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The microtubule-binding protein CLIP-170 coordinates mDia1 and actin reorganization during CR3-mediated phagocytosis.

Lewkowicz E, Herit F, Le Clainche C, Bourdoncle P, Perez F, Niedergang F - J. Cell Biol. (2008)

Bottom Line: CLIP-170 directly interacts with the formin homology 2 domain of mDia1.The interaction between CLIP-170 and mDia1 is negatively regulated during alphaMbeta2-mediated phagocytosis.Our results unravel a new microtubule/actin cooperation that involves CLIP-170 and mDia1 and that functions downstream of alphaMbeta2 integrins.

View Article: PubMed Central - PubMed

Affiliation: Institut Cochin, Université Paris Descartes, Centre National de la Recherche Scientifique, Unité Mixte de Recherche 8104, 75014 Paris, France.

ABSTRACT
Microtubule dynamics are modulated by regulatory proteins that bind to their plus ends (+TIPs [plus end tracking proteins]), such as cytoplasmic linker protein 170 (CLIP-170) or end-binding protein 1 (EB1). We investigated the role of +TIPs during phagocytosis in macrophages. Using RNA interference and dominant-negative approaches, we show that CLIP-170 is specifically required for efficient phagocytosis triggered by alphaMbeta2 integrin/complement receptor activation. This property is not observed for EB1 and EB3. Accordingly, whereas CLIP-170 is dynamically enriched at the site of phagocytosis, EB1 is not. Furthermore, we observe that CLIP-170 controls the recruitment of the formin mDia1, an actin-nucleating protein, at the onset of phagocytosis and thereby controls actin polymerization events that are essential for phagocytosis. CLIP-170 directly interacts with the formin homology 2 domain of mDia1. The interaction between CLIP-170 and mDia1 is negatively regulated during alphaMbeta2-mediated phagocytosis. Our results unravel a new microtubule/actin cooperation that involves CLIP-170 and mDia1 and that functions downstream of alphaMbeta2 integrins.

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Related in: MedlinePlus

CLIP-170 is important for CR3-mediated phagocytosis. (A) RAW264.7 cells were transfected with pEGFP–CLIP-170ΔH, encoding a dominant-negative mutant of CLIP-170 (bottom) or with pEGFP as a control (top). After 24 h, macrophages were labeled with the anti–CLIP-170/CLIP-115 antibodies (#2221 serum) and with anti-tubulin antibodies, and then analyzed by wide-field fluorescence microscopy. Insets with magnified images are shown on the right. Bar, 10 μm. (B) A dominant-negative form of CLIP-170 inhibits CR3-mediated phagocytosis. Macrophages were transfected with pEGFP–CLIP-170ΔH or with pEGFP as a control. After 24 h, macrophages were allowed to phagocytose C3bi-SRBCs for 3 and 60 min at 37°C, and then fixed and stained with Cy3–anti-rabbit IgG antibodies to detect SRBCs. Efficiencies of association and phagocytosis were scored in 50 cells expressing GFP–CLIP-170ΔH and 50 GFP-expressing control cells. Results are expressed as a percentage of control cells. The means ± SEM of at least three independent experiments are plotted. *, P < 0.0001. (C) RAW264.7 macrophages were transfected with pSUPER-115A, pSUPER-115B, or pSUPER-G (directed against giantin) as a control. After 48 h, lysates were prepared and Western blotting was performed with anti–CLIP-170/115 antibodies (top) or with anti-clathrin (bottom) as a loading control. (D) RAW264.7 macrophages were transfected with pSUPER-170A (directed against CLIP-170), pSUPER-AB (directed against CLIP-170 and CLIP-115), or pSUPER-G (directed against giantin) as a control. After 48 h, lysates were prepared and Western blotting was performed with anti–CLIP-170/115 antibodies (10 s [top] or 1 min [middle] of film exposure) or with anti-clathrin (bottom). In addition, cells were fixed, permeabilized, and labeled with anti–CLIP-170/115 antibodies followed by Cy3–anti-rabbit IgG antibodies. Cells were analyzed by wide-field fluorescence microscopy (right). The image shows a cell depleted of CLIP-170/115 with pSUPER-AB (asterisk). Bar, 10 μm. (E) Depletion of CLIP-170 but not CLIP-115 inhibits phagocytosis. Macrophages were transfected as described in C and D, and then allowed to phagocytose C3bi-SRBCs for 3 and 60 min at 37°C and fixed and stained with Cy2–anti-rabbit IgG antibodies and, after permeabilization, with anti–CLIP-170 or anti–CLIP-115 antibodies. The efficiencies of association and phagocytosis were calculated for 50 CLIP-115–depleted cells (115A and 115B), 50 CLIP-170–depleted cells (170A), 50 CLIP-170/115–depleted cells (AB), and 50 control cells. Results are expressed as a percentage of control cells. The means ± SEM of three independent experiments are plotted. *, P < 0.05; **, P < 0.005; ***, P < 0.001; ****, P < 0.0001.
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fig2: CLIP-170 is important for CR3-mediated phagocytosis. (A) RAW264.7 cells were transfected with pEGFP–CLIP-170ΔH, encoding a dominant-negative mutant of CLIP-170 (bottom) or with pEGFP as a control (top). After 24 h, macrophages were labeled with the anti–CLIP-170/CLIP-115 antibodies (#2221 serum) and with anti-tubulin antibodies, and then analyzed by wide-field fluorescence microscopy. Insets with magnified images are shown on the right. Bar, 10 μm. (B) A dominant-negative form of CLIP-170 inhibits CR3-mediated phagocytosis. Macrophages were transfected with pEGFP–CLIP-170ΔH or with pEGFP as a control. After 24 h, macrophages were allowed to phagocytose C3bi-SRBCs for 3 and 60 min at 37°C, and then fixed and stained with Cy3–anti-rabbit IgG antibodies to detect SRBCs. Efficiencies of association and phagocytosis were scored in 50 cells expressing GFP–CLIP-170ΔH and 50 GFP-expressing control cells. Results are expressed as a percentage of control cells. The means ± SEM of at least three independent experiments are plotted. *, P < 0.0001. (C) RAW264.7 macrophages were transfected with pSUPER-115A, pSUPER-115B, or pSUPER-G (directed against giantin) as a control. After 48 h, lysates were prepared and Western blotting was performed with anti–CLIP-170/115 antibodies (top) or with anti-clathrin (bottom) as a loading control. (D) RAW264.7 macrophages were transfected with pSUPER-170A (directed against CLIP-170), pSUPER-AB (directed against CLIP-170 and CLIP-115), or pSUPER-G (directed against giantin) as a control. After 48 h, lysates were prepared and Western blotting was performed with anti–CLIP-170/115 antibodies (10 s [top] or 1 min [middle] of film exposure) or with anti-clathrin (bottom). In addition, cells were fixed, permeabilized, and labeled with anti–CLIP-170/115 antibodies followed by Cy3–anti-rabbit IgG antibodies. Cells were analyzed by wide-field fluorescence microscopy (right). The image shows a cell depleted of CLIP-170/115 with pSUPER-AB (asterisk). Bar, 10 μm. (E) Depletion of CLIP-170 but not CLIP-115 inhibits phagocytosis. Macrophages were transfected as described in C and D, and then allowed to phagocytose C3bi-SRBCs for 3 and 60 min at 37°C and fixed and stained with Cy2–anti-rabbit IgG antibodies and, after permeabilization, with anti–CLIP-170 or anti–CLIP-115 antibodies. The efficiencies of association and phagocytosis were calculated for 50 CLIP-115–depleted cells (115A and 115B), 50 CLIP-170–depleted cells (170A), 50 CLIP-170/115–depleted cells (AB), and 50 control cells. Results are expressed as a percentage of control cells. The means ± SEM of three independent experiments are plotted. *, P < 0.05; **, P < 0.005; ***, P < 0.001; ****, P < 0.0001.

Mentions: We first set out to study the dynamics of microtubules during phagocytosis. For this, we either labeled microtubule plus ends by immunofluorescence using an anti–CLIP-170 antibody or expressed a YFP–CLIP-170 construct transiently in RAW264.7 cells. As in other cells (Perez et al., 1999; Komarova et al., 2002), CLIP-170 labeling appears as cometlike structures at the plus ends of microtubules (Fig. 1 A and Fig. 2 A). We followed YFP–CLIP-170 comet movement in resting cells using a tracking software. Although the majority of the comets arose from the microtubule-organizing center and moved toward the periphery, some comets seemed to be generated at a more distal site. The mean speed measured was 0.3 ± 0.016 μm/s (n = 1360 comets), which is in agreement with the speed measured in other cell types (Perez et al., 1999; unpublished data).


The microtubule-binding protein CLIP-170 coordinates mDia1 and actin reorganization during CR3-mediated phagocytosis.

Lewkowicz E, Herit F, Le Clainche C, Bourdoncle P, Perez F, Niedergang F - J. Cell Biol. (2008)

CLIP-170 is important for CR3-mediated phagocytosis. (A) RAW264.7 cells were transfected with pEGFP–CLIP-170ΔH, encoding a dominant-negative mutant of CLIP-170 (bottom) or with pEGFP as a control (top). After 24 h, macrophages were labeled with the anti–CLIP-170/CLIP-115 antibodies (#2221 serum) and with anti-tubulin antibodies, and then analyzed by wide-field fluorescence microscopy. Insets with magnified images are shown on the right. Bar, 10 μm. (B) A dominant-negative form of CLIP-170 inhibits CR3-mediated phagocytosis. Macrophages were transfected with pEGFP–CLIP-170ΔH or with pEGFP as a control. After 24 h, macrophages were allowed to phagocytose C3bi-SRBCs for 3 and 60 min at 37°C, and then fixed and stained with Cy3–anti-rabbit IgG antibodies to detect SRBCs. Efficiencies of association and phagocytosis were scored in 50 cells expressing GFP–CLIP-170ΔH and 50 GFP-expressing control cells. Results are expressed as a percentage of control cells. The means ± SEM of at least three independent experiments are plotted. *, P < 0.0001. (C) RAW264.7 macrophages were transfected with pSUPER-115A, pSUPER-115B, or pSUPER-G (directed against giantin) as a control. After 48 h, lysates were prepared and Western blotting was performed with anti–CLIP-170/115 antibodies (top) or with anti-clathrin (bottom) as a loading control. (D) RAW264.7 macrophages were transfected with pSUPER-170A (directed against CLIP-170), pSUPER-AB (directed against CLIP-170 and CLIP-115), or pSUPER-G (directed against giantin) as a control. After 48 h, lysates were prepared and Western blotting was performed with anti–CLIP-170/115 antibodies (10 s [top] or 1 min [middle] of film exposure) or with anti-clathrin (bottom). In addition, cells were fixed, permeabilized, and labeled with anti–CLIP-170/115 antibodies followed by Cy3–anti-rabbit IgG antibodies. Cells were analyzed by wide-field fluorescence microscopy (right). The image shows a cell depleted of CLIP-170/115 with pSUPER-AB (asterisk). Bar, 10 μm. (E) Depletion of CLIP-170 but not CLIP-115 inhibits phagocytosis. Macrophages were transfected as described in C and D, and then allowed to phagocytose C3bi-SRBCs for 3 and 60 min at 37°C and fixed and stained with Cy2–anti-rabbit IgG antibodies and, after permeabilization, with anti–CLIP-170 or anti–CLIP-115 antibodies. The efficiencies of association and phagocytosis were calculated for 50 CLIP-115–depleted cells (115A and 115B), 50 CLIP-170–depleted cells (170A), 50 CLIP-170/115–depleted cells (AB), and 50 control cells. Results are expressed as a percentage of control cells. The means ± SEM of three independent experiments are plotted. *, P < 0.05; **, P < 0.005; ***, P < 0.001; ****, P < 0.0001.
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Related In: Results  -  Collection

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Show All Figures
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fig2: CLIP-170 is important for CR3-mediated phagocytosis. (A) RAW264.7 cells were transfected with pEGFP–CLIP-170ΔH, encoding a dominant-negative mutant of CLIP-170 (bottom) or with pEGFP as a control (top). After 24 h, macrophages were labeled with the anti–CLIP-170/CLIP-115 antibodies (#2221 serum) and with anti-tubulin antibodies, and then analyzed by wide-field fluorescence microscopy. Insets with magnified images are shown on the right. Bar, 10 μm. (B) A dominant-negative form of CLIP-170 inhibits CR3-mediated phagocytosis. Macrophages were transfected with pEGFP–CLIP-170ΔH or with pEGFP as a control. After 24 h, macrophages were allowed to phagocytose C3bi-SRBCs for 3 and 60 min at 37°C, and then fixed and stained with Cy3–anti-rabbit IgG antibodies to detect SRBCs. Efficiencies of association and phagocytosis were scored in 50 cells expressing GFP–CLIP-170ΔH and 50 GFP-expressing control cells. Results are expressed as a percentage of control cells. The means ± SEM of at least three independent experiments are plotted. *, P < 0.0001. (C) RAW264.7 macrophages were transfected with pSUPER-115A, pSUPER-115B, or pSUPER-G (directed against giantin) as a control. After 48 h, lysates were prepared and Western blotting was performed with anti–CLIP-170/115 antibodies (top) or with anti-clathrin (bottom) as a loading control. (D) RAW264.7 macrophages were transfected with pSUPER-170A (directed against CLIP-170), pSUPER-AB (directed against CLIP-170 and CLIP-115), or pSUPER-G (directed against giantin) as a control. After 48 h, lysates were prepared and Western blotting was performed with anti–CLIP-170/115 antibodies (10 s [top] or 1 min [middle] of film exposure) or with anti-clathrin (bottom). In addition, cells were fixed, permeabilized, and labeled with anti–CLIP-170/115 antibodies followed by Cy3–anti-rabbit IgG antibodies. Cells were analyzed by wide-field fluorescence microscopy (right). The image shows a cell depleted of CLIP-170/115 with pSUPER-AB (asterisk). Bar, 10 μm. (E) Depletion of CLIP-170 but not CLIP-115 inhibits phagocytosis. Macrophages were transfected as described in C and D, and then allowed to phagocytose C3bi-SRBCs for 3 and 60 min at 37°C and fixed and stained with Cy2–anti-rabbit IgG antibodies and, after permeabilization, with anti–CLIP-170 or anti–CLIP-115 antibodies. The efficiencies of association and phagocytosis were calculated for 50 CLIP-115–depleted cells (115A and 115B), 50 CLIP-170–depleted cells (170A), 50 CLIP-170/115–depleted cells (AB), and 50 control cells. Results are expressed as a percentage of control cells. The means ± SEM of three independent experiments are plotted. *, P < 0.05; **, P < 0.005; ***, P < 0.001; ****, P < 0.0001.
Mentions: We first set out to study the dynamics of microtubules during phagocytosis. For this, we either labeled microtubule plus ends by immunofluorescence using an anti–CLIP-170 antibody or expressed a YFP–CLIP-170 construct transiently in RAW264.7 cells. As in other cells (Perez et al., 1999; Komarova et al., 2002), CLIP-170 labeling appears as cometlike structures at the plus ends of microtubules (Fig. 1 A and Fig. 2 A). We followed YFP–CLIP-170 comet movement in resting cells using a tracking software. Although the majority of the comets arose from the microtubule-organizing center and moved toward the periphery, some comets seemed to be generated at a more distal site. The mean speed measured was 0.3 ± 0.016 μm/s (n = 1360 comets), which is in agreement with the speed measured in other cell types (Perez et al., 1999; unpublished data).

Bottom Line: CLIP-170 directly interacts with the formin homology 2 domain of mDia1.The interaction between CLIP-170 and mDia1 is negatively regulated during alphaMbeta2-mediated phagocytosis.Our results unravel a new microtubule/actin cooperation that involves CLIP-170 and mDia1 and that functions downstream of alphaMbeta2 integrins.

View Article: PubMed Central - PubMed

Affiliation: Institut Cochin, Université Paris Descartes, Centre National de la Recherche Scientifique, Unité Mixte de Recherche 8104, 75014 Paris, France.

ABSTRACT
Microtubule dynamics are modulated by regulatory proteins that bind to their plus ends (+TIPs [plus end tracking proteins]), such as cytoplasmic linker protein 170 (CLIP-170) or end-binding protein 1 (EB1). We investigated the role of +TIPs during phagocytosis in macrophages. Using RNA interference and dominant-negative approaches, we show that CLIP-170 is specifically required for efficient phagocytosis triggered by alphaMbeta2 integrin/complement receptor activation. This property is not observed for EB1 and EB3. Accordingly, whereas CLIP-170 is dynamically enriched at the site of phagocytosis, EB1 is not. Furthermore, we observe that CLIP-170 controls the recruitment of the formin mDia1, an actin-nucleating protein, at the onset of phagocytosis and thereby controls actin polymerization events that are essential for phagocytosis. CLIP-170 directly interacts with the formin homology 2 domain of mDia1. The interaction between CLIP-170 and mDia1 is negatively regulated during alphaMbeta2-mediated phagocytosis. Our results unravel a new microtubule/actin cooperation that involves CLIP-170 and mDia1 and that functions downstream of alphaMbeta2 integrins.

Show MeSH
Related in: MedlinePlus