Animals and tissue collection for EM
Wistar rats were bred and housed in the animal facility of the Leibniz Institute for Neurobiology, Magdeburg. Experiments were conducted following ethical animal research standards defined by the German Law/European directive and approved by the Landesverwaltungsamt Saxony-Anhalt (Referat 203, Verbraucherschutz und Veterinärangelegenheiten). The competent authority follows the advice of an official animal welfare committee of the Federal State of Saxony-Anhalt, Germany.
For EM experiments, male adult Wistar rats were deeply anaesthetized using a mixture of Ketavet (Parke-Davis) and Domitor (Pfizer) and then transcardially perfused with a solution containing 0.9% NaCl for 1 min followed by 4% paraformaldehyde (PFA) and 0.05 glutaraldehyde in 0.1 M PBS for 20 min. Brains were removed and post-fixed in 4% PFA solution for 20 h. For pre-embedding immunogold labeling, brains were rinsed in 0.1 M PB and freeze-protected with 1 M sucrose in 0.1 M PB. Tissue was frozen at − 40 °C in iso-pentane and 25 µm frontal sections were prepared. For cryo-immunogold labeling, brains were rinsed in 0.1 M PB and sliced at 700 µm using a vibratome. Brain tissue was freeze-protected with 2.3 M sucrose. Samples of cerebral cortex (1 × 2 mm) were mounted on cryo holders and frozen by immersion in liquid nitrogen.
Pre-embedding immunogold labeling for EM
For immunoelectron microscopy, freely floating sections were rinsed in 0.01 mol/L phosphate buffered saline (PBS) at pH 7.4, treated for 15 min with 1% sodium borohydride in PBS and thoroughly washed in PBS. Thereafter, sections were pre-treated for 30 min in a blocking and permeabilizing solution consisting of 10% normal goat serum (NGS; Interchem, Bad Kreuznach, Germany), 0.1% saponine, in PBS at 22 °C. Rabbit anti-Jacob antibody (1:1000) was applied for 36 h in PBS containing 10% NGS, 0.1% saponine and 0.1% sodium azide at 4 °C. Sections were then thoroughly rinsed in PBS, pre-treated for 1 h with 0.1% acetylated BSA (Aurion, Wageningen, the Netherlands) and 0.1% Tween 20 in PBS and exposed for another 24 h to the secondary antibody, 1 nm colloidal gold-coupled goat anti-rabbit IgG (Auroprobe; Amersham, Little Chalfont, UK) diluted 1:50 in the same solution. After several PBS washing steps for at least 2 h, sections were post-fixed for 15 min with 2% glutaraldehyde at 22 °C and then thoroughly rinsed in 150 mmol/L aqueous sodium nitrate. Gold particles with a diameter of 1 nm were silver enhanced for 1–1.5 h using a commercial developer (Intense-M, Amersham) supplemented with one-third (v/v) gum arabic (100 g dissolved in 200 mL H2O; Sigma). Development was stopped by several washes in 150 mmol/L aqueous sodium nitrate. After three additional washes in 0.15 mol/L acetate buffer (pH 5.6), silver deposits were stabilized by a modified gold-toning procedure . Sections were treated for 10 min in 0.05% gold chloride (Sigma) in 150 mmol/L acetate buffer, pH 5.5, followed by a 5 min rinse in 150 mmol/L acetate buffer (pH 5.6) at 4 °C. Sections were transferred to 0.1 mol/L cacodylate buffer (CB), post-fixed for 30 min with 1% osmium tetroxide in 0.1 mol/L CB, and washed several times in CB. Finally, sections were dehydrated in a graded series of ethanol for 10 min each, including block staining with 2% uranyl acetate (Serva) in 70% ethanol and then flat embedded in Durcupan ACM (Fluka). Ultrathin sections were prepared with an Ultracut UCT (Leica), contrasted with uranyl acetate and lead citrate and analyzed with a Zeiss EM 900 equipped with a 1 K slow scan CCD camera.
Immunogold labeling of thin cryosections for electron microscopy
Thin cryosections were prepared and labeled according to a cryo-immunogold technique originally introduced by Tokuyasu  and modified by Griffiths . Briefly, thin cryosections of the parietal cerebral cortex were prepared at a thickness of 100 nm. Then, sections were collected on droplets of a mixture of 2.3 M sucrose and 2% methyl cellulose (1:1) and subsequently incubated on droplets of the following solutions: (1) 50 mM glycine in phosphate buffered saline, pH 7.4 (PBS) for 10 min; (2) 10% normal goat serum (NGS) in PBS (15 min); (3) rabbit anti-Jacob antibody (1:100) in PBS containing 10% NGS and 0.1% sodium azide for 20 hs; (4) PBS buffer (3 × 5 min); (5) 0.1% bovine serum albumin (BSA) in PBS (20 min), (6) gold-conjugated anti-rabbit antibody (British Biocell, 1:50) for 4 h; (7) PBS buffer (6 × 3 min); (8) 2.5% glutaraldehyde in PBS (10 min) for postfixation; (9) distilled water (4 × 2 min). Sections were then floated on drops of 1.5% methyl cellulose, containing 2% uranyl acetate (10 min on ice) for contrasting and analyzed with a Zeiss EM 900 equipped with a 1 K slow scan CCD camera.
Expression constructs and site directed mutagenesis
The following plasmids were obtained from AddGene: pmCherry-RanQ69L (#30309, ), hRanT24N-pmCherry (pTK21 #37396, ), mTagRFP-hLaminB1 (#58020, ), 3xFLAG-hCRM1 (Addgene #17647, ). For recombinant protein production, CRM1 and LaminB1 were further subcloned into Intein-pMXB10 vector between NdeI and XhoI restriction sites with the following primers: CRM1-fw 5′-AATTATCATATGCCAGCAATTATGACAATGTTA-3′, CRM1-rev 5′-TCCAGAAGAAATGTGTGATCTCGAGTAATAT-3′, LaminB1-fw 5′-ATTATTCATATGGCGACTGCGACCCCCGT-3′, LaminB1-rev 5′-ATGTGGCTCGAGCATAATTGCACA GCTTCT-3′. WT-Jacob-GFP, Jacob-ΔNLS-GFP, WT-Jacob-Myc, MBP-45–532-Jacob were described previously [14, 15]. Jacob NES mutant constructs (Jacob-L476A-GFP and MBP-45-532-Jacob-L476A) were generated using QuikChenge Site-Directed Mutagenesis (STRATAGENE) with the following primers: mNES-fw 5′ CGGGGAGAAGGCATTCCAGAACC 3′ and mNES-rev 5′ TTAGGGTTCCCCAGGATG 3'.
Neuronal and HEK293T cell culture and transfection
Rat hippocampal primary neuronal cultures were prepared from E18-E19 as described previously  and kept in Neurobasal (Gibco) neuronal medium supplemented with B-27 (Gibco) and 0.5 mM glutamine (Gibco). Neurons were transfected with Jacob mutant constructs at DIV16/17 using lipofectamine2000 (Invitrogen). HEK293T cells cultured in Dulbecco’s Modified Eagle’s Medium (DMEM, Gibco #41966-029) supplemented with 10% Fetal Calf Serum (FBS, Gibco #10270106) and an antibiotic penicillin/streptomycin cocktail were transfected the next day after splitting using the calcium phosphate method.
Compounds and treatment
Anisomycin (7.5 μM), 4-AP (2.5 mM) and leptomycin-B (LMB; 0.1 nM) were purchased from Sigma, D-APV (20 μM) and ifenprodil (5 μM), bicuculline (50 μM) were purchased from Tocris, and NVP-AAM077 (50 nM) was a kind gift from Dr. Auberson (Novartis). Treatments of hippocampal neurons with D-APV, 4-AP/bicuculline were performed in the presence of anisomycin. Cultures were fixed 30 min after D-APV, 4-AP/bicuculline or leptomycin-B treatment. Ifenprodil and NVP-AAM077 were applied for 28 h and all neurons were immunostained against Jacob and co-stained with anti-MAP2 antibodies. Hoechst or DAPI were used as a nuclear counterstain. Jacob immunoreactivity was measured as mean grey value in arbitrary units of pixel intensity using the Image J software and normalized to untreated conditions.
Custom-made, affinity-purified antibodies generated against two short amino acid stretches of rat Jacob (285–299 and 400–314, Jb150) have been described previously [14, 21, 22]. The following commercial primary antibodies were used in the present study: mouse monoclonal anti-MAP2 (Sigma-Aldrich, M4403), anti-MAP2, Alexa Fluor 488 conjugated (Merck, MAB 3418x), mouse monoclonal anti-LaminB1 (ProteinTeck, 3C10G12), rabbit polyclonal anti-CRM1 (ProteinTeck), mouse monoclonal anti-MBP (NEB), mouse monoclonal anti-GFP (BioLegend, B34), and rabbit polyclonal anti-tRFP (EVROGEN). For imaging and WB analysis, the following secondary antibodies were used: anti-rabbit, anti-mouse secondary antibodies conjugated with Alexa Fluor 488, 561 or 647 (Life Technologies), peroxidase-conjugated anti-mouse IgG (Dianova) and anti-rabbit IgG (Dianova).
Immunocytochemistry, proximity ligation assay and confocal microscopy
For immunocytochemical analysis, dissociated neuronal cultures were fixed with 4% PFA solution for 5 min at room temperature and after several washing steps with PBS, cells were permeabilized with 0.1% Triton X-100 in PBS for 10 min. Next, cells were incubated with blocking buffer containing 2% Glycine, 2% BSA, 0.2% gelatin and 50 mM NH4Cl for 1 h at RT. Incubation with primary antibodies was done in a blocking buffer at 4 °C overnight. Secondary antibodies were diluted in a blocking buffer 1:500 and applied for 1 h at RT. After washing in PBS, either DAPI or Hoechst was added for 10 min and coverslips were then rinsed in water and mounted with Mowiol 4–88 (Calbiochem/Merck Chemicals Ltd.).
For proximity ligation assay (PLA), neuronal cultures were treated as described above and after incubation with primary antibodies overnight at 4 °C, coverslips were washed with Duolink wash buffer A (Sigma-Aldrich) and incubated with Duolink PLA probes PLUS and MINUS in a humidity chamber for 1 h at 37 °C. For ligation of the probes, coverslips were washed with buffer A and then incubated with ligase for 1 h at 37 °C. For amplification step, coverslips were washed with buffer A and then incubated with polymerase and Duolink FarRed Detection Reagent for 100 min at 37 °C. Coverslips were washed two times with Duolink wash buffer B for 5 min and one time with 0.01 × buffer B for 1 min. Prior to incubation with other antibodies for immunocytochemistry or DAPI counterstaining, coverslips were washed with PBS and afterwards, mounted with Mowiol 4-88.
Images were acquired with a 63× (Leica) objective lens along the z-axis with 300 nm z-step in a 512 × 512 and 1024 × 1024-pixel formats at 8-bit and 16-bit image depth with at least two times frame average at 400 Hz laser frequency using either the SP8 -or SP5 CLSM system (Leica-Microsystems, Mannheim, Germany) equipped with white light laser (WLL). Nuclear Jacob immunoreactivity was measured as the mean grey value in arbitrary units of pixel intensity using Fiji software. For Jacob-LaminB1 co-expression analysis and PLA, raw confocal and STED images were deconvolved using Deconvolution wizard (Huygens Professional, SVI), where we first calculated the theoretical point spread function (PSF) based on optical microscopic parameters provided by original *.lif-files, and subsequently used optimized iteration mode.
STED images were acquired on a SP8 CLSM setup (Leica) using a 100x/1.4 oil objective (HC PL APO CS2, Leica). The STED laser (775 nm) was set to maximum power and used to deplete the white light laser excitations at 561 nm and 633 nm with optimized STED laser delay time. Gating of hybrid detectors was set to 0.5–6 ns. The line profiles from STED images were generated from an average of two optical sections.
Hippocampal primary neurons were transfected at DIV9 with the plasmid expressing WT-Jacob-GFP using lipofectamine 2000. Time-lapse imaging was performed as described previously [14, 30]. Z-stacks throughout the nucleus were acquired every 2 min and LMB was applied 10 min after recording as indicated with the timeline in the corresponding figure.
Heterologous co-immunoprecipitation (Co-IP) assay
HEK-293 T cells were co-transfected via the calcium phosphate precipitation method with the following plasmids: WT-Jacob-Myc and TagRFP-LaminB1 or tagRFP control. 24 h post-transfection cells were harvested and lysed in 1 ml of RIPA buffer containing 50 mM Tris–HCl pH 7.4, 150 mM NaCl and 1% Triton X-100 and 0.1% sodium dodecyl sulfate (SDS), PIC and PhosStop for 1 h at 4 °C. The cleared by centrifugation cell lysate was incubated with anti-Myc antibody-coated magnetic beads (MultiMACS GFP Isolation Kit, Miltenyi Biotec GmbH, Germany) and immunoprecipitation was performed following the manufacturer’s protocol (Mitenyibiotec, Bergisch-Gladbach, Germany) with subsequent WB analysis.
Induction and purification of fusion proteins for pull-down assay
Proteins were bacterially produced and purified as previously described . In brief, E. coli SHuffle (C3026) cells were transformed with either pMAL or LaminB1(PMXB10) vectors and E. coli ExpressLysY/Iq cells were transformed with CRM1 (PMXB10) vector. All bacterial cultures were grown at 30 °C until OD600 reaches 0.4–0.8 and subsequently 0.4 mM of IPTG was added for additional 16 °C. For purification of LaminB1 and CRM1, bacterial cell lysates were prepared in 20 mM Tris pH 8.5 500 mM NaCl, sonicated, centrifuged at 20.000g and then the supernatant was loaded onto chitin resin and bound to columns. After several washing steps, columns were incubated with the wash buffer containing 50 mM dithiothreitol (DTT) for 24 h at 10 °C. Proteins were eluted and run on the SDS-PAGE gel for the purity checking. For interaction assays between Jacob and LaminB1, and also between Jacob and CRM1, the matrix (amylose-MBP) alone or coupled with Jacob proteins (amylose-MBP-45-532-Jacob and amylose-MBP-45-532-Jacob-L476A) was washed with the buffer containing 20 mM Tris pH 7.5, 1 mM DTT, 3 mM EDTA, 100 mM NaCl, 0.3% TritonX-100, protease inhibitors (Complete, Roche). Subsequently, amylose resin was incubated with 20 μg of an untagged-LaminB1, untagged-CRM1 or with HEK293T cell extract expressing CRM1 overnight at 4 °C and all proteins were eluted and analyzed by WB.
Quantification and statistical analysis
Data in the manuscript is shown as mean ± S.E.M. Graphs and statistical analysis were made with GraphPad Prism (GraphPad Software). Statistical tests used are indicated in the figure legends. The number of subjects considered for statistical comparison is included in the graphs.