A Resealed-Cell System for Analyzing Pathogenic Intracellular Events: Perturbation of Endocytic Pathways under Diabetic Conditions

<div><p>Cell-based assay systems that can serve as cellular models of aberrant function in pathogenic organs would be novel and useful tools for screening drugs and clarifying the molecular mechanisms of various diseases. We constructed model cells that replicated the conditions in diabetic hepatocytes by using the cell resealing technique, which enables the exchange of cytosol. The plasma membrane of HeLa cells was permeabilized with the streptococcal toxin streptolysin O, and cytosol that had been prepared from wild-type or db/db diabetic mice was introduced into the resulting semi-intact cells. By resealing the plasma membrane by exposure to Ca²⁺, we created WT or Db model cells, in which the cytosolic conditions replicated those of healthy or diabetic liver. Interestingly, phosphorylation of p38 MAPK was promoted, whereas the level of endosomal phosphatidylinositol-3-phosphate was decreased, in Db cells. We investigated several endocytic pathways in WT and Db cells, and found that retrograde endosome-to-Golgi transport was delayed in a p38 MAPK-dependent manner in Db cells. Furthermore, the degradation pathway of the EGF receptor from endosomes to lysosomes was enhanced in Db cells, and this did not depend on the activation of p38 MAPK. The disease model cell system should become a powerful tool for the detection of aberrant processes in cells under pathogenic conditions and for therapeutic applications.</p> </div

Degradation of EGFR in WT and Db cells.

<p>A. HeLa cells were preincubated with DMEM without serum overnight, and were incubated without (WT and Db) or with (WT+SB and Db+SB) 2 µM SB203580 for 60 min. Semi-intact HeLa cells were incubated with WT or Db liver cytosol that contained Alexa546-conjugated dextran, and were resealed. After incubation with DMEM in the presence or absence of SB203580 for 30 min, the cells were treated with 10 ng/ml EGF, and then incubated with medium at 37°C for 0, 15, 30, and 60 min. The cells were fixed, and stained with anti-EGFR antibody. We were able to distinguish resealed cells, which were fluorescently labeled with dextran, from non-resealed cells easily under a fluorescence microscope. Bar  =  10 µm. B. HeLa cells were treated as described in A, lysed, and subjected to Western blotting using antibodies against EGFR and COX VI. C. Means and standard deviations for the band intensities of EGFR/COX VI are shown in the graph. We performed three independent experiments and verified the results by applying Student’s <i>t</i>-test. We found that the <i>P</i> value was > 0.05, which indicated that treatment with 2 µM SB203580 did not affect the enhanced degradation of EGFR by Db cytosol.</p

Intracellular PI3P was decreased in a p38 MAPK-dependent manner in Db cells.

<p>A. HeLa cells were pretreated with or without 2 µM SB203580 or 2 µM SB202190 at 37°C for 60 min. Semi-intact HeLa cells were incubated with 3 mg/ml WT or Db liver cytosol, an ATP regenerating system, GTP, and glucose in the presence or absence of 2 µM SB203580 or SB202190 at 32°C for 30 min, and then for a further 15 min at 32°C after the addition of 1 µg of GST-2xFYVE recombinant protein. The cells were fixed and GST-2xFYVE was visualized with Alexa488-conjugated antibodies against GST. Bar  =  10 µm. B. The fluorescence intensity of GST-2xFYVE was measured as described in Materials and Methods, and the means and standard deviations for the fluorescence intensity are shown in the graph. We performed three independent experiments and counted 100 cells in each experiment. Data were analyzed using one-way ANOVA and Dunnett’s post hoc test, and the <i>P</i> value was < 0.01 (**). C. Measurement of PI3P content in WT and Db cells by lipid blot. D. Western blotting of p38 MAPK and phosphorylated p38 MAPK in liver lysates from three WT or three Db mice. E. The intensities of the bands shown in C were measured and the relative proportion of phosphorylated p38 is shown as a percentage. Means and standard deviations for the relative proportion are shown in the graph. F. Western blotting of p38 MAPK and phosphorylated p38 MAPK in WT and Db cells.</p

Introduction of fluorescein-dextran of different molecular weights into resealed cells.

<p>A. HeLa cells were incubated with or without (2000 kDa dextran w/o SLO) 0.13 µg/ml SLO on ice for 5 min. After wash with PBS three times, the cells were further with transport buffer containing propidium iodide at 32°C for 5 min. Semi-intact HeLa cells were incubated with 1.5 mg/ml L5178Y cytosol, an ATP regenerating system, GTP, glucose, and 100 µg/ml fluorescein-dextran of 3, 10, 40, 70, or 2000 kDa at 32°C for 15 min, and then were resealed by treatment with 1 mM CaCl₂ at 32°C for 5 min. After incubation with DMEM supplemented with FCS for 30 min, the cells were observed by confocal microscopy. Since the cells without SLO treatment did not contain the fluorescence of propidium iodide, differential interference contrast (DIC) image was shown. Bar  =  10 µm. B. HeLa cells were treated as described in A, were trypsinized, and were subjected to flowcytometry. The histograms of fluorescein fluorescence of dextran with different molecular weight in PI-positive cells were shown.</p

Electron microscopic observation of WT and Db cells.

<p>Resealed WT (A, B, and C) and Db (D, E, and F) cells were observed by electron microscopy. G, M, or E indicates the Golgi apparatus, mitochondria, or endoplasmic reticulum, respectively. No significant difference in organelles morphology was observed between WT and Db cells. Bar  =  2 µm (A and D) or 1 µm (B, C, E, and F).</p

Permeabilization of HeLa cells with streptolysin O (SLO).

<p>A. Basic protocol for permeabilization of HeLa cells with SLO. B. HeLa cells were incubated with 0.10, 0.13, 0.20, or 0.40 µg/ml SLO on ice for 5 min. The cells were further incubated with transport buffer (TB) that contained propidium iodide (PI) at 32°C for 5 min, and were observed by confocal microscopy. Bar  =  10 µm. C. Dependency of permeabilization on SLO concentration. HeLa cells were treated as in B, and the means and standard deviations for the percentage of PI-positive permeabilized cells are shown in the graph. D. Dependency of permeabilization on number of cells. HeLa cells were grown on 3-cm dishes at a density of 1, 2, 3, 4, or 5×10⁵ cells per dish, and were permeabilized as in A. Means and standard deviations for the percentage of PI-positive permeabilized cells are shown in the graph. E. Dependency of permeabilization on incubation time on ice. HeLa cells were incubated with 0.13 µg/ml SLO on ice for 0.0, 1.0, 3.0, 5.0, or 7.5 min, and then further incubated with TB that contained PI at 32°C for 5 min. Means and standard deviations for the percentage of PI-positive permeabilized cells are shown in the graph. F. Dependency of permeabilization on incubation time at 32°C. HeLa cells were incubated with 0.13 µg/ml SLO on ice for 5 min, and then further incubated with TB that contained PI at 32°C for 0.0, 1.0, 3.0, 5.0, or 7.5 min. Means and standard deviations for the percentage of PI-positive permeabilized cells are shown in the graph.</p

Retrograde transport of Cholera toxin B subunit (CtxB) in WT and Db cells.

<p>A. Semi-intact HeLa cells were incubated with 3 mg/ml WT or Db liver cytosol in the presence of ATP/GTP/glucose and Alexa647-conjugated dextran (10 kDa, blue) at 32°C for 30 min, and then resealed by addition of 1 mM CaCl₂ for 5 min. After incubation with DMEM supplemented with 10% FCS for 30 min, the cells were treated with 2 µg/ml Alexa546-conjugated CtxB (red) on ice for 30 min, and then incubated with medium at 37°C for 0, 15, 30, and 45 min. The cells were fixed, were immunostained with antibodies against GM130 (green), and were observed by confocal microscope. Bar  =  10 µm. B. We counted the number of cells in which CtxB was accumulated at the Golgi on the basis of the colocalization of CtxB with GM130, after a 0, 15, 30, and 45 min chase in WT (○) and Db (•) cells with or without treatment of 2 µM SB203580 (△, WT+SB; ▴, Db+SB). The means and standard deviations for the percentages of these cells are shown in the graph. Three independent experiments were performed and we counted 300 cells in each experiment. C. CtxB transport assay was performed in WT or Db cells treated with or without 2 µM SB203580 or 2 µM SB202190. In 30 or 45 min after internalization of CtxB by incubating cells at 37°C, the cells were fixed and the number of cells in which CtxB was accumulated at the Golgi was counted. The means and standard deviations for the percentages of the cells are shown in the graph. Data were analyzed using one-way ANOVA and Dunnett’s post hoc test, and the <i>P</i> value was < 0.01 (**), which indicated that transport of CtxB to the Golgi was significantly delayed in Db cells compared to that in Db cells treated with SB203580 or SB202190.</p

<i>Ehrlichia</i> secretes Etf-1 to induce autophagy and capture nutrients for its growth through RAB5 and class III phosphatidylinositol 3-kinase

<p><i>Ehrlichia chaffeensis</i> is an obligatory intracellular bacterium that causes a potentially fatal emerging zoonosis, human monocytic ehrlichiosis. <i>E. chaffeensis</i> has a limited capacity for biosynthesis and metabolism and thus depends mostly on host-synthesized nutrients for growth. Although the host cell cytoplasm is rich with these nutrients, as <i>E. chaffeensis</i> is confined within the early endosome-like membrane-bound compartment, only host nutrients that enter the compartment can be used by this bacterium. How this occurs is unknown. We found that ehrlichial replication depended on autophagy induction involving class III phosphatidylinositol 3-kinase (PtdIns3K) activity, BECN1 (Beclin 1), and ATG5 (autophagy-related 5). <i>Ehrlichia</i> acquired host cell preincorporated amino acids in a class III PtdIns3K-dependent manner and ehrlichial growth was enhanced by treatment with rapamycin, an autophagy inducer. Moreover, ATG5 and RAB5A/B/C were routed to ehrlichial inclusions. <i>RAB5A/B/C</i> siRNA knockdown, or overexpression of a RAB5-specific GTPase-activating protein or dominant-negative RAB5A inhibited ehrlichial infection, indicating the critical role of GTP-bound RAB5 during infection. Both native and ectopically expressed ehrlichial type IV secretion effector protein, Etf-1, bound RAB5 and the autophagy-initiating class III PtdIns3K complex, PIK3C3/VPS34, and BECN1, and homed to ehrlichial inclusions. Ectopically expressed Etf-1 activated class III PtdIns3K as in <i>E. chaffeensis</i> infection and induced autophagosome formation, cleared an aggregation-prone mutant huntingtin protein in a class III PtdIns3K-dependent manner, and enhanced ehrlichial proliferation. These data support the notion that <i>E. chaffeensis</i> secretes Etf-1 to induce autophagy to repurpose the host cytoplasm and capture nutrients for its growth through RAB5 and class III PtdIns3K, while avoiding autolysosomal killing.</p

<i>Vps34</i> KO MEFs show a decrease in LC3 conjugation and LC3 puncta formation upon starvation.

<p>(<b>A</b>) Control and <i>Vps34</i> KO MEFs were cultured in normal medium (N) or HBSS (St) in the presence or absence of 50nM Bafilomycin (Nm+B or St+B, respectively) for 90 min. Right: Lysates were analyzed by immunoblotting using the indicated antibodies. Left: Relative LC3-II levels normalized to actin (n=4). (<b>B</b>) Control and <i>Vps34</i> KO MEFs were cultured in normal media (Nm) or HBSS (St) for 30 and 90 min, fixed and immunostained. Confocal analysis of LC3, p62 and GFP-Cre fluorescence, which is artificially shown in green, red and blue colors, respectively. Arrowheads indicate LC3 and p62 colocalization (yellow). Scale bar: 10 µm. (<b>C</b>) Quantification of the number of LC3 (left panel) and p62 puncta (middle panel) per cell. Colocalization of p62 with LC3 puncta is also shown (right panel) (colocalization was defined as the number of pixels overlapping in the p62 and LC3 channels normalized per cell) (n=35-45 cells).</p

Silencing class II PI3Ks decreases autophagy in both control and <i>Vps34</i> null MEFs.

<p>(<b>A</b>) Control MEFs were transfected with mock or PI3K-C2α/β siRNA as well as GFP-WIPI-1 for 48 hrs, cultured in normal media or HBSS for 90 min and fixed. Right: Confocal microscopy images of GFP-WIPI-1 fluorescence in mock or PI3K-C2α/β siRNA-treated control cells after HBSS starvation for 90 min. Scale bar: 10 µm. Left: Quantification of the number and size (arbitrary units) of GFP-WIPI-1 puncta observed after 90 min HBSS starvation (n=11-15 cells). (<b>B</b>) Cells prepared as in (A) were fixed and immunostained. Right: Confocal microscopy images showing endogenous LC3 (green) in cells cultured in HBSS in the presence of 50 nM Bafilomycin (St+B) for 30 min. DAPI is shown in blue. Scale bar: 10 µm. Left: Quantification of the number and size (arbitrary units) of LC3 puncta observed under normal media (Nm), HBSS (St) and HBSS in the presence of Bafilomycin (St+B) conditions (n=12-19, 23-40 and 26-53 cells for Nm, St and St+B conditions, respectively). Scale bars: 10 µm.(<b>C</b>) Control and <i>Vps34</i> KO MEFs were transfected for 48 hrs with mock or PI3K-C2α/β siRNA, cultured in normal medium (N), HBSS (St) or HBSS with 50 nM Bafilomycin (St+B) for 30 min, lysed and analyzed by immunoblotting using the indicated antibodies (n=3).</p