Crucial role for the LSP1-myosin1e bimolecular complex in the regulation of Fcγ receptor-driven phagocytosis

Abstract

Actin cytoskeleton remodeling is fundamental for Fcγ receptor-driven phagocytosis. In this study, we find that the leukocyte-specific protein 1 (LSP1) localizes to nascent phagocytic cups during Fcγ receptor-mediated phagocytosis, where it displays the same spatial and temporal distribution as the actin cytoskeleton. Down-regulation of LSP1 severely reduces the phagocytic activity of macrophages, clearly demonstrating a crucial role for this protein in Fcγ receptor-mediated phagocytosis. We also find that LSP1 binds to the class I molecular motor myosin1e. LSP1 interacts with the SH3 domain of myosin1e, and the localization and dynamics of both proteins in nascent phagocytic cups mirror those of actin. Furthermore, inhibition of LSP1-myosin1e and LSP1-actin interactions profoundly impairs pseudopodial formation around opsonized targets and their subsequent internalization. Thus the LSP1-myosin1e bimolecular complex plays a pivotal role in the regulation of actin cytoskeleton remodeling during Fcγ receptor-driven phagocytosis.

FIGURE 1:

Localization and dynamics of LSP1 during Fcγ receptor–mediated phagocytosis. (A) Wide-field fluorescence images showing the spatial distribution of LSP1 in macrophages during Fcγ receptor–mediated phagocytosis. After incubation with opsonized beads for 5 min, actin concentrated around internalization sites (left, arrow), where LSP1 also accumulated (right, arrow). Conversely, beads that were not being internalized, and therefore negative for actin accumulation (left, asterisk), were also LSP1 negative (right, asterisk). Scale bar, 5 μm. (B, C) Macrophages expressing LSP1-GFP or RFP-LSP1 were incubated with opsonized beads and immediately analyzed by time-lapse fluorescence video microscopy. LSP1 accumulated at the sites of internalization within seconds of cell–bead (B, asterisks) interactions and remained at these locations as phagocytosis proceeded until the internalization process was complete (B and C, arrows). Note that LSP1 localization at the cell periphery is probably due to the interaction of LSP1 with the cortical actin cytoskeleton. Arrowheads in insets point to opsonized particles being taken up. Numbers represent elapsed time in seconds. Scale bar, 5 μm.

FIGURE 2:

Down-regulation of LSP1 severely impairs Fcγ receptor–mediated phagocytosis. (A) shRNA-mediated down-regulation of LSP1 in macrophages. Cytosolic lysates from control untreated cells and cells stably transfected with LSP1-specific shRNAs (316 or 755) or a scrambled shRNA were analyzed for LSP1 expression by Western blotting. Compared with control cells, cells expressing si316 or si755 had much lower levels of LSP1. As expected, the scrambled control did not affect the expression of LSP1. Actin served as loading control. Numbers on the left indicate molecular weight markers in kilodaltons. (B) LSP1 deficiency greatly impairs actin remodeling during phagocytosis. In macrophages expressing si316 or si755 (see insets showing the GFP signal, which reflects shRNA expression), LSP1 levels were greatly reduced compared with control cells. At the sites of cell–bead contact, neither LSP1 nor actin accumulation could be detected (arrowheads; asterisks indicate the beads). This is in contrast with control cells or cells expressing the scrambled shRNA, in which LSP1 and actin colocalized at phagocytic cups (arrows). Scale bars, 5 μm (longer bar applies only to images on right). (C) Quantification of internalized RBCs in control and shRNA-expressing macrophages. Box-and-whiskers plots. The line in the middle of the box indicates the median, the top of the box indicates the 75th quartile, and the bottom of the box indicates the 25th quartile. Whiskers represent the 10th (lower) and 90th (upper) percentiles, respectively. Mann–Whitney U test, ***p < 0.0001; ns, nonsignificant difference. (D, E) LSP1 down-regulation impairs lamellipodium formation at Fcγ receptor internalization sites. Control macrophages (D) and LSP1-deficient macrophages (E) were incubated for 5 min at 37°C with opsonized RBCs (white asterisks), fixed, and processed for scanning electron microscopy. Control cells almost completely surrounded the opsonized targets with large lamellipodia (D, arrows ). By contrast, although LSP1-deficient cells were still able to bind the opsonized targets (white asterisks), they did not form lamellipodia around RBCs. Scale bar, 1 μm.

FIGURE 3:

The interaction of LSP1 with actin filaments is required for phagocytic cup formation. LSP1-deficient J774 macrophages expressing RFP-tagged LSP1-Δ30 were incubated with opsonized beads, fixed, and stained with Alexa 350–conjugated phalloidin. Note that, as expected, the LSP1-Δ30 did not colocalize with the actin rim at the periphery of J774 cells (A–C; arrows in insets). Moreover, cells expressing LSP1-Δ30 (D–F) did not accumulate actin and did not form phagocytic cups at the contact sites with opsonized beads (D, arrows ; asterisks indicate the beads), which were also devoid of LSP1-Δ30 (E, arrows ). Inset in E shows the expression of shRNA 316. Scale bars, 10 μm (A–C), 5 μm (D–F).

FIGURE 4:

Characterization of the direct binding between LSP1 and myosin1e. (A, B) LSP1 forms protein complex(es) with myosin1c and myosin1e in macrophages. (A) SDS–PAGE gel (after silver staining), showing proteins that coprecipitated with LSP1 from J774 cytosolic lysates. Control IP (ctrl IP) was done using beads coated with BSA. Arrows indicate the expected positions of myosin 1c (lower arrow) and myosin1e (upper arrow). MWM, molecular weight markers (in kilodaltons). (B) Western blotting showing the presence of myosin1c and myosin1e in both cytosolic lysates and LSP1 immunoprecipitates (LSP1 IP). Numbers on the left indicate molecular weight markers in kilodaltons. (C) LSP1 directly interacts with the SH3 domain of myosin1e. Purified LSP1 was incubated with either GST or GST-SH3. After incubating with glutathione beads (bound to GST), the presence of LSP1 in the supernatant (after centrifugation of beads), wash (after third washing step), and eluate (bound to beads) fractions was tested by Western blotting. LSP1 bound to GST-SH3 but not to GST, indicating a direct LSP1–SH3 domain interaction. J774 cytosolic lysate (lysate) served as positive control. Number on the left indicates molecular weight marker in kilodaltons. (D) Class I and atypical SH3 domain–binding sequences within LSP1 are dispensable for its interaction with the SH3 domain of myosin1e. The replacement of proline residues with alanine within the class I motif R/KxxPxxP (mut P>A) or arginine and lysine residues within the two atypical SH3 domain–binding motifs of LSP1 (mut RK>A) did not grossly affect the ability of LSP1 to interact with the SH3 domain of myosin1e. Number on the left indicates molecular weight marker in kilodaltons. (E) Schematic representation of LSP1 domain structure and binding sites. (F, G) Deletion of the novel SH3 domain–binding (SBS) site of LSP1 severely impairs its ability to interact with myosin1e. (F) In vitro binding assays between LSP1 deletion mutants and the SH3 domain of myosin1e. Numbers on the left indicate molecular weight markers in kilodaltons. Sup, input; bound, IPs. (G) Pull-down assays from NIH-3T3 and J774 cell lysates. Numbers at the bottom indicate the myosin1e/LSP1 ratio as determined by densitometry analysis using ImageJ. (H) Inhibition of LSP1-myosin1e interaction inhibits Fcγ receptor–driven phagocytosis. Phagocytosis of RBCs was quantified in LSP1-deficient cells expressing the LSP1-ΔSBS mutant. Mann–Whitney U test, **p < 0.0001. (I) Immunofluorescence labeling, showing the lack of actin, LSP1-ΔSBS, and myosin1e accumulation at the contact sites (arrows) with opsonized beads (asterisks). Inset, si316 expression. Scale bar, 5 μm. (L) J774 cells expressing LSP1-ΔSBS were still able to bind opsonized RBCs (white asterisks), but they did not form lamellipodia around them. Scale bar, 1 μm.

FIGURE 5:

LSP1 and myosin1e have the same spatial and temporal distribution during Fcγ receptor–mediated phagocytosis. (A) LSP1 and myosin1e colocalize at phagocytic cups. After incubation with opsonized beads, macrophages stably transfected with GFP-myosin1e were fixed and labeled with anti-LSP1 antibodies. The levels of both proteins (arrows) are highest around beads (asterisks) at internalization sites. Scale bars, 5 μm. (B) Dynamics of myosin1e during Fcγ receptor–mediated phagocytosis. J774 cells expressing GFP-myosin1e were incubated with opsonized beads and immediately analyzed by time-lapse fluorescence video microscopy (Supplemental Fig5B Video3). Myosin1e initially accumulated at the proximal side of a bead (arrows; asterisks indicate beads). As phagocytosis proceeded, myosin1e accumulation could be observed on the lateral sides of a bead and finally on its distal portion (arrows). The green arrowhead points to the proximal, already internalized, portion of a bead devoid of myosin1e. (C) Dynamics of myosin1e and LSP1 during Fcγ receptor–mediated phagocytosis. Cells expressing both GFP-myosin1e and RFP-LSP1 were incubated with opsonized beads and immediately analyzed by time-lapse fluorescence video microscopy. In agreement with the data in A and B, the proteins showed similar dynamics throughout particle internalization (arrows; asterisks indicate beads). The green arrowhead points to the bead in the corresponding phase contrast images. Numbers represent elapsed time in seconds. Scale bar, 5 μm.

FIGURE 6:

Fluorescence intensity analysis of LSP1, actin, and myosin1e during Fcγ receptor–mediated phagocytosis. (A, B, D, E) Plots showing the distribution of actin and myosin1e (A) or LSP1 and myosin1e (D) during phagocytosis in live cells. (B, E) Accumulation of myosin1e and actin (B) or LSP1 and myosin1e (E) around phagocytic cups in fixed cells. Arrows in B and E indicate the maximum intensity of LSP1 and myosin1e corresponding to the bead edges. (C, F) Plots for myosin1e-actin (C) and LSP1-myosin1e (F) pairs, showing a high correlation for the intensity values of these proteins at phagocytic cups.

FIGURE 7:

LSP1 deficiency impairs myosin1e accumulation at internalization sites. (A, B) After incubation with opsonized beads, LSP1-deficient J774 cells were fixed and labeled with Alexa 350–conjugated phalloidin and anti-myosin1e antibodies. Arrows point to the bead–cell contact sites and show the lack of actin and myosin1e accumulation at these locations. Asterisks indicate opsonized beads. In the merged image, actin and myosin1e are represented in red and green, respectively. Scale bar, 10 μm. (B) Dynamics of myosin1e Fcγ receptor–mediated phagocytosis in LSP1-deficient cells. Cells expressing GFP-myosin1e were incubated with opsonized RBCs and analyzed by time-lapse fluorescence video microscopy (Supplemental Fig7B Video6). In agreement with the data in A, myosin1e did not accumulate at the bead–cell contact site throughout the imaging time (white arrows point to RBCs, whereas green arrows highlight the lack of myosin1e accumulation at the RBC–cell interface). Numbers represent elapsed time in seconds. Scale bar, 10 μm. (C) Schematic model for the role of LSP1 and myosin1e during Fcγ receptor–mediated phagocytosis. After the engagement of Fcγ receptors, early events, including the activation of protein kinases (PTKs), will lead to the recruitment of LSP1, which, in turn, will interact with myosin1e at nascent phagocytic cups. At these locations, LSP1 and myosin1e will work in concert to coordinate actin assembly, lipid transport, and actin–membrane linkage, finally leading to phagocytic cup formation and closure.