Journal: The Journal of Biological Chemistry

Article Title: Constitutive Endocytosis of the Chemokine CX 3 CL1 Prevents Its Degradation by Cell Surface Metalloproteases *

doi: 10.1074/jbc.M109.045682

Figure Lengend Snippet: CX3CL1 undergoes temperature-sensitive internalization from the cell surface. A, ECV-CX3CL1 cells were fixed, permeabilized, and incubated with anti-CX3CL1 Ab (2.5 μg/ml), followed by Cy3-conjugated anti-goat-IgG. The cells were examined using a Leica DMIRE2 spinning disc confocal microscope with a Hamamatsu backthinned EM-CCD camera. The images were acquired using the appropriate excitation and emission filters and a 100× oil immersion objective. B, ECV-CX3CL1 cells were incubated with anti-CX3CL1 Ab (2.5 μg/ml) at 37 °C for 1 h, then fixed, permeabilized, and incubated with Cy3-conjugated anti-goat IgG. The cells were examined using a Leica DMIRE2 spinning disc confocal microscope at 100× magnification. Optical sections of 0.2–0.5 μm were obtained. The lower panel depicts x versus z optical section. C, cells were incubated with anti-CX3CL1 Ab (2.5 μg/ml) at 4 °C for 1 h, then fixed, permeabilized, and incubated with Cy3-conjugated anti-goat IgG. The cells were examined using a Leica DMIRE2 spinning disc confocal microscope at 100× magnification. Optical sections of 0.2–0.5 μm were obtained. The lower panel depicts x versus z optical section. D, from each of three separate experiments, at least 40 cells were examined. The fraction of cells demonstrating intracellular labeling of CX3CL1 was compared between samples from experiments performed at 37 and at 4 °C.

Article Snippet: The distribution of AP-2 ( A ) was compared with that of CX 3 CL1 ( B ) using a spinning disc DMIRE2 confocal microscope with a Hamamatsu backthinned EM-CCD camera (100×).

Techniques: Incubation, Microscopy, Labeling

Journal: The Journal of Biological Chemistry

Article Title: Constitutive Endocytosis of the Chemokine CX 3 CL1 Prevents Its Degradation by Cell Surface Metalloproteases *

doi: 10.1074/jbc.M109.045682

Figure Lengend Snippet: Endocytosis of CX3CL1 is dynamin-dependent. A–D, ECV-CX3CL1 cells were transfected with a dominant negative allele of dynamin-1 (K44A). To allow identification of transfected cells, cells were co-transfected with EGFP at a ratio of 1:10. A and B, cells were incubated with transferrin-rhodamine (30 μg/ml) at 37 °C for 1 h, then fixed, and examined using a Leica DMIRE2 inverted microscope at 63× magnification. The distribution of transferrin-rhodamine (B) was compared in cells expressing K44A (A) to transferrin-rhodamine distribution in untransfected cells. C and D, cells were incubated with anti-CX3CL1 Ab (2.5 μg/ml) at 37 °C for 1 h. The cells were washed, fixed, and permeabilized prior to incubating with Cy3-conjugated anti-goat IgG. The cells were examined using a Leica DMIRE2 microscope at 63× magnification. The distribution of CX3CL1 (D) was compared in cells expressing K44A (C) to CX3CL1 distribution in untransfected cells. E–H, to label the plasma membrane, cells were transfected with cDNA encoding red fluorescent protein-tagged K-Ras R-pre (E). After 48 h, the cells were incubated with anti-CX3CL1 Ab (2.5 μg/ml) at 37 °C for 1 h, then fixed, permeabilized, and incubated with Alexa488-conjugated anti-goat IgG (F). The cells were examined at 100× magnification using a Leica DMIRE2 spinning disc confocal microscope with a backthinned EM-CCD camera. The images were acquired using the appropriate excitation and emission filters. Optical sections of 0.2–0.5 μm were obtained. E, a representative x versus z image showing R-pre-RFP expression in a single cell is depicted. F, x versus z image showing CX3CL1 distribution in the same cell. G, merged image of E and F. H, cells were transfected with HA-tagged dominant negative (DN) dynamin-1 (K44A) together with R-pre-RFP. The cells were incubated with anti-CX3CL1 Ab at 37 °C for 1 h, then fixed, permeabilized, and incubated with an Alexa488-conjugated secondary Ab. To identify cells expressing the K44A mutant allele, the cells were incubated with anti-HA Ab followed by a Cy5-conjugated secondary Ab. Using Volocity™ co-localization software, the percentage of total CX3CL1 signal that co-localized with plasma membrane R-pre-RFP was calculated for at least 20 cells from three separate experiments. *, p < 0.0001.

Article Snippet: The distribution of AP-2 ( A ) was compared with that of CX 3 CL1 ( B ) using a spinning disc DMIRE2 confocal microscope with a Hamamatsu backthinned EM-CCD camera (100×).

Techniques: Transfection, Dominant Negative Mutation, Incubation, Inverted Microscopy, Expressing, Microscopy, Mutagenesis, Software

Journal: The Journal of Biological Chemistry

Article Title: Constitutive Endocytosis of the Chemokine CX 3 CL1 Prevents Its Degradation by Cell Surface Metalloproteases *

doi: 10.1074/jbc.M109.045682

Figure Lengend Snippet: Plasmalemmal CX3CL1 undergoes CME. A–D, ECV-CX3CL1 cells were grown on coverslips, fixed, permeabilized, and incubated with anti-clathrin mAb (A; 3 μg/ml) together with anti-CX3CL1 mAb (B; 2.5 μg/ml). The cells were washed and incubated with Cy2-conjugated anti-mouse IgG to visualize clathrin, and Cy3-conjugated anti-goat IgG to visualize CX3CL1. The cells were examined using a spinning disc Leica DMIRE2 confocal microscope. The images were acquired using the appropriate excitation and emission filters and a 100× oil immersion objective. The ruler bar is 10 μm. The distribution of clathrin (A) was compared with that of CX3CL1 (B). C, merged image of A and B. D, magnified view of the area indicated in C. E, ECV-CX3CL1 cells were electroporated with siRNA targeting clathrin or with control, nontargeting siRNA on Days 0 and 2. On Day 4, the cell lysates were harvested, and immunoblotting was performed using anti-clathrin Ab (0.3 μg/ml) and horseradish peroxidase-conjugated secondary Ab. To control for protein loading, blots were stripped and reprobed with anti-actin Ab and horseradish peroxidase-conjugated secondary Ab. A representative blot is depicted. Shown are the mean values ± S.E. for clathrin expression (normalized to actin) from four separate experiments. p < 0.05 versus untreated cells or versus control siRNA-treated cells. F–N, cells were electroporated with clathrin siRNA (G, J, and M) or control, nontargeting siRNA (H, K, and N) by nucleofection on Days 0 and 2. After the second electroporation, the cells were plated on glass coverslips and were analyzed on Day 4. The cells were incubated with transferrin-rhodamine (30 μg/ml; I–K) or anti-CX3CL1 Ab (2.5 μg/ml; L–N) at 37 °C for 1 h. The cells were washed, fixed, permeabilized, and incubated with anti-clathrin Ab (3 μg/ml) for 1 h. The cells were again washed and then incubated with Cy2-conjugated anti-mouse IgG. Cells that had been labeled with anti-CX3CL1 Ab were also incubated with Cy3-conjugated anti-goat IgG. In untransfected cells (F), transferrin was internalized (I). In cells in which clathrin expression was knocked down (G), transferrin labeled mainly the cell surface, with little internalization (J). In cells transfected with control siRNA (H), clathrin expression was similar to that of untransfected cells (compare H–F), and transferrin was internalized (K). In untransfected cells (L) and cells treated with control siRNA (N), anti-CX3CL1 Ab was internalized from the plasma membrane. In cells treated with clathrin siRNA, anti-CX3CL1 Ab labeled mainly the cell surface, with little internalization (M). From each of three separate experiments, at least 25 cells were examined. The fraction of cells demonstrating internal labeling of CX3CL1 was determined and compared for cells expressing control nontargeting siRNA and clathrin siRNA.

Article Snippet: The distribution of AP-2 ( A ) was compared with that of CX 3 CL1 ( B ) using a spinning disc DMIRE2 confocal microscope with a Hamamatsu backthinned EM-CCD camera (100×).

Techniques: Incubation, Microscopy, Western Blot, Expressing, Electroporation, Labeling, Transfection

Journal: The Journal of Biological Chemistry

Article Title: Constitutive Endocytosis of the Chemokine CX 3 CL1 Prevents Its Degradation by Cell Surface Metalloproteases *

doi: 10.1074/jbc.M109.045682

Figure Lengend Snippet: AP-2-binding motifs within the cytoplasmic tail of CX3CL1 enable internalization of the chemokine. A and B, ECV-CX3CL1 cells were grown on coverslips, fixed, permeabilized, and incubated with anti-AP-2 mAb (5 μg/ml; A) together with anti-CX3CL1 mAb (2.5 μg/ml; B). The cells were washed and incubated with Cy3-conjugated anti-goat IgG and Cy2-conjugated anti-mouse IgG secondary Ab to detect CX3CL1 and AP-2, respectively. The distribution of AP-2 (A) was compared with that of CX3CL1 (B) using a spinning disc DMIRE2 confocal microscope with a Hamamatsu backthinned EM-CCD camera (100×). The ruler bar is 10 μm. C, merged image of A and B. D, magnified view of the area indicated in C. E, ECV-304 cells stably expressing mutant CX3CL1 lacking the intracellular domain (ECV-CX3CL1–360) were fixed, permeabilized, and incubated with anti-CX3CL1 Ab (2.5 μg/ml), followed by Cy3-conjugated anti-goat-IgG. The cells were examined using a Leica DMIRE2 deconvolution microscope at 100× magnification. For comparison, the inset shows ECV-304 cell expressing full-length CX3CL1 (ECV-CX3CL1) labeled with anti-CX3CL1 Ab followed by Cy3-conjugated anti-goat-IgG. F, ECV-CX3CL1–360 cells were incubated with anti-CX3CL1 Ab (2.5 μg/ml) at 37 °C for 1 h, then fixed, permeabilized, and incubated with Cy3-conjugated secondary Ab. Cells were examined as described in E. G, Ab uptake experiments were performed in cells expressing CX3CL1–360 or full-length CX3CL1 as described in F. The cells were examined using a spinning disc Leica DMIRE2 inverted microscope with a Hamamatsu ORCAER charge-coupled device camera (100×) and Z-stacks obtained. Upper panel, x versus z image from cell expressing CX3CL1–360. Lower panel, x versus z image from cell expressing full-length CX3CL1. H, site-directed mutagenesis was used to generate cDNA constructs in which the critical tyrosine residue of each AP-2-binding motif was replaced by an alanine residue, separately or in combination (Y362A, Y392A, and Y362A-Y392A). H–K, ECV-304 cells were co-transfected with cDNA encoding RFP-tagged R-pre, a plasma membrane marker, together with full-length CX3CL1, CX3CL1-Y362A, CX3CL1-Y392A, or CX3CL1-Y362A-Y392A. I–L, cells were incubated with anti-CX3CL1 Ab (2.5 μg/ml) at 37 °C for 1 h, then fixed, permeabilized, and incubated with Cy5-conjugated secondary Ab to visualize uptake of the chemokine. The cells were examined at 100× magnification using a Leica DMIRE2 spinning disc confocal microscope with a backthinned EM-CCD camera. The images were acquired using the appropriate excitation and emission filters. Optical sections of 0.2–0.5 μm were obtained. I, cell expressing full-length CX3CL1. J, cell expressing CX3CL1-Y362A. K, cell expressing CX3CL1-Y392A. L, cell expressing CX3CL1-Y362A-Y392A. M, experiments were performed as in I–L. As described for Fig. 2 (E–H), using Volocity™ co-localization software, the proportion of total CX3CL1 that co-localized with the R-pre membrane marker was determined. The values represent the means ± S.E. for 25–30 cells/condition from three separate experiments. *, p < 0.01 versus full-length CX3CL1.

Article Snippet: The distribution of AP-2 ( A ) was compared with that of CX 3 CL1 ( B ) using a spinning disc DMIRE2 confocal microscope with a Hamamatsu backthinned EM-CCD camera (100×).

Techniques: Binding Assay, Incubation, Microscopy, Stable Transfection, Expressing, Mutagenesis, Labeling, Inverted Microscopy, Construct, Transfection, Marker, Software

Journal: The Journal of Biological Chemistry

Article Title: Constitutive Endocytosis of the Chemokine CX 3 CL1 Prevents Its Degradation by Cell Surface Metalloproteases *

doi: 10.1074/jbc.M109.045682

Figure Lengend Snippet: Constitutive CME of CX3CL1 protects the chemokine from proteolytic shedding by membrane-anchored metalloproteases. A, ECV-CX3CL1 cells were fixed, permeabilized, and labeled with anti-CX3CL1 Ab (2.5 μg/ml), followed by Cy3-conjugated anti-goat IgG. The cells were examined using a spinning disc Leica DMIRE2 confocal microscope at 100× magnification. Optical sections of 0.2–0.5 μm were obtained. Lower panel, x versus z image. B, ECV-CX3CL1 cells were fixed, permeabilized, and labeled with anti-TACE Ab (2.7 μg/ml), followed by Cy3-conjugated anti-mouse IgG. The cells were visualized as in A. C, ECV-CX3CL1 cells were fixed, permeabilized, and labeled with anti-ADAM10 Ab (10 μg/ml), followed by Cy3-conjugated anti-mouse IgG. The cells were visualized as in A. D, cells were incubated with Dynasore (160 μm) for 30 min at 37 °C, conditioned medium was collected, and cell lysates were harvested. In some experiments, the cells were pretreated with the metalloprotease inhibitor, GM6001 (20 μm for 4 h), prior to incubation with Dynasore. Protease inhibitor mixture was added to conditioned medium and to cell lysis buffer to prevent protein degradation. Supernatants and cell lysates were cleared by centrifugation. Using fresh samples, CX3CL1 was measured using a CX3CL1 ELISA Kit (R & D Systems), according to the manufacturer's specifications. Three independent experiments were performed, and the data were expressed as the means ± S.E. of the normalized soluble CX3CL1 released into the conditioned medium in relation to the total amount of cell-associated CX3CL1 in the corresponding well. **, p < 0.001; *, p < 0.05. E, cells were incubated with metalloprotease inhibitor, GM6001 (20 μm) for 4 h, followed by Dynasore for 30 min, and surface and total CX3CL1 expression was determined as in Fig. 2 E–H. As described for Fig. 2 (E–H), the fraction of total CX3CL1 that was present at the plasma membrane was determined for 10 cells from three separate experiments. The results are expressed as the mean values ± S.E. *, p < 0.01. F, ELISA experiments were performed as described in D, using conditioned medium harvested from cells expressing the native form of CX3CL1, CX3CL1-Y362A, CX3CL1-Y392A, or CX3CL1-Y362A-Y392A. Three independent experiments were performed, and the data were expressed as the mean ± S.E. of the soluble CX3CL1 released into the conditioned medium in relation to the total amount of cell-associated CX3CL1 in the corresponding well. *, p < 0.0005 versus CX3CL1; **, p < 0.005 versus CX3CL1.

Article Snippet: The distribution of AP-2 ( A ) was compared with that of CX 3 CL1 ( B ) using a spinning disc DMIRE2 confocal microscope with a Hamamatsu backthinned EM-CCD camera (100×).

Techniques: Labeling, Microscopy, Incubation, Protease Inhibitor, Lysis, Centrifugation, Enzyme-linked Immunosorbent Assay, Expressing