Dynamic partitioning into lipid rafts controls the endo-exocytic cycle of the alphaL/beta2 integrin, LFA-1, during leukocyte chemotaxis

Abstract

Cell migration entails the dynamic redistribution of adhesion receptors from the cell rear toward the cell front, where they form new protrusions and adhesions. This process may involve regulated endo-exocytosis of integrins. Here we show that in primary neutrophils unengaged alphaL/beta2 integrin (LFA-1) is internalized and rapidly recycled upon chemoattractant stimulation via a clathrin-independent, cholesterol-sensitive pathway involving dynamic partitioning into detergent-resistant membranes (DRM). Persistent DRM association is required for recycling of the internalized receptor because 1) >90% of endocytosed LFA-1 is associated with DRM, and a large fraction of the internalized receptor colocalizes intracellularly with markers of DRM and the recycling endocytic compartment; 2) a recycling-defective mutant (alphaL/beta2Y735A) dissociates rapidly from DRM upon being endocytosed and is subsequently diverted into a late endosomal pathway; and 3) a dominant negative Rab11 mutant (Rab11S25N) induces intracellular accumulation of endocytosed alphaL/beta2 and prevents its enrichment in chemoattractant-induced lamellipodia. Notably, chemokine-induced migration of neutrophils over immobilized ICAM-1 is abrogated by cholesterol-sequestering agents. We propose that DRM-associated endocytosis allows efficient retrieval of integrins, as they detach from their ligands, followed by polarized recycling to areas of the plasma membrane, such as lamellipodia, where they establish new adhesive interactions and promote outside-in signaling events.

Figure 1.

αL/β₂ partitions into lipid rafts upon chemokine stimulation in PMN. (A) Freshly isolated PMN were incubated in the absence or presence of 100 nM fMLF for 5 min at 37°C, followed by lysis in 1% Triton X-100 and floatation on a discontinuous step sucrose gradient. The relative distribution of αL and the raft marker flotillin-2 were analyzed by Western blotting. (B) Densitometric analysis of a representative experiment showing the distribution of αL and flotillin-2 over a sucrose gradient in resting (control) or activated (100 nM fMLF) PMN.

Figure 2.

αL/β₂ internalization is sensitive to cholesterol depletion in chemokine-stimulated PMN. Freshly purified PMN were preincubated in the absence (circles) or presence (closed triangles) of 5 μg/ml filipin, surface labeled with cleavable biotin, and incubated at 37°C for the indicated time points in absence (closed circles) or presence of 100 nM fMLF (open circles). Endocytosis (A) and recycling (B) were assessed as described in Materials and Methods. Data are expressed as the percentage of internalization over the total amount of labeled receptor (A) or as the percentage of recycling over the amount of internalized receptor (B). Values represent a mean ± SD of four separate experiments.

Figure 3.

The endocytosed fraction of αL/β₂ partitions into DRM in PMN. Freshly purified PMN were labeled at 4°C with cleavable biotin and stimulated with 100 nM fMLF for 5 min at 37°C before surface biotin cleavage, lysis, and floatation over a discontinuous step sucrose gradient, as described in Materials and Methods. αL was immunoprecipitated either from the pooled floating fractions 9–11 or from the individual bottom fractions 1–3 (see Figure 1). The immunoprecipitated, biotinylated receptor was revealed with avidin-HRP to assess DRM partitioning of the internalized versus total αL/β₂ (top). The total amount of αL/β₂ in each lane was determined by immunoblotting with anti-αL antibody.

Figure 4.

Cholesterol depletion inhibits fMLF-induced PMN migration. Freshly purified PMN were pretreated for 45′ at 37°C in the absence or presence of either 10 mM βMCD or 10 μg/ml filipin, plated on ICAM 1-coated glass-bottom dishes, and analyzed by time-lapse microscopy for 20 min. Then, 100 nM fMLF was added after 5 min. Spontaneous and fMLF-induced cell migration was quantified with Cell Tracker software, as described in Materials and Methods. From 30–100 cells for experiment were scored, and the average distance (in micrometers) covered during the observation period was assessed. Data represent the mean ± SD of four separate experiments.

Figure 5.

EM analysis of fMLF-induced endosomal localization of αL/β₂ in PMN. (A–D) Primary PMN were labeled at 4°C with anti-αL Fab′ fragment, incubated at 4°C (A and B) or 37°C (C and D) for 5 min, fixed, and processed for cryo-EM. Panels show ultrathin cryosections double labeled for αL/β₂ (10-nm gold) and either cholera toxin (15-nm gold; A–C) or EEA-1 (15-nm gold; D). Bar, 0.33 μm (A–C); 0.17 μm (D).

Figure 6.

Confocal microscopy analysis of fMLF-induced endosomal localization of αL/β₂ in PMN. Primary PMN were labeled at 4°C with a Fab′ fragment of an anti-αL mAb, followed by fMLF stimulation and incubation for the indicated time points. Cells were then fixed, permeabilized, and stained with a Cy3-conjugated goat anti-mouse immunoglobulin antiserum (left). Endogenous Rab11 was detected with an affinity-purified rabbit antiserum followed by an FITC-conjugated goat anti-rabbit antibody (top and middle center panels). Endogenous LAMP-1 was detected with an anti-LAMP-1 mAb (IgG_2b) followed by a FITC-conjugated, isotype-specific antiserum (bottom center). Nuclei were detected with Hoechst 33342 (blue fluorescence). Right, merged images.

Figure 7.

αL/β₂ internalization does not follow a clathrin-mediated pathway in CHO cells. HAfpR-CHO cells were transiently transfected with either αL/β₂ (A) or the TfR (B), and cotransfected with either mock cDNA or the indicated wild-type and dominant negative (DN) constructs. Twenty-four hours posttransfection, cells were surface labeled with cleavable biotin at 4°C and stimulated with fMLF (1 μM for 5 min), followed by processing as described in Figure 2 legend. Data are expressed as percentage of internalization over mock-transfected cells. Values represent a mean ± SD of three separate experiments.

Figure 8.

αL/β₂ is recycled via a Rab11-positive compartment. CHO cells stably expressing the fMLF receptor were transiently transfected with αL/β₂-GFP and either a wt or a Rab11^S25N mutant, expressed as monomeric DsRed chimeric constructs. Twenty-four hours posttransfection, cells were fixed and inspected by laser scanning confocal microscopy.

Figure 9.

αL/β₂ wt and αL/β₂^Y735A mutant internalization involve DRM inclusion and are sensitive to cholesterol depletion in CHO cells. (A) HAfpR-CHO cells were transiently transfected with either αL/β₂ wt or αL/β₂^Y735A and preincubated in the absence or presence of 5 μM filipin. Cells were then surface labeled with cleavable biotin at 4°C and processed as described in Figure 2 legend. Data are expressed as the percentage of internalization over the total amount of labeled receptor (A) or as the percentage of recycling over the amount of internalized receptor (B). Values represent a mean ± SD of three separate experiments. (C) HAfpR-CHO cells were transiently transfected with either αL/β₂ wt or αL/β₂^Y735A, labeled at 4°C with cleavable biotin, and stimulated with 1 μM fMLF for 5 min at 37°C before surface biotin cleavage, lysis, and floatation over a discontinuous step sucrose gradient, as described in Materials and Methods. αL was immunoprecipitated either from the pooled floating fractions 9–11 or from the individual bottom fractions 1–3 (see Figure 1). The immunoprecipitated, biotinylated receptor was revealed with avidin-HRP to assess DRM partitioning of the internalized versus total αL/β₂ (top). The total amount of αL/β₂ in each lane was determined by immunoblotting with anti-αL antibody.