Cortical actin limits ER accessibility to the plasma membrane.

(A) TIRF microscope images of Chinese hamster ovary cells expressing GFP-MAPPER-L (cyan) and mCherry-Lifeact (magenta). (B) Image from panel “A” with a line indicating the position of the line scan and the corresponding histogram. (C) Representative TIRF micrograph of CHO cells expressing GFP-MAPPER-L and mCherry-Lifeact in either control or RhoA activator-treated conditions. (D) Area occupied in the TIRFM field by Lifeact labeled F-actin, essentially stress fibers, and (E) the area occupied by MAPPER-L in control and Rho Activator treated cells. (F) TIRF micrographs of a RAW264.7 macrophage expressing GFP-MAPPER-L and mCherry-Lifeact at time 0 and 5 min after actin depolymerization induced by latrunculin B. Bottom row is the magnified inset. For GFP-MAPPER-L at time 0, the image is contrast-enhanced; at the 5 min timepoint, no such enhancement is required. (G) Quantitation of 10 individual movies collected on two separate experiments. For statistical analysis, starting intensities of MAPPER-L in individual movies were set equal to 1, and the data were analyzed using a one-sample t-test and a Wilcoxon test. Default GraphPad indicators for significance levels are used throughout the study; *** P < 0.005, ** P < 0.01, * P < 0.05. Scale Bar = 5 μm.

Focal actin disassembly during phagocytosis allows for the formation of ER-PM membrane contact sites.

(A) Schematic representation of frustrated phagocytosis. Cover glass is opsonized with human IgG and RAW264.7 macrophages are parachuted to initiate frustrated phagocytosis. (B) TIRF micrographs at the indicated times of frustrated phagocytosis. RAW264.7 cells stably expressing mCherry-actin (magenta) were transiently transfected with YFP-STIM1 (cyan). The white line in the merged image at 360 sec was used to generate the line scan in (C). F.I. represents fluorescent intensity. (D) mCherry-Lifeact expressing RAW 264.7 macrophages were transfected with GFP-MAPPER-L, GFP-E-Syt1 or GFP-E-Syt2 (all pseudo-colored cyan) and were also examined during frustrated phagocytosis. (E) Schematic representation of how complementary split GFP/YFP, β1-10 and YFP β11, can be expressed and reformed. Split GFP (or YFP) based contact site sensors (SPLICS) targeting the PM and ER, respectively, to monitor formation of new ER-PM contact sites. RAW 264.7 macrophages were electroporated to express mCherry-Lifeact with either SPLICS-ER-PM Short (F) or SPLICS-ER-PM Long (G) and monitored using TIRF microscopy during frustrated phagocytosis. The 16-color LUT is used for YFP signal. Scale bar 5 μm.

PTP1B co-localizes with FcγR in the actin-cleared zone.

(A) TIRF micrographs of paraformaldehyde fixed RAW 264.7 macrophages transiently transfected to express PTP1B-mCherry (magenta) and FcγRIIa-GFP (cyan) and stained with iFluor647 conjugated phalloidin (yellow). (B) the same as “A” except cells express the substrate trapping mutant mCherry-PTP1BD181A. (C) Pearson’s colocalization coefficient for FcγRIIa-GFP and either wild-type or substrate trapping D181A PTP1B-mCherry. Mean ± SD and analyzed by an unpaired t-test of individual values across n = 3 independent experiments, * = P < 0.05. (D) TIRF micrographs of paraformaldehyde fixed RAW 264.7 macrophages transiently transfected to express PTP1BD181A-mCherry (magenta) and FcγR2a-GFP or FcγR2a lacking amino acids 280-307, which constitute the ITAM, FcγR2a Δ3Y (cyan). (E) Representative co-immunoprecipitation immunoblots of stably transfected doxycycline-inducible RAW 264.7 cells transfected with the indicated GFP constructs treated with PBS or aggregated IgG for 10 min. Immunocapture was performed using GFP-Trap and blots probed for the gamma chain of Fc receptor or with an anti-GFP antibody. Scale Bar = 5 μm.

PTP1B interacts with and attenuates phospho-Syk in response to FcγR activation.

(A) TIRF micrographs of paraformaldehyde fixed RAW 264.7 macrophages during frustrated phagocytosis transiently transfected to express PTP1BD181A-mCherry (magenta) and GFP-Syk. Scale Bar = 5 μm. (B) Representative co-immunoprecipitation immunoblots of doxycycline-inducible RAW264.7 cells, as in Fig. 3E, treated with PBS or aggregated IgG for 10 min. Immunocapture was performed using GFP-nanotrap, and blots were probed for endogenous Syk and anti-GFP antibody. (C) Parental and PTP1Bko RAW 264.7 macrophages were incubated with aggregated IgG for the indicated times. The dotted box highlights a contrast-enhanced version of the panel above since the PTP1B knockouts have such intense bands. (D) Line chart of three individual experiments depicted in “C”. Given variability in the magnitude of the PTP1Bko cell response, the mean of the orange lines (PTP1Bko) is not significantly different from the mean of the blue lines (P ∼ 0.10).

Loss of PTP1B results in increased superoxide production without altering phagocytic efficiency.

(A) Representative immunoblot of a time course of RAW 264.7 cells incubated with IgG-opsonized sheep red blood cells (sRBC). (B) Quantitation of “A”. Data represent the means ± SD from 4 biological replicates. *** P < 0.005 and ** P < 0.01 by 2-way ANOVA with Bonferroni post-hoc test. (C) Confocal micrographs of parental and PTP1Bko RAW 267.4 cells after 10 min incubation with human IgG-opsonized latex beads. Cells are delineated with iFluor 647 phalloidin, all beads are labeled with rhodamine (magenta), and outside beads are labeled with Alexa 488 conjugated Donkey anti-human IgG. Scale Bar = 5 μm. (D) Quantitation of “C” from n=3 biological replicates. Data are the mean ± SD. Results are non-significant using an unpaired t-test. (E) Brightfield and fluorescent micrographs are RAW 264.7 macrophage 10 min post addition of TRITC-conjugated zymosan particles incubated with nitroblue tetrazolium. (F) Quantitation of NBT-positive phagosomes in parental and three PTP1Bko clones. Data are the mean ± SD from n=4 biological replicates. * P < 0.05, ** P < 0.01 as determined by one-way ANOVA and Tukey’s post-hoc test. (G) Quantitation of superoxide production in phagosomes following NBT solubilization and measurement of OD 630nm. Data are the mean ± SD from n=4 biological replicates. * P < 0.05, *** P < 0.005 as determined by one-way ANOVA and Tukey’s post-hoc test.

Protein kinase C activation and alterations in phospho-tyrosine signaling following aggregated IgG treatment.

(A) Representative immunoblots monitoring the increase in the phosphorylation of Protein Kinase C α and β isoforms following the addition of aggregated IgG for the indicated amounts of time. (B) Quantitation of “A”. Data are the mean ± SD from n=4 biological replicates. (C) Scatter plot comparing phosphotyrosine (pY) signaling responses to stimulation in wild-type (WT) and KO conditions. Each point represents an individual phosphoprotein (top 50 hits depicted). The x- and y-axes show the WT and KO stimulation responses, respectively, expressed as Log fold change (Log FC) relative to unstimulated controls. The dashed diagonal indicates equal fold change between conditions. Points are colored by classification: proteins upregulated upon stimulation in both conditions (Log FC > 0.5 in both; blue), proteins with a greater KO response than WT (KO − WT Log FC > 0.5; red), proteins with a greater WT response than KO (WT − KO Log FC > 0.5; green), and proteins with similar responses between conditions (gray). Full results available in Supplemental Table 1.

p52SHC1 is phosphorylated during phagocytosis downstream of SFKs and Syk.

(A) Schematic representation of the three primary isoforms of SHC1, p66, p52 and p46. PTB, phosphotyrosine-binding domain, SH2, Src homology domain 2, CB, cytochrome c binding domain, and CH1 and CH2, collogen homologous region 1 and 2. (B) immunoblot of parental RAW264.7 and SHC1ko cells probed with an anti-SHC1 antibody. The bands corresponding to p52-SHC1 and p46-SHC1 are absent from the three knockout clones. (C) Immunoblots of parental and PTP1Bko cells treated with IgG-opsonized zymosan, sheep red blood cells, or aggregated IgG probed with either anti-SHC1 or anti-phospho-SHC1. Red arrowheads indicate the expected positions for p66, p52 and p46, respectively. (D) Quantitation of “C”. Data represent the mean ± SD. * P < 0.05 using an unpaired t-test. (E) immunoblots using an anti-phospho-SHC1 antibody in parental and knockout cells stimulated for 5 min with aggregated IgG in the presence or absence of Src family kinase inhibitor (PP2) or the Syk inhibitor (BAY61-3606). (F) Representative co-immunoprecipitation immunoblots of doxycycline-inducible RAW264.7 cells, as in Fig. 3E, treated with PBS or aggregated IgG for 10 min. Immunocapture was performed using GFP-nanotrap, and blots were probed anti-GFP and anti-SHC1 antibodies. The GFP blots provided for reference are re-used from Fig. 3E.

Loss of SHC1 reduces superoxide production.

(A) Quantitation of superoxide production in parent and SHC1 knockout RAW264.7 cell lines. Macrophages were incubated with IgG-opsonized zymosan for 15 min in the presence of NBT. NBT was solubilized using methanol, and the absorbance was measured at 620 nm. Data represent the mean ± SD from independent biological replicates, n=3. * P < 0.05, ** P < 0.01 as determined by one-way ANOVA and Tukey’s post hoc test. (B) Collapsed Z-slices of parental and PTP1Bko RAW267.4 macrophages grown on cover glass or parachuted onto IgG-coated cover glass and examined using proximity ligation assay. Specifically using a rabbit anti-p47phox and a mouse anti-SHC1. Puncta indicate areas where the antibodies are proximal (magenta), and the nucleus stained with DAPI (70% transparent, white) is included. Scale bar = 5 μm. (C-D) Quantitation of the relevant head-to-head comparisons in “B”. Data are the means ± SD from n=3 (C) and n = 4 (D) biological replicates. ** P < 0.01 using an unpaired t-test. (E) Quantitation of the ∼250 nm adjacent to the coverslip. Data are the means ± SD from n=3 (C) biological replicates. ** P < 0.01 using an unpaired t-test.

Fcγ Receptor signaling cascade leading to NOX2 activation and negative regulation by PTP1B.

Engagement of Fcγ Receptors (FcγRIII and FcγRIIα) by IgG-opsonized phagocytic targets initiates a signaling cascade that culminates in NOX2 assembly and superoxide production. Receptor clustering leads to phosphorylation of immunoreceptor tyrosine-based activation motifs (ITAMs) by Src family kinases (SFKs) (red dotted arrows, tyrosine phosphorylation). Phosphorylated ITAMs recruit and activate Syk kinase, which phosphorylates downstream effectors, including PLCγ (leading to PI4,5P hydrolysis and DAG production) and PKC α/β activation and phosphorylation of NOX2 components (cyan dotted arrow, serine phosphorylation) and the adaptor protein Shc1. Tyrosine-phosphorylated Shc1 interacts with p47phox, facilitating assembly of the NOX2 complex at the membrane. The NOX2 complex comprises the membrane-bound subunits gp91 and p22, along with the cytosolic regulatory subunits p47phox, p67phox, p40phox, and the small GTPase Rac. Upon assembly and activation, NOX2 generates superoxide. The signaling cascade is negatively regulated by protein tyrosine phosphatase 1B (PTP1B), which dephosphorylates Syk at MCS, thereby attenuating the pathway and limiting NOX2 activation. Created with BioRender.com.