Dynamin 2 is required for phagocytosis in macrophages

Abstract

Cells internalize soluble ligands through endocytosis and large particles through actin-based phagocytosis. The dynamin family of GTPases mediates the scission of endocytic vesicles from the plasma membrane. We report here that dynamin 2, a ubiquitously expressed dynamin isoform, has a role in phagocytosis in macrophages. Dynamin 2 is enriched on early phagosomes, and expression of a dominant-negative mutant of dynamin 2 significantly inhibits particle internalization at the stage of membrane extension around the particle. This arrest in phagocytosis resembles that seen with inhibitors of phosphoinositide 3-kinase (PI3K), and inhibition of PI3K prevents the recruitment of dynamin to the site of particle binding. Although expression of mutant dynamin in macrophages inhibited particle internalization, it had no effect on the production of inflammatory mediators elicited by particle binding.

Figure 1

Dynamin 2 is enriched on murine macrophage phagosomes. (A) 20 μg of total protein extract from RP macrophages (PMφ), RAW-TT10 macrophages, or brain was separated on a 10% SDS-PAGE gel, transferred to polyvinylidene difluoride membrane, and probed with an anti-dynamin 2 (Dyn 2), or an anti-dynamin 1 (DG2) antibody as indicated. A protein of the expected 100-kD size was detected in macrophages by the anti-dynamin 2 antibody but was not detected by the anti-dynamin 1 antibody. (B) Synchronized phagosomes containing either zymosan particles (i–vi), complement-opsonized RBCs (vii), or IgG-opsonized RBCs (viii) were created in RP macrophages (i, ii, iii, vii, viii) or in transiently transfected RAW-TT10 cell expressing V5 epitope–tagged dynamin 2 (iv–vi). 5 min after particle binding, cells were fixed and prepared for immunofluorescence. Dynamin 2 was detected with the anti-dynamin 2 antibody Dyn 2 (i, vii, and viii) or the pan anti-dynamin antibody MC63 (iii); V5 epitope–tagged dynamin was detected with anti-V5 antibody (iv and v); actin was stained with rhodamine-phalloidin (vi); and zymosan was visualized directly (ii). Dynamin enrichment on phagocytic cups and phagosomes (v) colocalized with F-actin (vi).

Figure 2

Dyn^K44A inhibits macrophage phagocytosis. Transiently transfected RAW-TT10 macrophages internalized the indicated particles for 10 min. Uninternalized particles were removed, and the cells were analyzed by flow cytometry. (A) Dyn^K44A inhibited phagocytosis in a dose-dependent manner. Dyn^K44A expression, as determined by GFP fluorescence, is plotted on a logarithmic scale on the x-axis. Phagocytosis, expressed as the percentage of cells internalizing particles relative to the percentage of nonexpressing control cells internalizing particles is plotted on the y-axis. The SEM from four independent experiments is shown, but the error bars do not extend beyond the symbols. (B) A typical experiment is shown. RAW-TT10 cells transfected with dyn^K44A-pTIGZ2 were incubated with TRITC-zymosan and analyzed. The level of transgene expression is expressed on the x-axis (GFP fluorescence), and the phagocytosis of labeled particles is shown on the y-axis (TRITC fluorescence). (C) RAW-TT10 cells were transiently transfected with either pTIGZ2 vector alone (control) or dyn^K44A-pTIGZ2, and phagocytosis of TRITC-loaded IgG-opsonized SRBCs, TRITC-loaded complement-opsonized SRBCs, or TRITC-zymosan was assessed. Phagocytosis is expressed as the percentage of transfected cells internalizing particles relative to the percentage of untransfected cells internalizing particles. Control and dyn^K44A cells expressing the same level of GFP were compared. The data shown represent a minimum of three independent experiments, and error bars reflect SEM.

Figure 3

Dynamin 2 blocks receptor-mediated endocytosis in macrophages. RAW-TT10 cells, transfected with the TIGZ control vector (thin solid line) or the dyn^K44A vector (thick solid line), were incubated with 20 μg/ml of DiI-labeled acetylated LDL (DiI AcLDL) for 30 min at 37°C. As a control, untransfected cells were incubated with the same concentration of DiI AcLDL at 4°C (dashed line). Cells were collected and analyzed by flow cytometry. The amount of intracellular DiI AcLDL is indicated on the x-axis in log fluorescence units. The histograms were generated by gating on the highly expressing transfected cells (see Fig. 2 B for sample gates). The gates used in this experiment were the same as those used to assay phagocytosis.

Figure 4

Dyn^K44A colocalized with F-actin on phagocytic cups. RAW-TT10 cells transfected with V5 epitope–tagged dyn^K44A were incubated with zymosan for 10 min, fixed, and stained with the anti-V5 antibody. Actin was visualized with rhodamine-phalloidin. Arrows indicate where tagged dyn^K44A colocalized with actin on partially formed phagocytic cups.

Figure 5

Dyn^K44A inhibits membrane extension around the forming phagosome. Transiently transfected cells were sorted by FACS^®, and cells expressing the highest levels of GFP were studied by scanning electron microscopy. Sorted cells were incubated with IgG-coated SRBCs for 10 min (A and B) or for 1 h (C and D). After a 10-min internalization, control pTIGZ2–transfected cells demonstrated various stages of particle internalization (A), and after 1 h, >90% of the particles were internalized (C). By contrast, cells expressing dyn^K44A could only extend membrane partially around the bound particles at both time points (B and D). The size bar shown in D applies to A–D.

Figure 6

Dynamin is not recruited to the site of particle binding in macrophages treated with the PI3K inhibitor, wortmannin. Murine RP macrophages were treated with wortmannin for 1 h and incubated with zymosan for 10 min. Arrows indicate the site of zymosan binding. Actin polymerized under the bound particles (A), but dynamin was not enriched in the actin pedestals (B).

Figure 7

Dyn^K44A does not inhibit particle-induced inflammatory signaling. TNF-α production induced by IgG-opsonized particles or by zymosan was analyzed by intracellular cytokine staining in RAW-TT10 cells transiently transfected with either control vector or dyn^K44A. Complement-opsonized particles do not stimulate TNF-α production in macrophages and therefore were not analyzed. (A) TNF-α production is expressed as the percentage of transfected cells producing the cytokine. (B) The amount of intracellular TNF-α produced per cell is expressed on the x-axis as log fluorescence units. Unstimulated cells are represented by the dotted line; nonexpressing control cells exposed to zymosan are shown by the bold solid line; and cells expressing high levels of dyn^K44A that were stimulated with zymosan are represented by the thin solid line.