μMap-Interface: Temporal Photoproximity Labeling Identifies F11R as a Functional Member of the Transient Phagocytic Surfaceome

Abstract

Phagocytosis is usually carried out by professional phagocytic cells in the context of pathogen response or wound healing. The transient surface proteins that regulate phagocytosis pose a challenging proteomics target; knowledge thereof could lead to new therapeutic insights. Herein, we describe a novel photocatalytic proximity labeling method: "μMap-Interface", allowing for spatiotemporal mapping of phagocytosis. Utilizing photocatalyst-conjugated IGG-opsonized beads and initiating phagocytosis in a synchronized manner, we capture phagocytic interactome "snapshots" at the interface of the phagocyte and its target. This allows profiling of the dynamic surface proteome of human macrophages during the engulfment process. We reveal previously known phagocytic mediators as well as potential novel interactors and validate their presence with super-resolution microscopy. This includes F11R, an important cancer target yet to be investigated in the context of phagocytosis. Further, we demonstrate that knocking down F11R leads to an increased degree of phagocytosis; this insight could contribute to explaining its oncogenic activity. Lastly, we show capture of orthogonal phagocytic surfaceomes across different cells, using a neutrophil-like model. We believe this method will enable new insights into phagocytic processes in a variety of contexts.

Figure 1:

Overview of phagocytosis importance and the new photocatalytic μMap method to probe the phagocytic surface proteome.

Figure 2:

(a) Schematic for functionalization of carboxy beads, first with human IGG antibody for opsonization, then with Iridium-photocatalyst for labeling experiments. (b) Temperature gated activation of phagocytosis of macrophages in a synchronized manner; description of temporal optimization workflow. (c) Microscopy images showcasing timepoint optimization of phagocytosis labeling, with 20x widefield images captured in 1-minute intervals at 1–4 minutes, highlighting precise temporal control, and then at 10 and 30 minutes. The selected “before”, “during” and “after” stages of active phagocytosis and their corresponding timepoints are highlighted on the bottom at 60x magnification. (d) Streptavidin staining via microscopy reveals larger cross-section of phagocytic cup labeling consistent with larger radius probe by microscopy. (e) Western blot labeling validation of labeling for both probes.

Figure 3:

(a) Overview of TMT-based proteomics workflow: 1) labeling, 2) lysis and labeled protein enrichment, 3) peptide digestion/labeling, and 4) bottom-up TMT proteomics analysis. Timepoints analyzed are described as well as the two probes used for each timepoint. (b) Proteomics data comparing of both pre- vs. active phagocytosis states and active vs. post phagocytosis states. Datasets were generated for both the diazirine (narrow radius probe) and phenyl-azide (wider radius probe). Pre-phagocytosis states contained few enriched proteins, active phagocytosis states contained proteins important to early phagocytosis (FC receptor etc.), and post phagocytosis data contained lysosomal components (as expected for the phagolysosome) Probe comparison reveals 8 shared proteins among the top hits, most of which are phagocytosis/macrophage associated. Comparative gene ontology between the two probes in their “active” timepoints reveals that diazirine leads to higher direct phagosome enrichment, as well as other processes such as cytokine signaling, while Phenyl-Azide data shows a higher enrichment for cell-adhesion related processes.

Figure 4:

(a) Identification of phagosome proteins via gene ontology reveals average protein intensity increase across timepoints (shown in heatmap), consistent with capture of the “growing phagosome”. (b) Super resolution microscopy reveals presence of proteins of interest in phagocytic cup region (bead-cell interface). CD36, ACE, Vimentin, and AHNAK are validated as phagocytic surfaceome members detected via STED. (c) Among the top enriched hits in the Phenyl-Azide data is F11R, an adhesion protein with known important roles in cancer but not connected with phagocytic processes. Validation of F11R in the surfaceome via STED. (d) Confirmation of F11R knockdown via qPCR to conduct functional studies. (e) Overview of quantitative flow cytometry phagocytosis assay. Analysis reveals an increase in fluorescence upon F11R knockdown, consistent with increased phagocytosis. This confirms the functional role of F11R in FC-mediated phagocytosis. Jurkats were used as a negative control.

Figure 5:

(a) Overview of HL60 cells as a neutrophil-like cell line to probe phagocytosis within another type of professional phagocytes. (b) Kinetics microscopy studies conducted as described previously revealed slightly slower phagocytosis, with active engulfment at 5 minutes and post-phagocytosis at 20 minutes. (c) Proteomics results on HL-60 cells revea known phagocytosis proteins enriched in the “active” timepoint, as well as neutrophil specific and novel proteins. Post-phagocytosis timepoint shows enrichment of lysosomal components but also TPR which is a known neutrophil-specific protein involved in pathogen killing via super oxides. Known top-hit phagosome proteins, as well as novel ones, are tabulated. (d) Comparative analysis between macrophage and HL-60 surfaceomes reveal 5 common interactors. Comparative gene ontology reveals enrichment of neutrophil-characteristic processes in the HL-60 data, consistent with their neutrophil-like character. STRING analysis of HL-60 data unveils network of 4 proteins found enriched (MPO, PRTN3, CTSG, ELANE), which are associated with neutrophils.