) were collected in the neighbourhood of Siena and dissected in phosphate buffered saline (PBS; 171 mM NaCl, 6 mM Na phosphate, 3 mM KCl, 2 mM EDTA, 2 mM EGTA, pH 7.4). Midguts were then processed for electron microscopy, protein analysis, RNA extraction and immunomicroscopy as described below.
Midgut cytoskeletal insoluble fractions were obtained following the procedure described by Pruss et al. . Briefly, 300-500 midguts were homogenized in a volume of 1.5 mL of PBS added with a cocktail of protease inhibitors (P2714, Sigma, NY, USA) and centrifuged at 10,000 g for 10 min at 4°C. The sediment was then extracted in 1.5 mL of PBS containing 0.6 M KCl and 0.5% Triton X-100 and centrifuged as above. The resulting pellet was washed twice in PBS and then either fixed for electron microscopy analysis or denatured for successive sodium dodecyl sulphate (SDS)-acrylamide gel electrophoresis.
Gel electrophoresis and immunoblot
Electrophoretic analysis of cytoskeletal fractions was carried out on 8% or 12% SDS-polyacrylamide gels according to the method used by Laemmli . Gels were either stained using standard Coomassie blue staining procedures or transferred onto nitrocellulose for subsequent immunoblot analysis.
Electrophoretic transfer of proteins onto nitrocellulose was performed as described by Towbin et al. , using a modified transfer buffer, consisting of 50 mM Tris, 38 mM glycine and 5% methanol. Transferred protein bands were immunostained using the affinity-purified anti-isomin polyclonal antibody (see below) and a peroxidase-labelled anti-rabbit secondary antibody (Cappel, PA, USA). Blots were then processed for ECL detection (GE Healthcare, NJ, USA), with exposure times from 5 s to 5 min.
Protein analysis by mass spectrometry (LC-ESI-MS/MS)
Analysis by mass spectrometry (LC-ESI-MS/MS) of the main tryptic fragments obtained by digestion of the isomin band was performed by the Swiss-2D Service (Geneva, Switzerland); the obtained peptide aminoacid sequences are listed in Additional File 1, Table S1.
RNA preparation and RT-PCR
Total RNA was isolated from adults of I. maculatus frozen in liquid nitrogen and homogenized using a Polytron homogenizer (Kinematica AG, Littau, Switzerland) according to the method described by Chomczynski and Sacchi . RNAs (1 μg) were converted to cDNAs with an oligo(dT)18 primer and SuperScript™ II Reverse Transcriptase (Invitrogen, CA, USA) according to manufacturer's instructions. Forward and reverse degenerate primers were designed on the basis of three amino acid sequences obtained by mass spectrometry: (B1-B2) YEAEAAK, (C1-C2) ESTGDAE and (D1-D2) FGHADQEK. Their sequences are listed in Additional File 1, Table S1. A partial 360 bp fragment of the isomin protein was amplified by RT-PCR using the combination B2/D1 of degenerate primers. Reactions were performed using Expand High Fidelity PCR System (Roche, Basel, Switzerland) under the following conditions: 95°C for 10 min; cycles 1-5: 94°C for 1 min, 35°C for 1 min, 72°C for 1 min; cycles 6-46: 94°C for 1 min, 54°C for 1 min, 72°C for 1 min and 72°C for 10 min. PCR products were purified using Wizard® SV Gel and PCR Clean-Up System (Promega, WI, USA), cloned into a TA cloning vector (Invitrogen) and sequenced using a CEQ 8000XL automated DNA Analysis System (Beckman Coulter, CA, USA) on both strands. The 360 bp deduced amino acid sequence showed the complete sequences used to design B2 and D1 degenerate primers plus an additional one corresponding to the mass spectrometry fragment 5 (Additional File 1, Table S1).
Cloning of full length cDNA by rapid amplification of the complementary DNA ends (RACE)
In order to obtain the full-length cDNA for isomin, a 5' and 3' RACE was performed on I. maculatus total RNA using a GeneRacer™ Kit (Invitrogen) and following manufacturer's instructions. Isomin specific primers - D1_FEcoRI, Iso112_for (3' nested primer; Additional File 1, Table S1) used in 3' amplification, B2_RBamHI and iso577_rev (5' nested primer; Additional File 1, Table S1) used in 5' amplification - were designed based on the partial 360 bp sequence mentioned above. Both nested PCR reactions gave a single product of ≈1200 bp and ≈ 500 bp, respectively. The 5' and 3' RACE products were purified, cloned and sequenced as described above. The complete isomin cDNA sequence has been deposited in GenBank under accession number FJ264504.
Homology searches for nucleotide and amino acid sequences were performed using the BLAST suite of programs (http://blast.ncbi.nlm.nih.gov/Blast.cgi). Programs employed for the prediction of molecular domains implicated in coiled coils formation were Coils (http://www.ch.embnet.org/software/COILS_form.html) and Marcoil (http://bioinf.wehi.edu.au/folders/mauro/Marcoil/index.html); sequence alignments were obtained using ClustalW (http://www.ebi.ac.uk/Tools/clustalw2/index.html). Prediction of phosphorylated sites and of sumoylated residues was performed using NetPhos 2.0 (http://www.cbs.dtu.dk/services/NetPhos/) and SUMOsp 2.0 (http://sumosp.biocuckoo.org/prediction.php), respectively. Distance analysis of protostome cytoplasmic IF sequences was carried out using the Protdist and Neighbor programs of the Phylip package (JTT matrix, 100 bootstrap replicates) available at the Pasteur Institute web server (http://mobyle.pasteur.fr/cgi-bin/portal.py).
Bacterial expression of recombinant protein
For the preparation of the glutathione S-transferase (GST) fusion constructs, either a portion (360 bp, coding the protein region from Phe11 to Lys131) or the whole isomin protein coding region (1236 bp) were amplified by PCR. Oligonucleotide pairs containing sites for in-frame directional cloning in the pGEX-6P-2 vector (GE Healthcare, NJ, USA) were: D1_FBamHI/B2_RSmaI for the 360 bp fragment and isoml_BamHI/isoml_SmaI for the 1236 bp one (Additional File 1, Table S1). PCR cycling, for the short fragment amplification, included an initial denaturation at 94°C for 5 min followed by 40 cycles of 94°C for 1 min, 52°C for 1 min, 72°C for 1 min and a final extension step at 72°C for 10 min. The 1236 bp region was amplified under the same PCR conditions but with an annealing temperature of 56°C. The PCR products were cloned into the BamHI and SmaI sites of the bacterial expression vector pGEX-6P-2 (GE Healthcare, NJ, USA) in frame with the upstream gene GST. Expression of the fusion protein was induced in E. coli by addition of Isopropyl-β-D-thiogalactopyranoside (IPTG; see below).
Purification of the fusion protein and production of specific antibodies
Recombinant bacterial strains expressing the fusion protein - either the whole isomin molecule or its 120 aa fragment from Phe11 to Lys131 (see Additional File 2, Figure S1) - were grown at 37°C in 400 mL of Luria-Bertani (LB) medium added with 50 μg/mL of ampicillin, until an OD600 of about 0.6-0.8 is achieved. After the addition of IPTG to a final concentration of 0.1 mM, the culture was incubated at 37°C for 2 h under shaking. Cells were harvested by centrifugation at 3500 g for 10 min, resuspended by vortexing in 25 mL of B-PER™ reagent (Pierce) and shaken at room temperature for 10 min; soluble proteins were separated by insoluble material by centrifugation at 27000 g for 15 min.
Most part of the fusion protein was contained in inclusion bodies. Only in the case of the 120 aa fragment it was possible to recover an amount of soluble protein sufficient for the subsequent purification by affinity chromatography on Gluthatione Sepharose 4B (GE Healthcare, NJ, USA). This procedure was carried out according to manufacturer's instructions. Briefly, the recovered soluble fraction was added with 1 mL of a 50% slurry of resin and incubated for 1 h with gentle agitation. Resin was then collected by centrifugation at 500 g for 5 min and washed twice in PBS. Protein bound to the resin was eluted with 10 mM glutathione in 50 mM Tris-HCl pH 8.0, dialyzed against 0.9% NaCl and used for rabbit immunization following standard procedures. The resulting polyclonal antibodies were blot-affinity purified according to Tang (36), using nitrocellulose membrane fragments containing the isomin 40 kDa protein band.
Isomin in vitroreassembly
Recombinant isomin was essentially recovered in the insoluble inclusion bodies. For their purification, the insoluble pelleted material obtained from a 50 mL-culture after cell lysis was resuspended by vortexing in 5 mL B-PER™, added with 200 μg/mL lysozime and incubated at room temperature for 5 min. The suspension was then added with 15 mL of B-PER™ diluted 1:10 in 20 mM Tris pH 7.5 and centrifuged for 15 min at 27000 g. Pelleted inclusion bodies were washed twice in B-PER™ diluted 1:10 in 20 mM Tris pH 7.5, then twice in 2% TRITON 10 mM EDTA. The final pellet essentially consisted of a band of about 66 kDa, corresponding to the recombinant protein (Additional File 5, Figure S4A). For cleavage of the GST moiety, pelleted inclusion bodies were thoroughly resuspended at about 75 μg/mL in a buffer consisting of 50 mM Tris-HCl, 150 mM NaCl, 1 mM EDTA, 1 mM DTT, pH 7.0, added with 10 U of PreScission Protease (GE Healthcare, NJ, USA) and incubated at 5°C for 20 h. After centrifugation at 8000 g for 20 min, both supernatant and sediment fractions were analysed by SDS-page (Additional File 5, Figure S4B); most part of the recombinant protein was shown to be cleaved and still to be contained in the insoluble fraction, while the GST protein was recovered in the soluble fraction.
The insoluble pellet recovered after cleavage was dissolved in 10 mM Tris, 9.5 M urea, pH 7.0 and incubated for 1 h at room temperature. After centrifugation for 1 h at 150000 g, analysis by SDS-page of the soluble and insoluble fractions revealed that urea treatment solubilised about 50% of the isomin protein (Additional File 5, Figure S4C). As described by Herrmann et al. , urea-solubilized protein was then dialyzed against a series of solutions (1 h each) containing decreasing concentrations (8 M, 6 M, 4 M, 2 M) of urea dissolved in 5 mM Tris, 0.1 mM EGTA, 1 mM EDTA, 1 mM DTT, pH8.4 and, finally, against the same buffer containing no urea. After dialysis, the sample was added with an appropriate volume of 10× reassembly buffer (0.2 M Tris, 0.5 M NaCl, pH 7.0), incubated for 1 h at room temperature and then centrifuged at 150000 g for 1 h. Sediment and supernatant were analysed by both SDS-page and negative staining.
For immunofluorescence microscopy, midguts were fixed in absolute ethanol for 1 h at 4°C and then paraffin-embedded. After rehydration, sections were treated with 1% NaBH4 for 30 min to quench autofluorescence and washed twice for 5 min with PBS. Successively, sections were incubated for 1 h in PBS containing 3% bovine serum albumine and 0.5% Tween-20, and for 2-4 h in the affinity-purified primary antibody. After two 10-min washes in PBS containing 0.1% Tween-20, sections were incubated for 1 h in the secondary antibody (fluorescein-conjugated anti-rabbit antibodies, Cappel), washed again in PBS-T and finally mounted in 90% glycerol.
Whole midguts or midgut TRITON-resistant fractions were fixed with glutaraldehyde and tannic acid, post fixed with uranyl acetate and embedded in an Epon/Araldite mixture as previously described in . Before observation by TEM, thin sections were routinely stained with uranyl acetate and lead citrate.
For post-embedding electron microscopy immunolocalization, whole midguts were fixed at 4°C for 2 h in 0.1 M phosphate buffer (PB) pH 7.2, containing 0.2% glutaraldehyde and 2% p-formaldehyde, rinsed overnight in PB, dehydrated at 4°C in a graded ethanol series and embedded in Lowicryl K4M as described in . For immunogold staining, sections were saturated with 3% bovine serum albumin (BSA), 15% normal serum in PBS (2 h), treated with 20 mM glycine in PBS (20 min), and then incubated overnight at 4°C in the primary antibody diluted in PBS containing 0.2% BSA. After one 10-min wash with PBS containing 0.5% Tween 20 and five 10-min washes in PBS, sections were incubated for 1 h at room temperature in the secondary antibody (GAM IgG-G10, Biocell, Cardiff, Wales, UK). Grids were then washed 5 × 10 min in PBS and 5 × 10 min in distilled water, then counterstained as above reported. All the ultrathin sections were observed at a Philips CM10 EM operating at 80 kV.