The authors have declared that no competing interests exist.
Conceived and designed the experiments: AR IJ GAH AP. Performed the experiments: AR SH AS MH. Analyzed the data: AR MH AS. Contributed reagents/materials/analysis tools: IJ GAH AP. Wrote the paper: AR IJ.
Exoenzyme C3 from
Cellular uptake of bacterial protein toxins such as diphtheria toxin or cholera toxin takes place via receptor-mediated endocytosis
Sensitivity of various cell lines towards C3 differs dramatically. Low concentrations of C3 are sufficient to selectively intoxicate cultured macrophage-like murine J774A.1 cells
The interaction of a bacterial protein toxin with its cellular receptor is the key step for cell entry. The nature of toxin receptors is very broad, covering numerous lipids or lipid derivatives or membranous proteins or glycoproteins
Murine hippocampal HT22 cells, which were a generous gift from Prof. Dr. Carsten Clumsee (Institute for Pharmacology and Toxicology, Philipps University Marburg, Germany)
Pronase was from Roche (Roche Applied Science, Mannheim, Germany). For pronase treatment cells 300,000 HT22 cells were seeded onto 3.5 cm plates and grown for 24 h at 37°C and 5% CO2. The medium was removed and cells were washed with PBS. Cells were cultivated in DMEM with 1% FCS and preincubated with 500 µg/ml of pronase for 30 min at 4°C. Serum (5% FCS) was added to detached cells to block pronase activity and cells were then washed with PBS and incubated with C3 (500 nM) for 1 h at 4°C.
For Western blot analysis the following primary antibodies were used: RhoA was identified using a mouse monoclonal IgG from Santa Cruz Biotechnologies (Santa Cruz, USA). Identification of C3 was achieved by a rabbit polyclonal antibody (affinity purified), which was raised against the full length toxin C3bot (accession no. CAA41767). Vimentin was identified using rabbit monoclonal anti-vimentin antibody (Epitomics, Carlifornia, USA). A polyclonal antiserum against GFAP used to label astrocytes and Actin (used as loading control) was purchased from Sigma (Sigma-Aldrich Chemie GmbH, Munich, Germany). Western blot analyses were performed as described previously
HT22 cells were washed and scraped into Triton X-100 buffer. The obtained suspension was shaken at 37°C for 10 min. Ultrasonic disruption was performed in a cycle of 10×5 sec, 5×10% sonic energy using a sonotrode (Bandelin Electronic, Berlin, Germany). Lysates were centrifuged at 2000 g for 10 min at 4°C. The particulate fraction was resuspended in 50 µl Triton X-100 buffer [150 mM NaCl, 50 mM Tris and 1% Triton-X 100] and samples were separated by 10% SDS-PAGE and transferred to nitrocellulose membranes. After incubation with blocking buffer [5% powdered milk, in Tris-Buffered Saline with Tween (TBST)], membranes were probed with 10 µg/ml purified C3 in blocking buffer overnight at 4°C. After extensive washing with TBST, membranes were incubated with anti-C3 antibody, followed by HRP-conjugated secondary antibody, and detected by ECL. As a positive control, whole cell lysates with 10–20 ng C3 was loaded in the same nitrocellulose membranes and immunoblotted with C3 antibody. For the chemiluminescence reaction, ECL Femto (Pierce, Thermo Fisher Scientific Inc., Rockford, IL, USA) or Immobilon (Millipore, Schwalbach, Germany) was used.
HT22 cells were washed and scraped into Triton X-100 buffer. The obtained suspension was shaken at 37°C for 10 min. Ultrasonic disruption was performed in a cycle of 10×5 sec, 5×10% sonic energy using a sonotrode (Bandelin Electronic, Berlin, Germany). Lysates were centrifuged at 2000 g for 10 min at 4°C. The pellets were resuspended in 50 µl Triton X-100 buffer and separated by Nonequilibrium pH gradient electrophoresis (NEPHGE)/SDS-PAGE. NEPHGE/SDS-PAGE and protein blotting was carried out by WITA protolays (WITA GmbH, Germany). 2D-gels were subjected to either protein electro transfer onto PVDF membrane, or colloidal coomassie brilliant blue (CBB-G250) staining. The PVDF membrane was used for a C3 overlay assay and the colloidal CBB stained gel for mass spectrometry analysis. For alkylation of cysteine residue, dehydrated gel pieces were incubated with 20 µl 15 mM DTT (Sigma-Aldrich Chemie GmbH, Munich, Germany) for 45 min at 56°C. After dehydration with acetonitrile, gel pieces were incubated with 100 mM iodoacetamide (Sigma-Aldrich Chemie GmbH, Munich, Germany) for 15 min at room temperature in the dark. Subsequently, gel pieces were extracted with 200 µl 50% (v/v) acetonitrile in 0.2% (v/v) TFA for 30 min at 400 rpm followed by dehydration with 100 µl of 100% acetonitrile. Peptide containing supernatants were pooled and dried using vacuum evaporation.
HT22 cells were washed and scraped into Triton X-100 buffer [150 mM NaCl, 50 mM Tris and 1% Triton-X 100]. The obtained suspension was shaken at 37°C for 10 min. Ultrasonic disruption was performed in a cycle of 10×5 sec, 5×10% sonic energy using a sonotrode (Bandelin Electronic, Berlin Germany).
50 µg of recombinant C3 protein were separated by native 10% PAGE and transferred to nitrocellulose membranes. After Western blot the transfer efficiency was checked with ponceau staining and as a negative control the membrane was cut below the C3 protein band. After incubation with blocking buffer [5% powdered milk, in Tris-Buffered Saline with Tween (TBST)], membranes were probed with HT22 whole cell lysate for 2 h at 4°C. After extensive washing with TBST, membranes were incubated with 20 mM NH4HCO3, 10% (v/v) acetonitrile and digestion by trypsin gold (Promega GmbH, Mannheim, Germany) was continued over night at 37°C. The reaction was stopped with 50 µl 50% (v/v) acetonitrile in 0.5% (v/v) TFA for 10 min and shaking at 400 rpm. Peptide containing supernatants were dried using vacuum evaporation.
Peptides of 1D gels, 2D gel spots and the HT22 cell lysate probed membranes were analyzed using MALDI-MS/MS as described previously
Peptides of 1D gel digestion were analyzed using LC-MS analysis. Therefore, dried samples were resuspended in 50 µl 2% (v/v) ACN, 0.1% (v/v) TFA. After centrifugation for 2 min at 13 500 g, the supernatant was transferred to an LC sample vial and an appropriate amount of each sample was injected into the LC system (Ultimate 3000 RSLC system, Dionex). Peptides were loaded on a trap column (C18 material, 2 cm length, 75 µm ID, Acclaim PepMap, Dionex) with 6 µl/min and washed with 0.1% (v/v) TFA loading buffer. After 5 min the trap column was switched in line with the nano flow separation column (C18 material, 50 cm length, 75 µm ID, Acclaim PepMap, Dionex) and peptides were eluted with a flow of 250 nl/min and a linear gradient of elution buffer A (0.1% (v/v) formic acid) and elution buffer B (80% (v/v) acetonitrile, 0.1% (v/v) formic acid) from 4–50% elution buffer B in 65 min, from 50–90% elution buffer B in 5 min. Then the column was flushed isocratically with 90% elution buffer B for 10 min, 90–4% elution buffer B in 5 min and finally equilibrated for 15 min in 4% elution buffer B. The LC system was online connected to the nanoESI source of an LTQ Orbitrap velos (Thermo Fisher Scientific, USA). MS precursor scans were acquired from 300–1700 m/z in profile mode with a resolution of 60,000 at 400 m/z in the orbitrap mass analyzer. The top five most intense ions of charge state ≥2 and a minimum signal threshold of 1,800 counts were selected for HCD fragmentation with normalized collision energy of 38%. Fragments were scanned out in the orbitrap mass analyzer in centroid mode with a resolution of 7,500 at 400 m/z. Data were analyzed with Proteome Discoverer 1.2 (Thermo Fisher Scientific, USA). Spectra were searched with Mascot search algorithm and the Swissprot database with maximum of two missed cleavage sites, propionamide of cysteine as static and oxidation of methionine, acetylation of lysine and phosphorylation of serine and threonine as fixed modification. Peptide mass tolerance was set to ±5 ppm and fragment mass tolerance to 0.8 Da.
C3 wild type and
For the binding assays cultivated cells were seeded onto 3.5 cm plates at a concentration of 300,000 cells/ml and grown for 24 h at 37°C and 5% CO2. The medium was removed and cells were washed with PBS. 300,000 cells/ml were exposed to 100 or 500 nM of C3 for 1 h at 4°C. Subsequently, cells were stringent washed three times with PBS. Cells were scraped into Laemmli sample buffer. The obtained suspension was shaken at 37°C for 10 min. Ultrasonic disruption was performed in a cycle of 10×5 sec, 5×10% sonic energy using a sonotrode (Bandelin Electronic, Berlin, Germany). The lysate was then incubated at 95°C for 10 min and submitted to SDS-PAGE and Western blot analysis against α-C3 and β-actin.
Plasmids of mouse vimentins provided by Prof. Dr. Yi-Ling Li, Institute of Biomedical Sciences, Genomics Research Center, Academia Sinica, Taipei, Taiwan were used
Purified recombinant vimentin (2 µg/ml) and C3 exoenzyme (2 µg/ml) were incubated in 1 ml immunoprecipitation buffer (20 mM Tris HCl pH 7.2, 50 mM NaCl, 3 mM MgCl, 1% NP40, 100 µM PMSF, 1% protease inhibitors) for 2 h at 4°C under rotation. Immunoprecipitation of C3-vimentin complex were done with C3 antibody followed by incubation with 50 µl protein A/G PLUS-agarose beads (Santa Cruz Biotechnology, Inc.) for 45 minutes. Agarose beads were spun down at 10,000 g for 5 min, washed 2 times with immunoprecipitation buffer and re-suspended in SDS-PAGE sample buffer. Beads proteins and supernatant from the last wash step were separated by 10% SDS-PAGE followed by Western blot analysis with anti-C3 and anit-vimentin (Epitomics, Carlifornia, USA).
An equal amount of purified vimentin head (amino acids 1 to 101), rod (amino acids 102 to 410), and tail (amino acids 411 to 466) domains in the His-tagged forms were separated by 15% SDS-PAGE and transferred to nitrocellulose membranes. After incubation with blocking buffer [5% powdered milk, in TBST], membranes were probed with 10 µg/ml purified C3 in blocking buffer overnight at 4°C. After extensive washing with TBST, membranes were incubated with anti-C3 antibody, followed by HRP-conjugated secondary antibody, and detected by ECL. For the chemiluminescence reaction, ECL Femto (Pierce, Thermo Fisher Scientific Inc., Rockford, IL, USA) or Immobilon (Millipore, Schwalbach, Germany) was used.
The surface membrane biotinylation of cells surface proteins and isolation was performed using the Cell Surface Protein Isolation Kit (Pierce, Rockford, IL) as directed by the manufacturer. Cultured HT22 cells grown in 75 cm2 culture flasks. Conditioned medium was removed; cells were washed with 2×2 ml of PBS, and surface proteins were labeled with a non-cell-permeable sulfo-NHS biotin analog (500 µl at 500 µg/ml PBS, Pierce) under gentle shaking at 4°C for 1 h. After washing, cells were incubated with 500 µl quenching solution. Washed cells were lysed with 500 µl of lysis buffer, collected with a cell scraper, and clarified by centrifugation (10,000×g, 2 min, 4°C). To precipitate biotin-labeled cell surface proteins, lysate was added to immobilized NeutrAvidin gel and incubate for 1 h under gentle shaking. Complexes were analyzed by 10% SDS-PAGE and Western blot analysis. Further biotinylated samples were resolved by SDS-PAGE. The gel was stained with Coomassie blue (Bio-Rad Laboratories GmbH, Munich, Germany) and bands at 55 kDa were excised and analyzed by mass spectrometry.
Cells (2×105 cells/ml DMEM) were incubated at 37°C, 5% CO2 for 24 h. The medium was removed and cells were washed with PBS and incubated with accutase (1 ml per 3.5 cm plates) (Sigma-Aldrich Chemie GmbH, Munich, Germany) for 3 min at 37°C. Then accutase activity was blocked with ice cold PBS. Detached cells were centrifuged and cell pellet was resuspended in PBS with 5% FCS and incubated for 15 min on ice. Cells were washed in PBS and incubated for 60 min on ice with rabbit monoclonal anti-vimentin antibody (Epitomics, Carlifornia, USA). Oregon green-488 conjugated goat anti-rabbit antibody alone was used as negative control. Cells were washed in PBS and incubated for 30 min on ice with Oregon Green 488-conjugated goat anti-rabbit antibody (Molecular Probes, Life Technologies GmbH, Darmstadt, Germany). Cell surface expression was analyzed using a FACScan (FACScan flow cytometer, Becton Dickinson). Ten thousand events were monitored per condition.
For testing the interaction of C3 with HT22 and J774A.1 cells using FACS cytometry, recombinant C3-A1C/E174Q/K211C protein was labeled with 5-flourescein isothiocyanate (FITC) microscale protein labeling kit (AnaSpec Inc, Fremont, Ca, USA) according to the manufacturer’s instructions. For the flow cytometry analysis we used a recombinant enzyme deficient C3-A1C/E174Q/K211C with two free amines to conjugate fluorescein- 5-isothiocyanate to C3bot. C3 deficient enzyme was chosen to avoid morphological changes even after prolonged incubation time.
Cells (2×105 cells/ml DMEM) were incubated at 37°C, 5% CO2 for 24 h. Cells were harvested in cold PBS and blocked with PBS containing 5% FCS for 15 min on ice. Cells were washed in PBS and incubated for 60 min on ice with 500 nM C3-E174Q-FITC. Cells were washed three times with PBS and the C3 binding was analyzed using a FACScan (FACScan flow cytometer, Becton Dickinson). Ten thousand events were monitored per condition.
HT22 and J774A.1 cells were plated at 1.5×105 onto 3.5 cm plates and grown for 24 h at 37°C and 5% CO2. The medium was removed and cells were washed with PBS. Cells were transfected with Vim siRNA (On-TARGETplus Smart pool, Dharmacon, USA) at a final concentration of 100 or 200 nM with jetPRIME siRNA transfection reagent (Peqlab Biotechnologie GmbH, Erlangen, Germany) according to the manufacturer’s instructions. A scrambled siRNA was used as a control. Scrambled siRNAs contained a random sequence content and with no calculated target gene specificity.
For pulse chase experiment with C3, HT22 cells were 48 h post-transfectional incubated with 500 nM C3 for 60 min by 4°C. Afterwards C3 containing medium was removed. Cells were washed three times with PBS and fresh medium was added. Cells were cultivated for 48 h and then harvested.
For RhoA gel-shift assay, J774A.1 cells were 48 h post-transfectional incubated with 500 nM C3 for 4 h at 37°C. After this incubation, cells were washed with PBS and scraped into Laemmli sample buffer.
HT22 and J774A.1 cells seeded on cover slips were washed with PBS and subsequently fixed in 4% formaldehyde in phosphate buffered saline (PBS) (pH 7.4) at room temperature for 20 min. Cells were then washed and permeabilized with 0.3% (w/v) Triton X-100 in PBS supplemented with 5% BSA. Vimentin was stained by α-vimentin antibody and Oregon green 488 conjugated secondary antibody for 1 h at room temperature either. F-actin staining was performed using rhodamine-conjugated phalloidin (30 µg/ml) for 30 min at room temperature. Then, a 0.1 µg/ml solution of DAPI in phosphate-buffered saline supplemented with 0.1% (w/v) Tween-20 was used for nuclei staining for 30 min at 37°C. Cells were analyzed by fluorescence microscopy using a Zeiss Axiovert 200 M and Leica TCS 2 inverted confocal microscope.
In the case of co-localization of C3-FITC and vimentin, cells were treated with 1000 nM C3-FITC for 2 h in serum-free DMEM and washed with PBS, then fixed with 4% PFA for 20 min at room temperature. Cells were then washed and permeabilized with 0.02% saponin in PBS supplemented with 5% BSA. Vimentin was stained by α-vimentin antibody and Alexa-555 conjugated secondary antibody for 1 h at room temperature either. DAPI was used for nuclei staining. Cells were analyzed using a Leica TCS 2 inverted confocal microscope.
Primary astrocytes were fixed and stained as described previously
For image acquisition a Leica TCS SL confocal laser scanning microscope using a 63× oil immersion objective was used. Fluorescent dyes were excited at a wavelength of 488 nm (green fluorescence), 543 nm (red fluorescence), and 405 nm (blue fluorescence) respectively. Images were captured at a resolution of 1024×1024 pixels.
All experiments were performed independently at least three times. Results from representative experiments are shown in the figures. Values (n ≥3) are means ± SEM. The two-sided unpaired Student's
To check whether C3 binding to intact HT22 cells was dependent on proteinaceous cell surface structures or not, cells were treated with pronase prior to binding to C3. Detached cells were washed and pronase activity was inhibited by addition of serum (
A) Pronase pre-incubated HT22 cells were exposed to 100 or 500 nM of C3 for 1 h at 4°C. Subsequently, β-actin and bound C3 were detected by Western blot. NC = negative control without C3, PC = positive control lysate with 10 ng C3. One representative experiment is shown (n = 3 independent experiments). B) Pronase-treated HT22 cells were exposed to 500 nM of C3-E174Q-FITC for 1 h at 4°C and bound C3- E174Q-FITC was analyzed by FACS.
To identify the putative surface receptor, cell lysates and the particulate cell fractions from HT22 cells were separated by SDS-PAGE followed by electroblotting onto nitrocellulose. The nitrocellulose was then overlaid with C3, washed and bound C3 was detected by anti-C3. Bound C3 was detected at a molecular mass range of about 110 kDa and of 45–55 kDa (cell lysates), at about 55 kDa as one distinct twin band (particulate fraction) and at about 55 and 110 kDa (cytosolic fraction) (
A) Whole cell lysate, cytosolic fraction or particulate fraction from HT22 cells were generated as described in material and methods followed by separation through SDS-PAGE and transfer onto nitrocellulose. Nitrocellulose was incubated with 10 µg/ml of C3 for 60 min at 4°C. After washing bound C3 was detected by anti-C3. Arrows indicate the protein of interest (55 kDa). B) The right panel shows the anti-C3 Western blot without C3-overlay. M = molecular mass marker, WCL = whole cell lysate, PF = particulate fraction, CF = cytosolic fraction, WCL +10 ng C3 = C3 was added to whole cells lysate prior to SDS-PAGE and blotting to generate a positive C3 signal.
To dissolve the 55 kDa bands, the particulate fraction of HT22 cells was separated by two-dimensional gel electrophoresis (2DE), transferred to PVDF membrane and overlaid with C3 followed by probing with anti-C3. Seven spots were detected in the overlay-blot (
To confine the putative C3 binding partners a reverse experiment was set up. C3 separated by non-denaturating SDS-PAGE was blotted onto nitrocellulose membrane and overlaid with HT22 lysates. After intensive washing proteins bound to the immobilized C3 (25 kDa band) were digested and identified by LC-MS. The proteins identified were: histone H2B type 1F (H2B1F), 40S ribosomal protein S1 (RS19), heterogeneous nuclear ribonucleoproteins A2/B1 (HNRNPA2B1), β-actin and vimentin. Notably, β-actin and vimentin were also identified in the 2D gel experiment. A complete list of identified proteins including accession No, Mascot identification score, number of identified peptides, mass, isoelectric point and sequence coverage is available as
To confirm the interaction between C3 and vimentin, additional blot overlay assays with recombinant vimentin were performed. Using the
His-tagged vimentin domains (A) head: amino acids 1 to 101; (B) rod: amino acids 102 to 410; (C) tail: amino acids 411 to 466 were expressed in
Next the role of extracellular vimentin in C3 cell binding and uptake into intact HT22 cells was analyzed. HT22 cells were incubated with C3 or a combination of C3 with vimentin. Bound C3 was analyzed by Western blot analysis against anti-C3.
A) The Western blot shows the binding of C3 in presence and absence of extracellular added vimentin (n = 3 independent experiments). B) Densitometric evaluation of C3 (from A) and adjustment to the corresponding actin band are shown. C) The Western blot shows the degradation of RhoA as marker for C3 uptake and Rho-ADP-ribosylation. HT22 cells were treated with C3 (500 nM) alone or C3 (500 nM) plus vimentin (1 ng/µl) for 48 h. Cell lysates were submitted to Western blot analysis probing RhoA and β-actin. One representative Western blot experiment is shown (n = 3 independent experiments). D) Densitometric evaluation of RhoA (from C) and adjustment to the corresponding actin band are shown; the bars give the relative RhoA level. E) HT22 cells were incubated with C3 (500 nM) or C3 (500 nM) plus 1 ng/µl of either vimentin head-, rod- or tail-domain for 48 h. Cell lysates were submitted to Western blot analysis probing RhoA and β-actin. Decreased signal of RhoA reflects degradation of RhoA after ADP-ribosylation and thus, enhanced C3 uptake. One representative Western blot experiment is shown (n = 3 independent experiments). F) Densitometric evaluation of RhoA (from E) and adjustment to the corresponding actin band are shown; the bars give the relative RhoA level. G) J774A.1 macrophages were treated with C3 (500 nM) alone or C3 (500 nM) plus vimentin (1 ng/µl) for 2 h. Cell lysates were submitted to Western blot analysis probing RhoA and β-actin. One representative Western blot experiment is shown (n = 3 independent experiments). H) Primary astrocytes were exposed to C3 (300 nM) alone or a combination of C3 (300 nM) with different concentrations of vimentin (0.2, 2 and 20 ng/µl) for 6 h at 37°C. After incubation time the astrocytes were stained for the intermediate filament protein GFAP to visualize morphological changes.
To study the influence of vimentin on the uptake of C3, a concentration of 1 ng/µl of vimentin (whole protein) was used because this vimentin concentration seemed to have no effect on mere C3 binding. HT22 cells were incubated with C3 alone or with C3 plus vimentin. Cells were lysed and subjected to Western blot analysis against RhoA and β-actin (loading control). The combination of full length vimentin with C3 caused increased internalization as detected by ADP-ribosylation-induced RhoA degradation (
To rule out that added vimentin per se did not accelerate endocytosis, the influence of vimentin on the endocytosis of
Next the cell surface expression of vimentin was studied. As first step the monoclonal anti-vimentin V9 antibody was characterized using Western blot analysis of HT22 cell lysates and subfractions. As shown in
A) Intact HT22 cells were biotinylated for 1 h at 4°C. Whole cell lysates, cytosolic and particulate fractions were prepared. In addition, cell surface biotinylated proteins were enriched by precipitation with NeutrAvidin beads. The fractions and precipitation, respectively, were immunoblotted and probed with anti-vimentin. Biotinylation fraction represents the extracellular proteins exclusively. One representative Western blot experiment is shown (n = 3 independent experiments). Presence of vimentin at the cell surface of HT22 cells (B) and J774A.1 cells (C) was analyzed by FACS cytometry using anti-vimentin. Oregon green-488 conjugated goat anti-rabbit antibody alone served as negative control. Untreated cells were used as control.
RNA interference technique was applied to further clear up the role of vimentin in C3 binding and uptake. Compared to control and scramble siRNA, vimentin siRNA transfection significantly reduced the expression of vimentin at protein level in HT22 cells (
A) HT22 cells were transfected with siRNA for 48 h (scr = scrambled, Vim = vimentin). Vimentin and β-actin were detected by Western blot analysis of cell lysates. B) After siRNA transfection for 48 h, HT22 cells were exposed to C3 (100 nM) for 1 h at 4°C. Bound C3 was detected in Western blot with anti-C3. β-actin was used as internal control. C) Densitometric evaluation of bound C3 (from B) and adjustment to the corresponding actin band are shown; the bars give the relative C3 binding. D) HT22 cells transfected with siRNA for 48 h were incubated with C3-E174Q-FITC (500 nM) for 1 h at 4°C and bound C3-E174Q-FITC was analyzed by FACS cytometry. E – G) Same experiments for J774A.1 macrophages. E) Knock down of vimentin. F) Binding of C3 to cells with vimentin knock down. G) Densitometric evaluation of F. H) Binding of C3-E174Q-FITC to cells with vimentin knock down and FACS analysis.
Cell surface expression of vimentin was checked 48 h after transfection; in fact, a slight increase of vimentin at the cell surface was detected (
A) Vimentin was detected by anti-vimentin at the cell surface of HT22 cells transfected with siRNA for 48 h at 37°C by FACS analysis. B) Confocal microscopy of vimentin in HT22 cells transfected for 48 h at 37°C. The green (oregon green 488) anti-vimentin, DNA staining in blue (Dapi), rhodamine red-staining for actin and a merge image are shown for each panel. In the enlarged images the cell boundaries are shown. Significant difference between the vimentin distribution was detected for the transfected cells (lower panel) in comparison to the control (upper panel). Scale bar = 20 µM. C) Detection of vimentin at the cell surface of J774A.1 cells transfected with siRNA. D) Confocal microscopy of vimentin distribution in J774A.1 cells transfected for 48 h.
Next the effect of vimentin knock down on the cellular uptake of C3 was studied. In a pulse-chase experiment exposure of HT22 cells to C3 caused degradation of RhoA as a marker for ADP-ribosylation. In Vim siRNA transfected cells C3- induced RhoA degradation is delayed (
A) Influence of Vim-siRNA knock down (for 48 h) on the uptake of C3 into HT22 cells detected as RhoA degradation (induced by C3-catalysed ADP-ribosylation). In a pulse-chase experiment, HT22 cells were incubated with C3 (500 nM) at 4°C for 60 min. Afterwards unbound C3 was removed by washing the cells three times with PBS and fresh medium was added. Cells were then cultivated for further 48 h. Cell lysates were generated and separated by SDS-PAGE followed by Western blot analysis probing RhoA and β-actin. One representative experiment is shown (n = 3 independent experiments). B) Cellular levels of RhoA proteins were quantified by densitometric evaluation of RhoA (from A) and adjusted to the corresponding actin band. C) HT22 cells were pre-treated with acrylamide (5 mM) for 30 min followed by incubation with C3 (500 nM) for 24 h. Cells were lysed and submitted to Western blot analysis probing RhoA and β-actin. C3 alone causes a complete mol weight shift of RhoA in SDS-PAGE. Western blot analysis of one representative experiment is shown (n = 3 independent experiments). D) RhoA shift (indicative of Rho-ADP-ribosylation) by quantified by densitometric evaluation of RhoA (from C) and adjusted to the corresponding β-actin signal. E) Influence of Vim-siRNA knock down (for 48 h) on the uptake of C3 into J774A.1 cells detected as incomplete RhoA ADP-ribosylation. J774A.1 macrophages were incubated with C3 (500 nM) at 37°C for 4 h. Cell lysates were generated and separated by SDS-PAGE followed by Western blot analysis probing RhoA and β-actin. One representative experiment is shown (n = 3 independent experiments). F) J774A.1 cells were pre-treated with acrylamide (5 mM) for 30 min followed by incubation with C3 (500 nM) for 4 h. Cells were lysed and submitted to Western blot analysis probing RhoA and β-actin.
Taken together, these results strongly indicate that uptake of C3 in HT22 and J774A.1 cells is dependent on vimentin network.
C3 exoenzyme is still used as cell biological tool because of its high selective inactivation of RhoA/B/C GTPases in intact cells
Poor cell accessibility has been overcome by generation of fusion constructs enhancing cell entry. However, some cell types are per se sensitive such as macrophages, neutrophils, astrocytes and neurons. C3 at nanomolar concentrations is able to efficiently enter the cells and cause ADP-ribosylation of Rho resulting in morphological changes
Pre-tests revealed the proteineous nature of the putative receptor. Therefore, Western blot binding overlays were employed to identify distinct bands. Mass spectrometry analysis of one exemplarily SDS-PAGE band resulted in the identification of more than 100 proteins. To confine the receptor candidates the membrane extract was therefore resolved by 2D gel electrophoresis and the overlay was repeated after blotting. Now seven major spots (C3 binding to proteins) were visualized and identified by mass spectrometry. To further confine the candidates the inversed experiment was performed using C3 as immobilized bait. C3 bound proteins were again identified by mass spec. Now the lists of identified C3-binding proteins were compared and the intersection of both lists was determined. Two proteins, namely vimentin and β-actin, appeared in all identification approaches. Vimentin is an intermediate filament protein appearing in different posttranslational modifications and forms. Vimentin, the most abundant component of intermediate filaments is mainly involved in structural processes, such as wound healing
The next step was to check whether vimentin is indeed present at the extracellular surface of HT22 cells. Two different approaches were applied: selective labeling of extracellular membrane proteins by biotinylation of intact cells, followed by an enrichment step and detection of vimentin by anti-vimentin in the fraction of biotinylated proteins. In addition, vimentin was identified in the immune-reactive band by mass spec. Second approach used the labeling of intact cells with anti-vimentin and analysis by flow cytometry. A small percentage of cells were in fact labeled with monoclonal anti-vimentin. Moreover, this result was confirmed by flow cytometry analysis of J774A.1 macrophages. HT22 cells revealed a much lower percentage of positive staining for surface vimentin expression in comparison to J774A.1 cell line. Thus, vimentin is present at the plasma membrane of intact HT22 and J774A.1 cells. Mor-Vaknin et al. reported on vimentin expression at the cell surface of activated macrophages and on vimentin secretion into the extracellular milieu
To investigate the role of vimentin in detail we used siRNA technology to knock down vimentin because we were able to detect vimentin by Western blot analysis in two described vimentin negative cancer cell lines like HT29
However, after siRNA vimentin knock down an enhanced binding of C3 in Western blot analysis and flow cytometry was observed. Confocal immunofluorescence microscopy of transfected cells detected different vimentin fragments at the cell periphery and flow cytometry revealed slight increase of vimentin at cell surface. Another study reports that vimentin undergoes phosphorylation resulting in disassembling of vimentin and expression of tetrameric vimentin at the cell surface
Vimentin is a three domain protein consisting of the head (aa1–101), rod (aa102–410) and tail (aa411–466) domain. Applying overlay technique we showed that C3 exclusively binds to the rod domain. Vimentin reportedly interacts with various proteins (see
It is known that vimentin is initially expressed in neuronal precursors where expression is necessary for neurite extension. It is replaced by neurofilaments shortly after the immature neurons become post-mitotic. In adult brain, vimentin expression is largely restricted to vascular endothelial cells and certain subpopulations of glial cells
In conclusion, our findings strongly indicate that vimentin is expressed at the cell surface of hippocampal HT22 cells and J744A.1 macrophages and gets internalized upon binding to C3. After binding to surface rod-domain of vimentin C3 is endocytosed via vimentin-mediated endocytosis. Further analysis will be required to explore the exact molecular mechanism of C3-vimentin interaction. Because C3 lacks a translocation domain, demonstration of the interaction of C3 either with vimentin at cell surface or uptake and subsequent association of C3 with the cellular intermediate filament protein vimentin represent important steps in understanding how C3 may access its intracellular target Rho. The nature of this interaction is not fully elucidated but represents a starting point for future studies on the property of vimentin as a target structure for bacterial toxins.
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We would like to thank Prof. Dr. Ralf Gerhard (Institute of Toxicology, Hannover Medical School, Germany) for providing the recombinant toxin A and Prof. Dr. Yi-Ling Li (Institute of Biomedical Sciences, Genomics Research Center, Academia Sinica, Taipei, Taiwan) for providing the vimentin constructs.