In contrast, sheep red blood cells opsonized with complement appeared to sink directly into the macrophage without the generation of membrane extensions, although, in some images, membrane ruffles can be seen in the immediate vacinity of the sinking particles. Phagocytic cup formation required actin polymerization since it was abrogated by the cell-permeable fungal toxin cytochalasin B, known to block actin dynamics 3. Kaplan showed by means of scanning electron microscopy that mouse peritoneal macrophages ingested an IgG-opsonized sheep red blood cell using thin membrane extensions which reached up and tightly enveloped the particle, initially giving rise to a cup-like structure. More than 100 years later, a pioneering study by Kaplan suggested that there were at least two morphologically distinct mechanisms of phagocytosis 2. He explicitly described membrane protrusions enveloping the particles, which were subsequently taken up into the cytoplasm and accumulated around the cell nucleus. Die Radiolarien (Rhizopoda radiaria): Eine Monographie Druck und Verlag von Georg Reimer, Berlin, 1862). Twenty years before Metchnikoff's observation of phagocytic mesenchyme cells in starfish larvae, in 1882, and subsequent development of his theory of phagocytosis 1, Ernest Haeckel described, in 1862, the engulfment of insoluble dye particles by blood cells of Thetis fimbris ( Tethys fimbria), a species of predatory sea slug (Ernest Haeckel. In addition, we describe assays to unambiguously image Fcγ receptor- or complement receptor-mediated phagocytosis. Here we describe phagocytic assays, using time-lapse spinning disk confocal microscopy, that allow 3D imaging of single phagocytic events. More commonly used approaches for the study of phagocytosis, such as end-point assays, miss the opportunity to understand what is happening at the interface of particles and phagocytes. With the availability of high resolution imaging techniques, phagocytosis assays are required that allow real-time 3D (three dimensional) visualization of how specific phagocytic receptors mediate the uptake of individual particles. However, the distinctions between the two modes have become blurred by reports that complement receptor-mediated phagocytosis may induce various membrane protrusions. These two modes of phagocytosis, phagocytic cup formation and sinking in, have become well established in the literature. In contrast, complement opsonized particles appear to sink into the phagocyte following binding to complement receptors. Binding of immunoglobulin G (IgG)-opsonized particles to FcγRs triggers the protrusion of thin membrane extensions, which initially form a so-called phagocytic cup around the particle before it becomes completely enclosed and retracted into the cell. Four decades ago, two distinct mechanisms of phagocytosis, exemplified by Fcγ receptor (FcγR)- and complement receptor (CR)-mediated phagocytosis, were identified using scanning electron microscopy. Although phagocytic receptors, downstream signaling pathways, and effectors, such as Rho GTPases, have been identified, the dynamic cytoskeletal remodeling of specific receptor-mediated phagocytic events remain unclear. Phagocytosis plays a key role in host defense, as well as in tissue development and maintenance, and involves rapid, receptor-mediated rearrangements of the actin cytoskeleton to capture, envelop and engulf large particles.
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