Journal: Redox Biology

Article Title: Coronin 1a-mediated F-actin disassembly controls effector function in murine neutrophils

doi: 10.1016/j.redox.2025.103618

Figure Lengend Snippet: Impaired ROS production in neutrophils deficient for the actin-regulatory protein Coro1a (A ) Confocal microscopy of WT and Coro1a −/− (KO) neutrophils showing the subcellular localization of Coro1a. Neutrophils were fixed, permeabilized and stained for Coro1a together with DAPI (nucleus). DIC, differential interference contrast. Scale bar, 3 μm. (B – J) ROS generation in WT and Coro1a −/− (KO) neutrophils. (B, D) Production of intracellular ROS in response to ingestible, serum opsonized (B) C. albicans yeast (SO–CaY) or (D) zymosan particles (SO-Z) measured with luminol chemiluminescence in the presence of superoxide dismutase (SOD) and catalase. (C, E, F) Production of extracellular ROS in response to non-ingestible (C) serum-opsonized C. albicans hyphae (SO–CaH), (E) serum-opsonized whole glucan particles (SO-WGP; filtered to have Ø ≥ 20 μm), or (F) immobilized BSA/anti-BSA immune complexes using ( C, E ) lucigenin or ( F ) luminol chemiluminescence assays. Left: ROS response kinetics; Right: normalized integrated ROS signals. (G – J) Production of (G) total, (H) intracellular, and (I) extracellular ROS in response to stimulation with PMA (100 ng/ml) measured with chemiluminescence assays. (J) Normalized integrated PMA-induced ROS signals. (K) Western-blot showing PMA-induced phosphorylation of p40phox in WT and Coro1a −/− (KO) neutrophils. (B–I) Data show mean ± SEM (n = 2–4). Unpaired Student's t-test: ∗ <0.05, ∗∗ <0.01, ∗∗∗∗ <0.0001. Data are representative for at least 4 independent experiments.

Article Snippet: Co-IP was conducted by incubation of cell lysates with magnetic beads conjugated to either rabbit polyclonal anti-Coro1a antibody (Proteintech) or isotype matched control antibody in an orbital rotator Intelli-Mixer RM-2M for 2 h at 4 °C.

Techniques: Confocal Microscopy, Staining, Western Blot, Phospho-proteomics

Journal: Redox Biology

Article Title: Coronin 1a-mediated F-actin disassembly controls effector function in murine neutrophils

doi: 10.1016/j.redox.2025.103618

Figure Lengend Snippet: F-actin disassembly promotes ROS production in neutrophils (A) Cellular F-actin content detected by intracellular phalloidin staining and flow cytometric analysis. Left: representative FACS histogram; Right: relative F-actin level (n = 5). Unpaired Student's t-test. (B) Confocal microscopy of WT and Coro1a −/− (KO) neutrophils showing staining for F-actin (phalloidin) and Coro1a. Nuclear staining: DAPI. Scale bar, 3 μm. (C – E) PMA (100 ng/ml)-induced ROS production in WT and Coro1a −/− (KO) neutrophils pretreated for 60 min with the indicated concentrations of (C) jasplakinolide (Jas; actin-stabilizing agent), (D) latrunculin B (Lat; actin-depolymerizing agent); or (E) benproperine phosphate (Benp; Arp2/3-complex inhibitor). ROS production was measured with luminol chemiluminescence. Left: ROS response kinetics; Right: normalized integrated ROS signals. Data are mean ± SEM (n = 2). (C – E) 2-way ANOVA test (Tukey). ns > 0.05, ∗∗ <0.01, ∗∗∗ <0.001, ∗∗∗∗ <0.0001. Data are representative for at least 3 independent experiments.

Article Snippet: Co-IP was conducted by incubation of cell lysates with magnetic beads conjugated to either rabbit polyclonal anti-Coro1a antibody (Proteintech) or isotype matched control antibody in an orbital rotator Intelli-Mixer RM-2M for 2 h at 4 °C.

Techniques: Staining, Confocal Microscopy

Journal: Redox Biology

Article Title: Coronin 1a-mediated F-actin disassembly controls effector function in murine neutrophils

doi: 10.1016/j.redox.2025.103618

Figure Lengend Snippet: Enhanced F-actin levels in Coro1a -deficient neutrophils are associated with impaired granule release (A-E) Granule exocytosis (degranulation) by WT and Coro1a −/− (KO) neutrophils. (A, B) Release of myeloperoxidase (MPO) from primary granules into culture supernatants upon stimulation of neutrophils with (A) serum-opsonized whole glucan particles (WGP) or (B) serum-opsonized C. albicans hyphae (SO–CaH). (C) Upregulation of CD63 (Lamp3) cell surface expression upon stimulation with WGP. (D, E) Release of (D) neutrophil gelatinase-associated lipocalin (NGAL) from secondary granules or (E) matrix metalloproteinase-9 (MMP-9) from tertiary granules into culture supernatants upon stimulation of neutrophils with TNFα. (F) Total amount of MPO, NGAL, and MMP-9 in WT and Coro1a −/− (KO) neutrophils expressed in % of WT (n = 5). Unpaired Student's t-test. (G – I) PMA (100 ng/ml)-induced release of MMP-9 by WT and Coro1a −/− (KO) neutrophils pretreated for 60 min with (G) 16 μM jasplakinolide (Jas; actin-stabilizing agent), (H) 1 μM latrunculin B (Lat; actin-depolymerizing agent); or (I) 30 μM benproperine phosphate (Benp; Arp2/3-complex inhibitor). Data are mean ± SEM ( A, D-E : n = 3; B, G-H : n = 2; C : n = 7). (A-E, G-H) 2-way ANOVA. ns > 0.05, ∗ <0.05, ∗∗ <0.01, ∗∗∗ <0.001, ∗∗∗∗ <0.0001. Data are representative for at least 3 independent experiments.

Article Snippet: Co-IP was conducted by incubation of cell lysates with magnetic beads conjugated to either rabbit polyclonal anti-Coro1a antibody (Proteintech) or isotype matched control antibody in an orbital rotator Intelli-Mixer RM-2M for 2 h at 4 °C.

Techniques: Expressing

Journal: Redox Biology

Article Title: Coronin 1a-mediated F-actin disassembly controls effector function in murine neutrophils

doi: 10.1016/j.redox.2025.103618

Figure Lengend Snippet: Local F-actin destruction is required for neutrophil granule exocytosis ( A ) Transmission electron microscopy (TEM) images of WT and Coro1a −/− (KO) neutrophils either unstimulated or challenged with TNFα (20 ng/ml; 30 min). Micrograph labels: primary (1°G), secondary – quaternary (2°G – 4°G) granules, mitochondria (Mt). TNFα treated condition: non-primary granules are labeled with yellow asterisks. Scale bar, 2 μm; each micrograph is 4.5 μm × 4.5 μm, x22000 magnification. Images shown are representative of at least n = 50 neutrophils being analyzed from each condition. ( B ) Airyscan-enhanced confocal micrographs of WT and Coro1a −/− (KO) neutrophils in basal state (left panel) or stimulated with 20 ng/ml of TNFα for 30 min (right panel). Neutrophils were fixed permeabilized and stained for F-actin (phalloidin) and MMP-9 together with DAPI (nucleus). Scale bar: 3 μm. Images shown are representative of neutrophils observed in 3 independent microscopy experiments. Each experiment consisted of analysis of 8–12 fields for every condition, with each field containing ca. 10–30 cells.

Article Snippet: Co-IP was conducted by incubation of cell lysates with magnetic beads conjugated to either rabbit polyclonal anti-Coro1a antibody (Proteintech) or isotype matched control antibody in an orbital rotator Intelli-Mixer RM-2M for 2 h at 4 °C.

Techniques: Transmission Assay, Electron Microscopy, Labeling, Staining, Microscopy

Journal: Redox Biology

Article Title: Coronin 1a-mediated F-actin disassembly controls effector function in murine neutrophils

doi: 10.1016/j.redox.2025.103618

Figure Lengend Snippet: Identification of Coro1a-associated protein complexes in neutrophils (A) (Top left) Total scatter plot from co-immunoprecipitation with mass-spectrometry (Co-IP/MS) assay displaying proteins found co-precipitating with Coro1a in 2 biological replicates (WT-1 and WT-2 IP) of neutrophil lysates (plot was generated in Perseus software). (Top right) Magnification of the upper right quadrant of the scatter plot showing the most abundant proteins in the Coro1a-precipitates. (Bottom panels) Selected proteins from the Coro1a-precipitates are divided and color-coded into three groups (green: Ras proteins/NADPH oxidase; blue: neutrophil intragranular compounds (and granule subtype: primary – tertiary (1°–3°) granules); magenta: cytoskeletal proteins. IQGAP1, IQ motif-containing GTPase activating protein 1; Ncf4, neutrophil cytosolic factor 4 ( alias p40 phox ); Wdr1, WD-repeat protein ( alias AIP1, actin-interacting protein 1); Arp, actin-related protein; Arpc, Arp 2/3 complex proteins. (B, C) WT and Coro1a −/− (KO) neutrophils were stimulated with PMA (100 ng/ml) for the indicated times. Cellular lysates were subjected to Coro1a-immunoprecipitation (IP) followed by Western blot analysis with the indicated Abs. ( p -Ser (PKC): phospho-Serine (PKC substrate)). H/C: Ig heavy chain of immunoprecipitating Ab.

Article Snippet: Co-IP was conducted by incubation of cell lysates with magnetic beads conjugated to either rabbit polyclonal anti-Coro1a antibody (Proteintech) or isotype matched control antibody in an orbital rotator Intelli-Mixer RM-2M for 2 h at 4 °C.

Techniques: Immunoprecipitation, Mass Spectrometry, Co-Immunoprecipitation Assay, Generated, Software, Western Blot

Journal: Redox Biology

Article Title: Coronin 1a-mediated F-actin disassembly controls effector function in murine neutrophils

doi: 10.1016/j.redox.2025.103618

Figure Lengend Snippet: Coro1a-mediated activation and membrane translocation of Rac2 controls neutrophil ROS production (A) WT and Coro1a −/− (KO) neutrophils stimulated with PMA (100 ng/ml) for the indicated times were analyzed for Rac1 and Rac2 activation by pull-down assay. Cell extracts were also analyzed for total amounts of Rac1 and Rac2. (B) PMA (100 ng/ml; 10 min)-induced translocation of Rac2 from the cytosol to the membrane was analyzed by subcellular fractionation assay. Gp91phox and HSP70 were used as loading controls for membrane and cytosolic fractions, respectively. (C, D) PMA (100 ng/ml)-induced ROS production in WT and Coro1a −/− (KO) neutrophils pretreated for 60 min with the indicated concentrations of (C) NSC23766 (Rac inhibitor), or (D) ML-099 (pan Ras activator). ROS production was measured with luminol chemiluminescence. Left: ROS response kinetics; Right: normalized integrated ROS signals. (C, D) Data are mean ± SEM (n = 2). 2-way ANOVA test (Tukey). ns > 0.05, ∗∗ <0.01, ∗∗∗ <0.001, ∗∗∗∗ <0.0001. Data are representative for at least 3 independent experiments.

Article Snippet: Co-IP was conducted by incubation of cell lysates with magnetic beads conjugated to either rabbit polyclonal anti-Coro1a antibody (Proteintech) or isotype matched control antibody in an orbital rotator Intelli-Mixer RM-2M for 2 h at 4 °C.

Techniques: Activation Assay, Membrane, Translocation Assay, Pull Down Assay, Fractionation