Munc13-4 Is a Rab11-binding Protein That Regulates Rab11-positive Vesicle Trafficking and Docking at the Plasma Membrane

Abstract

The small GTPase Rab11 and its effectors control trafficking of recycling endosomes, receptor replenishment and the up-regulation of adhesion and adaptor molecules at the plasma membrane. Despite recent advances in the understanding of Rab11-regulated mechanisms, the final steps mediating docking and fusion of Rab11-positive vesicles at the plasma membrane are not fully understood. Munc13-4 is a docking factor proposed to regulate fusion through interactions with SNAREs. In hematopoietic cells, including neutrophils, Munc13-4 regulates exocytosis in a Rab27a-dependent manner, but its possible regulation of other GTPases has not been explored in detail. Here, we show that Munc13-4 binds to Rab11 and regulates the trafficking of Rab11-containing vesicles. Using a novel Time-resolved Fluorescence Resonance Energy Transfer (TR-FRET) assay, we demonstrate that Munc13-4 binds to Rab11a but not to dominant negative Rab11a. Immunoprecipitation analysis confirmed the specificity of the interaction between Munc13-4 and Rab11, and super-resolution microscopy studies support the interaction of endogenous Munc13-4 with Rab11 at the single molecule level in neutrophils. Vesicular dynamic analysis shows the common spatio-temporal distribution of Munc13-4 and Rab11, while expression of a calcium binding-deficient mutant of Munc13-4 significantly affected Rab11 trafficking. Munc13-4-deficient neutrophils showed normal endocytosis, but the trafficking, up-regulation, and retention of Rab11-positive vesicles at the plasma membrane was significantly impaired. This correlated with deficient NADPH oxidase activation at the plasma membrane in response to Rab11 interference. Our data demonstrate that Munc13-4 is a Rab11-binding partner that regulates the final steps of Rab11-positive vesicle docking at the plasma membrane.

Keywords: Munc13-4; NADPH oxidase; Rab; Rab11; Rab27; docking; intracellular trafficking; neutrophil; recycling endosome; super-resolution microscopy.

FIGURE 1.

Munc13-4 binds to wild type Rab11 but not to the point-mutant, dominant negative, Rab11S25N. A, schematic representation, TR-FRET assay. Flag-tagged Munc13-4 and EGFP-tagged Rabs are co-expressed in 293T cells, and lysates are prepared under non-denaturing conditions as described under “Experimental Procedures.” The reactions are started by addition of the terbium cryptate-conjugated anti-Flag antibody (Cisbio) and carried out at 21 °C. The emission ratio of the acceptor (GFP, 520 nm)/donor (Tb, 495 nm) is indicative of protein binding. B, binding of Munc13-4 to the indicated Rab GTPases was analyzed as described in A. The results are mean ± S.E. from three to seven experiments performed in triplicates. *, p = 0.02; **, p = 0.01; ***, p = 0.0003; and ****, p < 0.0001 versus GFP control. Inset, GFP-Rab protein expression. C, binding of Munc13-4 to wild type Rab11 is competed by GTPγS-loaded recombinant Rab11. In these assays 0.27 nmol of the indicated recombinant proteins were used in 15 μl of TR-FRET reactions. **, p < 0.01. D and E, homologous competitive binding experiments for Rab11 using the TR-FRET assay. Time- (D) and dose-response (E) competitive binding assays were performed using the indicated concentrations of GTPγS-loaded GST-Rab11. Specific binding of a constant concentration of EGFP-Rab11 in the presence of various concentrations of GST-Rab11 was measured. IC₅₀ values were determined using a concentration of EGFP-Rab11 of 79 nm (determined by Western blot), so that the concentration of EGFP-Rab11 was less than half the IC₅₀. K_d was then calculated using the homologous competitive binding curve fitted to a built-in equation of one-site competition (GraphPad Prism). The assay assumes that GST-Rab11 and EGFP-Rab11 have similar affinity for Munc13-4. Error bars correspond to S.E. of three (D) or six biological replicates from two independent experiments (E). F, left panel, the binding of Munc13-4 to wild type Rab11 or to the point-mutants constitutively active Rab11Q70L and dominant negative Rab11S25N (DN) was analyzed by TR-FRET as described under “Experimental Procedures.” Mean ± S.E. of three independent experiments. **, p = 0.01. The level of protein expression in the lysates was analyzed by Western blot (right panel). G, binding of Munc13-4 to Rab11 or Rab11S25N was analyzed by co-immunoprecipitation assay (IP). The data is representative of two independent experiments with similar results. WB, Western blot. H, co-immunoprecipitation analysis of endogenous proteins. Human neutrophil lysates (1 mg of total protein) were incubated in the presence of specific anti-Rab11a or IgG control rabbit mAbs as described under “Experimental Procedures,” and the pull-downs were analyzed for the presence of Munc13-4 by Western blot (WB). Similar amounts of specific and control mAbs were visualized by detection of the antibody light chain (LC).

FIGURE 2.

Identification of molecular determinants in Munc13-4 that are important for Rab11 binding. A, the indicated Munc13-4 mutants and truncations were used in the TR-FRET binding assays together with Rab11, and the reactions were carried out as described under “Experimental Procedures.” C2A*, includes point mutations Asp-127 and Asp-133 to alanines in the Munc13-4 C2A domain, which knocks out the Ca²⁺-binding sites in this domain; C2B*, includes point mutations Asp-941 and Asp-947 to alanines to knock out the Ca²⁺-binding sites in the C2B domain. C2A*C2B* includes 4 Asp → Ala mutations corresponding to both C2 domains. SDM (site-directed mutagenesis). For truncation of the C2A domain (Flag-C2A-deletion) or the C2B domain (Flag-C2B-deletion), amino acids 1–301 or 901–1090 are deleted from Munc13-4, respectively. Equal loading was evaluated by Western blot (data not shown). Data are expressed as mean ± S.E. from three independent experiments. ***, p = 0.008. B, binding of Munc13-4 mutants and truncations to Rab27a was evaluated by the TR-FRET assay as described in A. Data are expressed as mean ± S.E. from three independent experiments. C, left panel, Rab11 competes with Rab27a for binding to Munc13-4. TR-FRET reactions were performed using lysates from EGFP-Rab27a-expressing cells co-transfected with Flag-Munc13-4 and myc-Rab11 or control empty vectors. **, p < 0.01. n = 3. Right panel, control of protein expression. D, competitive binding assays were performed in reactions containing 400 nm Flag-Munc13-4 and increasing concentrations of the Rab11 effector myc-Rip11. Mean ± S.E. from three independent experiments. *, p < 0.05. E, competitive Rab11-Munc13-4 binding reactions were carried out as described in D except that increasing concentration of the Rab27a effector myc-JFC1, used here as a negative myc-tagged control, were included instead of Rip11 in the reactions.

FIGURE 3.

Super-resolution microscopy shows adjacent distribution of endogenous Munc13-4 and Rab11 in primary neutrophils. A, subcellular localization of endogenous Munc13-4 (red) and Rab5 (A) or Rab11 (B) (green) was analyzed by direct STORM as described under “Experimental Procedures.” Single-molecule Munc13-4 was detected adjacent to Rab11 but rarely to Rab5. Scale bars: whole cell images, 1 μm; magnified images, 200 nm. B, quantification of the distance between Rab11 and Munc13-4 centroids was performed as described under “Experimental Procedures,” and results were expressed by binning the distance between pairs in 25- nm increments and plotted as a percentage of total pairs at the given distance for each cell. A total of 9121 and 11703 Munc13-4-Rab5 and Rab11 pairs were analyzed, respectively, from 7 independent cells from three independent experiments. Mean ± S.E. *, p < 0.05 and **, p < 0.01. C, example of drift correction. dSTORM images were processed using Nikon software to correct for drift during acquisition as described under “Experimental Procedures.”

FIGURE 4.

Immunofluorescence analysis of endogenous proteins identify Munc13-4 and p22^phox vesicles as Rab11-positive organelles in neutrophils. A, immunofluorescence analysis and quantification of the distribution of endogenous Munc13-4 and Rab11 in mouse primary neutrophils showing that Munc13-4 colocalizes with Rab11. Scale bars: 5 μm. B, immunofluorescence analysis and quantification showing lack of colocalization of Rab11 and gelatinase B (MMP-9) in either wild type (upper panel) or Munc13-4 KO (Jinx) (lower panel) cells. C, immunofluorescence analysis showing colocalization of Rab11 with a subpopulation of p22^phox-positive granules in both wild type (upper panel) and Munc13-4-KO (Jinx) (lower panel) neutrophils. Arrows indicate areas of colocalization. A–C, quantification of colocalization index was performed using ZEN-LSM software. A, 29 cells, two independent experiments; B, 28 WT and 40 Jinx cells from three independent experiments and C, 30 WT and 34 Jinx, two independent experiments. Mean ± S.D.

FIGURE 5.

Rab11 regulates the NADPH oxidase activity at the plasma membrane in neutrophils. Neutrophils were permeabilized using streptolysin O and incubated in the presence of anti-Rab11 (Rab11 mAb and Rab11 rabbit polyclonal, Rp) or control antibodies (IgG). Neutrophils were then washed and stimulated with fMLP either in the absence (blue symbols) or the presence of the TLR4 agonist LPS, a priming agent (red symbols), and extracellular superoxide anion production was evaluated by the cytochrome c reduction assay as described under “Experimental Procedures.” A, representative kinetics of the reactions are shown. B, results are presented as the mean ± S.E. of three independent experiments with similar results. Rab11mAb, anti-Rab11 monoclonal antibody; Rab11Rp, anti-Rab11 rabbit polyclonal antibody. IgG, rabbit IgG control. B, superoxide anion production was evaluated as described in A. *, p < 0.05 versus IgG control for the same stimuli.

FIGURE 6.

Recycling of CXCR2 receptor is not regulated by Munc13-4 in neutrophils. A, WT or Munc13-4-KO (Jinx) neutrophils were incubated in the presence or absence of human IL-8 for 1 h followed by removal of the stimuli and subsequent incubation of the cells in stimuli-free medium for an additional period of 30 (30 recov) or 60 min (60 recov) at 37 °C. The expression of CXCR2 at the plasma membrane was analyzed by flow cytometry as indicated in “Experimental Procedures.” n = 8 mice per condition were analyzed in four independent experiments. *, p < 0.05. b, WT or Munc13-4-KO (KO) neutrophils were incubated in the presence of fluorescently labeled BSA and stimulated with fMLP or left untreated (NS). Extracellular fluorescent BSA was quenched with trypan blue and the cells were analyzed by flow cytometry. Representative histograms are shown. Unstained control shown to the left (black line).

FIGURE 7.

Munc13-4 colocalizes with Rab11 in a spatio-temporal manner and regulates vesicular trafficking of Rab11-positive vesicles. A, live-cell microscopy analysis of the dynamics of Rab11- and Munc13-4-positive vesicles. Representative images of the subcellular distribution of mCherry-Munc13-4 and EGFP-Rab11 evaluated by TIRF microscopy in RPE cells. B, live-cell microscopy analysis of the dynamics of Rab11-positive vesicles and the Munc13-4 mutant Munc13-4-C2A*B* (described in Fig. 2). A and B, lower panels show examples of the tracking analysis performed for both channels, confirming identical spatio-temporal distribution of Rab11 and either Munc13-4 or Munc13-4-C2A*B*. C, histograms representing the speeds of Rab11-containing organelles in cells expressing either wild type Munc13-4 (blue bars) or the mutant Munc13-4-C2A*B* (red bars). The speeds for the independent vesicles were binned in 0.02-μm/s increments and plotted as a percentage of total vesicles for a given cell. Results are represented as mean ± S.E. from 18 cells expressing wild type Munc13-4 and 19 cells expressing the Munc13-4-C2A*B* mutant. The statistically significant differences between the two groups are indicated in the figure.

FIGURE 8.

Munc13-4 regulates Rab11-positive vesicular dynamics and tethering in primary neutrophils. Neutrophils from WT or Munc13-4 KO (Jinx) mice were transfected for the expression of EGFP-Rab11 and vesicular dynamics was analyzed by TIRFM. A, representative images of the subcellular distribution of Rab11 in WT and KO cells. The arrows indicate Rab11-positive vesicles that appear associated with the plasma membrane in vesicular dynamic studies (see associated supplemental movies S3 and S4). B, quantitative analysis of Rab11 dynamics. Histograms representing the speeds of Rab11-containing organelles in wild type (blue bars) or Munc13-4 KO neutrophils (red bars) are shown. The speeds for the independent vesicles were binned in 0.05-μm/s increments and plotted as a percentage of total vesicles for a given cell. Results are represented as mean ± S.E. from 85 wild type and 93 Munc13-4-KO cells. *, p < 0.05. C, neutrophils from WT or Rab27a KO (ashen) mice were transfected for the expression of EGFP-Rab11, and vesicular dynamics was analyzed by TIRFM. Results from the analysis of 101 wild type and 131 Rab27a KO (ashen) cells are depicted. No significant differences in Rab11 vesicular dynamics was observed between wild type and Rab27a KO neutrophils.

FIGURE 9.

Mobilization of Rab11-positive organelles and docking/retention at the plasma membrane in response to PKC activation is impaired in the absence of Munc13-4 expression. Neutrophils were stimulated with the PKC agonist PMA, the cells were fixed and stained for the detection of endogenous Rab11 and the distribution of Rab11-positive vesicles at the PM (exocytic active zone, ∼100 nm) was analyzed by TIRFM. A, representative images of the distribution of endogenous Rab11-positive vesicles in PMA-stimulated or unstimulated WT or Munc13-4-KO (Jinx) cells. B, quantitative analysis of the distribution of Rab11-positive vesicles. A total of 118, 191, 115, and 219 cells were analyzed for the conditions WT, unstimulated (UN); WT, PMA; Jinx, unstimulated and Jinx, PMA, respectively, in four independent experiments. ***, p < 0.0001. NS, not significant. C, Rab11 and Rab27a localize at independent subcellular compartments in neutrophils. Wild type or KO neutrophils were left untreated or stimulated with PMA as described above, fixed, and stained for endogenous Rab11 and Rab27a using specific antibodies. The magnified images show that Rab11 and Rab27a localize at different compartments in both stimulated and untreated, wild type, and Munc13-4 KO cells. Quantitative analysis of 28 wild type and 27 Munc13-4 KO (Jinx) cells was performed using the ZEN-LSM software. Three independent experiments are shown.