Figure 1.
Generation of filamentous M13 phages displaying cell-penetrating 3D8 VL transbody (3D8 VL-M13) and TAT peptide (TAT-M13).
As a control, anti-DR4 hAY4 scFv without cell-penetrating ability was also employed; the phage particles are designated as hAY4 scFv-M13. (A) Phage titers obtained from 100-ml culture supernatants of recombinant phagemid-transformed bacteria by VCSM13 helper phage superinfection under optimal culture conditions as described in the text. Phage titers were determined by CFU assay. Data represent mean ± S.E. (error bars) of 5 independent experiments. (B) Western blot analysis of display efficiency of fusion proteins (insert-pIII) (filled arrow) versus full-length pIII (open arrow) from VCSM13 helper phage on the recombinant M13 filamentous phage particles. Equal titers (109 or 1010 CFU) of phage particles prepared as described in (A) were western blotted with anti-myc antibody to detect only pIII-fusion proteins (3D8 VL-pIII, and TAT-pIII, and hAY4 scFv-pIII) from the phagemid vectors or anti-M13 pIII antibody to detect both pIII-fusion proteins from the phagemid vectors and full-length pIII from the helper phage. The positions of molecular size marker are indicated. (C) Phage ELISA on DR4 or DNA to examine antigen-binding specificity of the recombinant phages. Various titers (107∼1011 CFU) of recombinant phages or VCSM13 helper phage were applied to each well, precoated with the indicated antigen. Bound phages were detected with HRP-conjugated anti-M13 antibody. Data represent mean ± S.E. (error bars) of three independent experiments carried out in triplicate.
Figure 2.
3D8 VL-M13 and TAT-M13 phages penetrate living cells in an energy-dependent manner and in the presence of serum proteins, and can be rescued in their infective form in proportion to the input titer.
Unless otherwise specified, HeLa cells (1×106 cells) in serum-free medium were treated at 37°C with 1012 CFU of VCSM13, 3D8 VL-M13, or hAY4 scFv-M13 for 6 h or 1013 CFU of TAT-M13 for 2 h. (A) Internalization and subcellular localization of phage particles in HeLa cells, untreated (‘control’) or treated with VCSM13 helper phage or the indicated recombinant phages and analyzed by confocal immunofluorescence microscopy. Internalized phages were detected by primary anti-pVIII antibody and secondary FITC-anti-mouse antibody. Images show the merging of phages (green) and DAPI-stained nuclei (blue) at the centered single confocal section. (B) Effect of input phage titer on the recovery of internalized recombinant phages. HeLa cells were incubated with 1010, 1011, or 1012 CFU of phages (input phage titer), thoroughly washed with low pH glycine buffer to remove surface bound phages, and lysed. Bacteria were infected with the cell lysates to determine output phage titer by CFU assay. Data represent mean ± S.E. of 3 independent experiments. (C) Plasmid DNA analysis from the recovered phage particles. Cell lysates prepared as described in (B) were transformed into bacteria and then plasmid DNA was extracted from randomly chosen two colonies (#1, #2). The recovered plasmid DNA was digested with SfiI to excise the DNA insert [3D8 VL (366 bp) or TAT (62 bp)] prior to agarose gel electrophoresis and visualization by ethidium bromide staining. ‘C’ indicates the original phagemid vector carrying the 3D8 VL or TAT gene. ‘M’, DNA size marker. (D) Effects of incubation time, temperature, and presence of serum proteins on internalization of 3D8 VL-M13 and TAT-M13 phages. HeLa cells were incubated with 3D8 VL-M13 or TAT-M13 for the indicated periods (left panels), at 4°C or 37°C (middle panels), or at 37°C in the presence or absence of 10% fetal bovine serum (right panels). Internalized phages were quantified by the CFU assay and represented as mean ± S.E. of 2 independent experiments as described in (B) and visualized by confocal immunofluorescence microscopy as described in (A). In (A) and (D), image magnification, ×400; scale bar, 5 µm.
Figure 3.
3D8 VL-M13 is internalized by caveolae-mediated endocytosis, whereas TAT-M13 by clathrin- and caveolae-mediated endocytosis.
Unless otherwise specified, HeLa cells (1×106 cells) in serum-free medium were treated at 37°C with 1012 CFU of 3D8 VL-M13 for 6 h or 1013 CFU of TAT-M13 for 2 h. (A) Effect of pre-treatment of specific endocytosis inhibitors on the internalization of 3D8 VL-M13 or TAT-M13. HeLa cells were pre-treated with CPZ (1 µg/ml), MβCD (5 mM), or Cyt-D (1 µg/ml) for 30 min and then incubated with 3D8 VL-M13 or TAT-M13. Internalized phages were visualized by confocal immunofluorescence microscopy using primary anti-pVIII antibody and secondary TRITC-anti-mouse antibody or quantified by the CFU assay and represented as mean ± S.E. (error bars) of 3 independent experiments. Images show the merging of phages (red) and DAPI-stained nuclei (blue) at the centered single confocal section. (B) Co-localization of internalized 3D8 VL-M13 or TAT-M13 with intracellular endocytosis markers. Cells were co-treated for 2 h with the recombinant phages and endocytosis markers, 10 µg/ml Alexa 488-transferrin (TF, green), Alexa 488-choleratoxin-B (Ctx-B, green), or FITC-dextran (Dextran, green), and then analyzed by confocal microscopy after staining for internalized phages (TRITC, red) as described in (A). The lower panels show enlarged images of the boxed region in the upper panels. (C) Knockdown of clathrin, caveolin-1, or dynamin by specific siRNA, monitored by Western blotting (left panels), and the effects on internalization of 3D8 VL-M13 and TAT-M13 (right panels). HeLa cells were transfected with the indicated siRNA for 48 h and then incubated with 3D8 VL-M13 and TAT-M13 prior to phage visualization by confocal immunofluorescence microscopy as described in (A). ‘Control siRNA’ means a scrambled siRNA used as a control. In (A–C), image magnification, ×400 and scale bar, 5 µm.
Figure 4.
3D8 VL-M13 and TAT-M13 mainly interact with HSPG and CSPG, respectively, as a cell surface receptor for the cellular internalization.
In all experiments, wild-type CHO-K1 and the mutant cells (1×106 cells) in serum-free medium were treated at 37°C with 1012 CFU of 3D8 VL-M13 for 6 h or 1013 CFU of TAT-M13 for 2 h. (A) Effects of the presence of soluble GAGs on internalization of 3D8 VL-M13 and TAT-M13. CHO-K1 cells were pre-treated with 100 IU/ml of heparin, or 50 µg/ml of CS-A, CS-B, and CS-C for 30 min and then incubated with 3D8 VL-M13 or TAT-M13. (B) Effects of treatment of cells with GAG lyases on the internalization of 3D8 VL-M13 and TAT-M13. CHO-K1 cells were pre-treated with 5 mIU/ml of heparinase III (Hep III) or 20 mIU/ml of chondroitinase ABC (Chon ABC) for 2 h at 37°C and then incubated with 3D8 VL-M13 or TAT-M13. (C) Internalization of 3D8 VL-M13 and TAT-M13 into CHO-K1 mutant cells genetically defective in GAG biosynthesis. Wild-type CHO-K1, HS-deficient pgsD-677 (no HS, 3 times more CS), or HS/CS-deficient pgsA-745 (no proteoglycans) cells were incubated with 3D8 VL-M13 or TAT-M13. In (A–C), internalized phages were visualized by confocal immunofluorescence microscopy using primary anti-pVIII antibody and secondary TRITC-anti-mouse antibody (A and B, red) or FITC-anti-mouse antibody (C, green) or quantified by the CFU assay and represented as mean ± S.E. (error bars) of three independent experiments. Image magnification, ×400; scale bar, 5 µm.
Figure 5.
Internalized 3D8 VL-M13 phage routes to the cytosol and remains stable without further trafficking to other subcellular compartments, whereas TAT-M13 phage is routed to other subcellular compartments before rapid degradation in the lysosome.
In the following pulse-chase experiments, HeLa cells (1×106 cells) in serum-free medium were treated at 37°C with 1012 CFU of 3D8 VL-M13 for 2 h or 1013 CFU of TAT-M13 for 30 min. Then surface bound phages were removed by multiple washes with low pH glycine buffer and then internalized phages were chased at 0, 2, 6, and 18 h. (A) Time-course intracellular localization of internalized phages was visualized by confocal immunofluorescence microscopy or time-course output phages were quantified by the CFU assay and represented as mean ± S.E. (error bars) of three independent experiments. (B) Time-course intracellular trafficking of internalized phages monitored by co-localization with transferrin (TF, a), caveolin-1 (b), early endosome marker EEA-1 (c), late endosome/lysosome tracker LysoTracker (d), ER marker calnexin (e), or Golgi marker 58K Golgi protein (f), as visualized by confocal immunofluorescence microscopy. In (A) and (B), internalized phages were visualized by confocal immunofluorescence microscopy with primary anti-pVIII antibody and secondary FITC-anti-mouse antibody (A and B, a, green) or TRITC-anti-mouse antibody (B, b-f, green). In (A) and (B), magnification, ×400; scale bar, 5 µm.