Die Rolle von Rho GTPasen und Zytoskelettdynamik bei der Erfassung und Phagozytose von Partikeln durch Makrophagen
Zusammenfassung der Projektergebnisse
Macrophages (from Greek, meaning “big eaters”) are widely distributed in the body and are typically the first cells of the immune system to respond to tissue damage and infection. Phagocytosis is commonly studied in end-point assays, in which the number of ingested particles are scored after a certain incubation time. In this project, we addressed the longstanding issue of whether there are two distinct modes of phagocytosis, sinking phagocytosis and phagocytic cup formation, mediated by complement receptors and Fcγ receptors, respectively. We developed real-time 3D imaging assays which enabled the analysis of individual phagocytic events spanning from initial contacts between macrophages and particles through to complete particle ingestion. As phagocytic targets, we used freshly isolated human red blood cells (hRBCs) since these cells are deformable and unlabeled cells are not ingested, providing negligible background activity. We could specifically study Fcγ receptor-mediated phagocytosis after labeling hRBCs with mouse IgG antibodies. Complement receptor-mediated mediated phagocytosis could be studied by labeling hRBCs with mouse IgM antibodies followed by incubation with mouse serum. Alternatively, complement receptor-mediated phagocytosis could be studied in macrophages isolated from mutant (NOTAM (No ITAM) or Fcer1g knockout) mice which were not capable of Fcγ receptormediated phagocytosis. In this case, IgG-opsonized hRBCs treated with mouse serum could be used to isolate complement (C3b/iC3b)-induced phagocytosis. We found that Fcγ receptormediated phagocytosis was highly efficient and phagocytic cups could arise from any site of particle contact, including the tips of filopodia. Knockout mouse models revealed that phagocytic cup formation was critically dependent on a functional immunoreceptor tyrosinebased activation motif (ITAM) and the tyrosine kinase Syk. However, surprisingly, Fcγ receptor-mediated phagocytic cups became highly elongated and cup closure was delayed in macrophages lacking complement receptor 3. Independent of Fcγ receptors, complement receptors could also induce phagocytic cups, albeit with low efficiency and the cups typically emanated from membrane ruffles, rather than de novo at an arbitrary contact point. Deletion of both ITAM adapters (Fc receptor γ chain and DAP12) blocked this mode of complement receptor-mediated phagocytosis, but did not impair slow sinking phagocytosis. In summary, we found that (i) there is indeed a slow sinking mode of phagocytosis mediated by complement receptor 3, an integrin, (ii) complement receptor 3 can induce phagocytic cup-like structures through ITAM adapters (Fc receptor γ chain and DAP12) and Syk signaling, albeit with low efficiency, and (iii) complement receptor 3 is involved in shaping and closing phagocytic cups induced by stimulation of Fcγ receptors.
Projektbezogene Publikationen (Auswahl)
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(2017). Multiple roles of filopodial dynamics in particle capture and phagocytosis, and phenotypes of Cdc42 and Myo10 deletion. Journal of Biological Chemistry 292, 7258-7273
Horsthemke, M., Bachg, A. C., Groll, K., Moyzio, S., Müther, B., Hemkemeyer, S. A., Wedlich-Söldner, R., Sixt, M., Tacke, S., Bähler, M. & Hanley, P. J.
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(2018). Time-lapse 3D imaging of phagocytosis by mouse macrophages. Journal of Visualized Experiments 140, e57566
Horsthemke, M., Wilden, J., Bachg, A. C. & Hanley, P. J.
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(2019). Phenotypic Analysis of Myo10 Knockout Mice Selectively Lacking Full-length (Motorized) But Not Brain-Specific Headless Myosin X. Scientific Reports 9, 597
Bachg, A. C., Horsthemke, M., Skryabin, B. V., Klasen, T., Nagelmann, N., Faber, C., Woodham, E., Machesk, L. M., Bachg, S., Stange, R., Jeong, H.-W., Adams, R. H., Bähler, M. & Hanley, P. J.
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(2021). Complement receptor 3 mediates both sinking phagocytosis and phagocytic cup formation via distinct mechanisms. Journal of Biological Chemistry 296: 100256
Walbaum, S., Ambrosy, B., Schutz, P., Bachg, A. C., Horsthemke, M., Leusen, J. H. W., Mócsai, A. & Hanley, P. J.
