vector company modification/annotation insert

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1 Supplemental information Plasmids Table 1. List of constructs used in the study. vector company modification/annotation insert pegfp-c1 Mikhaylova et al Caln1 (NM_ ) pegfp-c1 Caln1 ΔC (aa 1-190) pegfp-c1 Caln1 CT (aa ) pegfp-c1 Caln1 23aa (aa ) pegfp-c1 Caln1 17aa ( ) pegfp-c1 Mikhaylova et al Caln2 (NM_ ) pegfp-c1 EGFP substituted by VC155 with HA-tag as linker Caln1 pegfp-c1 BD Biosciences, Heidelberg, EGFP substituted by VN173 with myc-tag as linker Caln1 pegfp-c1 Germany EGFP substituted by Rluc Caln1 pegfp-n1 Caln1 pegfp-n1 Mikhaylova et al NCS-1 (NM_ ) pegfp-n1 EGFP substituted by VC155 with HA-tag as linker Caln1 pegfp-n1 EGFP substituted by VN173 with myc-tag as linker Caln1 peyfp-c1 Caln1 peyfp-c1 Navarro et al Calmodulin peyfp-n1 Caln1 ptagrfp-c1 Evrogen,Moscow, Russia Caln1 pcdna3.1 Caln1 pcdna3.1 Caln2 pet-sumo Invitrogen, Darmstadt, Germany Mikhaylova et al Caln1 pet-sumo Caln1 ΔC pet-sumo Caln1 CT pmal-c2x NEB, Frankfurt, Germany Mikhaylova et al Caln1 vector company annotation pac-gfpc1-sec61beta Addgene plasmid by Tom Rapoport pbifc-vc155 Addgene, Cambridge USA Addgene plasmid by Chang-Deng Hu pbifc-vn173 Addgene plasmid by Chang-Deng Hu prluc-gfp2 PerkinElmer, Rodgau, Germany pdsred-monomer-golgi BD Biosciences, Heidelberg, Germany vector from annotation peyfp-trc40 F.Vilardi and B. Dobberstein, Vilardi et al Heidelberg, Germany Favaloro et al pgex-trc40 pvsv-g-gfp M.M. Kessels and B. Qualmann Antibodies Primary antibodies used in this study: Anti-ß-Actin mouse (A5441, SIGMA, Hamburg, Germany), Anti-Asna1 mouse (ab54843, Abcam, Cambridge, UK), Anti- CaBP7 (N-19) (sc-86350, Santa Cruz Biotechnology, Heidelberg, Germany), Anti- Calneuron-1 rabbit ( AP, Acris, Herford, Germany), Anti-Calneuron1 rabbit (produced in the lab and characterized previously), Anti-GFP mouse (MMS-118R, HiSS Diagnostics, Freiburg, Germany), Anti-GFP rabbit (Abcam, Cambridge, UK), Anti-GFP rabbit (generated in the lab), Anti-GM130 rabbit (ab , ab52649, Abcam, Cambridge, UK), Anti-Syntaxin 6 rabbit (110062, Synaptic Systems, Göttingen, Germany), Anti-TGN38 mouse (610899, BD Bioscience, Heidelberg, Germany). Secondary antibodies were:goat anti-mouse Immunoglobulins HRP conjugated (P0447, Dako, Hamburg, Germany), goat anti-rabbit IgG HRP conjugated (#7074, Cell Signaling, Frankfurt am Main, Germany), Alexa Fluor 568 goat antimouse IgG, Alexa Fluor 568 goat anti-rabbit IgG (A11031, A11036, Invitrogen,

2 2 Darmstadt, Germany), Cy5-conjugated AffiniPure goat anti-mouse IgG ( , Dianova, Hamburg, Germany). Subcellular fractionation HeLa cells were harvested and homogenized in a buffer containing 10 mm HEPES- NaOH, ph 7.4, 150 mm NaCl, 2mM MgCl 2, 100 µm DTT and protease inhibitors (Complete TM, Roche). The cell homogenate (H) was centrifuged at 1000xg for 10 min to spin down the nuclei and cell debris, followed by the ultracentrifugation of the supernatant (SN1) at 400,000 x g for 2 hrs to pellet down the microsomal fraction containing fragments of ER, PM, Golgi and vesicular structures (P2). The protein concentration of the resulting supernatant (SN2) as well as that of the fractions, H, SN1 and P2 were determined by amido-black assays and 20 µg protein of each of these fractions was loaded on SDS-PAGE in replicates. The gels were then subjected to immunoblotting using either anti-cabp7 (N-19) rabbit polyclonal antibody (Santa Cruz Biotechnology Inc., 1:200 dilution), anti-syntaxin 6 rabbit polyclonal antibody (Synaptic Systems,1:1000 dilution) or anti-β-actin mouse monoclonal antibody (Sigma, 1: 2000 dilution). Golgi localization assay To compare the efficiency of Golgi localization for the five different EGFP- Calneuron-1 constructs, COS-7 cells were transfected with the desired plasmids for 24 hours, followed by an ICC with GM130 (1:1000) and TGN38 (1:500). The analysis of the cells was done using the ImageJ software (NIH, USA). Therefore maximum intensity projections of the obtained z-stacks were created on which for each cell 3 ROI s (1 st the nucleus; 2 nd the non nucleus overlapping GM130- immunoreactive area as Golgi; 3 rd the complete cell area subtracted by 1 st and 2 nd ) were defined. EGFP fluorescence was measured as Integrated density for the complete cell ROI and the Golgi ROI. The ratio of Golgi/complete cell were plotted for each EGFP-Calneuron-1 construct and compared to EGFP transfected cells. BRET assays HEK-293T cells were transiently co-transfected with a constant amount of cdna encoding for the protein fused to Rluc and increasing amounts of cdna corresponding to the protein fused to YFP. To quantify protein-yfp expression cells (20 µg protein) were distributed in 96-well microplates (black plates with a transparent bottom) and fluorescence was read in a FLUOstar Optima Fluorimeter (BMG Labtechnologies, Offenburg, Germany) using an excitation filter at 400nm. Protein-fluorescence expression was determined as fluorescence of the sample minus the fluorescence of cells expressing the BRET donor alone. For BRET measurements, cell suspensions (20 µg protein) were distributed in 96-well microplates (Corning 3600, white plates; Sigma) and 5µM coelenterazine H (Molecular Probes, Eugene, OR) was added. After 1 minute, the readings were collected using a Mithras LB 940 that allows the integration of the signals detected in the short-wavelength filter at 485 nm ( nm) and the long-wavelength filter at 530 nm ( nm). To quantify protein-rluc luminescence readings were also collected after 10 minutes of adding coelenterazine H. The net BRET is defined as [(long-wavelength emission)/(short-wavelength emission)]-cf where Cf corresponds to [(longwavelength emission)/(short-wavelength emission)] for the donor construct expressed alone in the same experiment. BRET is expressed as mili BRET units, mbu (net BRET x 1000). Digitonin permeabilization assay COS-7 cells were co-transfected with equal amounts of pegfp-c1-calneuron-1 and ptagrfp-c1. 24 hours after transfection cells were washed with KD buffer (125 mm

3 NaCl, 2,5 mm KCl, 2 mm MgSO 4, 2 mm Ca 2+, 10 mm glucose, 30 mm Hepes, ph 7.3) and placed under the microscope. After baseline recording, 40 µm of digitonin was added to the buffer. Images were acquired with 10 sec per frame. To obtain digitonin extracted fraction of cytosol for immunoblotting COS-7 cells were transfected with pegfp-c1-calneuron-1 or pegfp-c1-calneuron-1_δc. 24 hours after cells were washed with KD buffer and incubated for 30 or 60 seconds with a new KD buffer containing 40 µm of digitonin. Control cells were kept with a regular KD buffer for 60 sec. After this treatment KD buffer was collected and cells were harvested and analysed by immunoblotting with anti-gfp mouse, anti-syntaxin 6 rabbit and anti-β-actin mouse antibody. 3

4 4 Supplemental Figures Figure S1. Overexpressed Calneuron-1 is mainly present in the membrane-bound form with a small fraction in cytosol. (A) Live imaging of COS-7 cells co-transfected with EGFP-Calneuron-1 and tagrfp. 5 min after the baseline recording cells were pemeabilized with 40 µm of digitonin. Images were taken with 20 sec per frame. (B) Examples of fluorescence intensity curves before and after digitonin treatment, measured in the Golgi, the cytosol, the nucleus and the area outside of the cell. Permeabilization and diffusion of soluble cytosolic proteins was confirmed by the loss in TagRFP fluorescence quickly after the addition of digitonin. EGFP-Calneuron-1 fluorescence was not changed at the perinuclear Golgi area but was reduced in the nucleus and cytosol. (C) Digitonin permeabilization assay in COS-7 cells transfected with full length EGFP-Calneuron-1 or the deletion construct lacking the C-terminal transmembrane region. Growth medium was replaced by KD buffer and cells were either untreated (for the control group) or treated with 40 µm of digitonin for 30 or 60 seconds. Then the buffer was collected and centrifuged (SN) in order to remove cell debris. COS-7 cells attached to the wells were harvested in the same volume of fresh KD buffer (P). Both the SN and P fractions from each experiment were subjected to SDS-PAGE and analyzed with anti-gfp, anti-syntaxin 6 and anti-β-actin antibody. EGFP-Calneuron-1_ΔC is found in SN fraction already after 30 seconds of digitonin treatment whereas the full length EGFP-Calneuron-1 is detectable only after 60 seconds. Figure S2. EGFP-Calnueuron-1 co-localizes with the Golgi marker DsRed- Monomeric-Golgi (left panel). The reticular distribution of Calneuron-1 subfraction probably represents the protein localized at the ER (visualized by co-transfection with the ER marker GFPC1-Sec61β / right panel). Figure S3. Characterization of the antibody used fort he PLA assay. Anti-GFP mouse, anti-asna-1 mouse and anti-calneuron-1 rabbit (ProteinTech) antibodies were tested on COS-7 cells overexpressing or lacking the respective proteins that were fused to a fluorescent tag (GFP/RFP/YFP). Immunostaining was observed only in cells that overexpress the respective protein. The staining pattern of untagged Calneuron (pcdna-caln1) observed with the anti-caln1 rabbit antibody is comparable to the GFP fluorescence pattern of pegfp-caln1. Figure S4. (A) GFP-Calneuron-1 constructs expressed in COS-7 cells are also showing higher molecular weight complexes compared to GFP in a semi-native PAGE. Complete denaturation of the sample results in a pattern comparable to the pull down inputs of Fig. 4C. (B) Semi-native PAGE for different GFP-tagged constructs of Calneuron-1 solubilized in the native loading dye. Next to the appearance of the bands corresponding to the molecular weight of each monomeric construct, double (indicated by *) size bands can be detected in all GFP-Calneuron-1 constructs. Especially the full length Calneuron-1 construct seems to be to a majority in at least a dimmer form. The weak binding of the full length construct to ASNA-1 in the pull down experiment shown in Fig. 4C can be therefore a result of a relatively small pool of monomeric Calneuron-1. (C) Calibration of Superdex 200 column using protein standards in the presence of 1 mm EDTA (left) and 1 mm Ca 2+ (right).

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