Joel A. Swanson

Phagocytosis by zippers and triggers

Two mechanisms have been considered for particle phagocytosis. According to the zipper mechanism, ingestion occurs by sequential engagement of a phagocytes membrane against the particle surface, and pseudopod advance proceeds no further than receptor-ligand interactions permit. In contrast, in the trigger mechanism particle binding initiates an all-or-none phagocytic response. Although the weight of experimental evidence has favoured the zipper mechanism, recent observations of bacterial entry into epithelial cells and macrophages indicate an indiscriminate, triggered response. This prompts a reconsideration of the underlying mechanisms.

Fluorescent labeling of endocytic compartments.

Publisher Summary This chapter discusses the fluorescent labeling of endocytic compartments. Endocytosis is that cellular process in which plasma membrane invaginates to form a closed vesicle within the cytoplasm. Extracellular solutes and surface-bound molecules enclosed by endocytic vesicles may encounter a variety of fates, depending upon both the engulfed molecule and the engulfing cell. The usual target of endocytosed solutes is the lysosome, an acidic compartment of degradative hydrolysis, and the repository for many nondigestible and membrane-impermeant molecules. Lucifer Yellow CH is a low molecular weight, water soluble, and membrane-impermeant marker for fluid phase pinocytosis. Macromolecules labeled with rhodamine often adsorb to cell surfaces. The efficient internalization of many physiologically important macromolecules is mediated by receptors in the plasma membrane. The best method at present for labeling lysosomes is to incubate cells for a long period in culture medium containing a nondigestible fluorescent probe, followed by a1-to 2-hour chase in unlabeled medium. The long chase should deplete probe from compartments such as pinosomes and endosomes, leaving only lysosomes fluorescent.

The efficiency of antigen delivery from macrophage phagosomes into cytoplasm for MHC class I-restricted antigen presentation

Macrophages can present exogenous antigen in association with MHC class I molecules. Indirect evidence indicates that antigens internalized by phagocytosis can enter cytoplasm before following the conventional MHC class I presentation pathway. However, little is known about how common such entry is, or to what extent it depends on the kind of particle ingested. This study reports quantitative and morphological characterization of antigen delivery from phagosomes into cytoplasm for MHC class I-restricted antigen presentation. Ovalbumin (OVA) was associated with polystyrene particles (PS), biodegradable poly-e-caprolactone particles (PCL), and sheep red blood cells (SRBC), and its delivery into macrophage cytoplasm, via phagocytosis was assessed with a T hybridoma assay for MHC class I-restricted presentation of OVA-derived peptides. Although direct introduction of antigen into cytoplasm by scrape-loading produced the most efficient presentation, comparable signals could be obtained after phagocytosis of PCL or PS. Phagocytosis of OVA-loaded SRBC, and OVA internalized by pinocytosis, did not deliver efficiently. MHC class I-restricted presentation of phagosome-derived OVA required cytoplasmic processing, as it was inhibited by proteasome inhibitors and brefeldin A. Morphological studies showed that biotinylated OVA originating in PCL phagosomes could be delivered into the cytoplasm of 90% of the macrophages. These results indicate that phagocytosis per se is not sufficient to deliver antigen into cytoplasm, but that phagocytosis of solid, synthetic polymeric particles delivers antigen efficiently into cytoplasm for MHC class I processing.

MICROTUBULES CAN MODULATE PSEUDOPOD ACTIVITY FROM A DISTANCE INSIDE MACROPHAGES

Microtubules are thought to influence cell shape as structural components of an integrated cytoskeletal matrix. Here we show that microtubules can affect the dynamics of macrophage pseudopodia without being integrated into their structure. Macrophages landing on glass surfaces spread within 15 min into flattened circular cells with radial symmetry, and the radial distribution of microtubules reflected this symmetry. Depolymerization of microtubules using nocodazole, colchicine, or vinblastine did not inhibit macrophage spreading or the early establishment of radial symmetry. Shortly after spreading, however, macrophages without microtubules gradually became asymmetric, assuming irregular, lobed profiles. The asymmetry resulted from exaggerated protrusion and retraction of pseudopodia, with net retraction overall. This loss of radial symmetry could be inhibited by treatment of initially spread cells with cytochalasin D, indicating that the change in cell shape was mediated by the actin cytoskeleton. Intact microtubules suppressed the exaggerated pseudopod movements, even when they were separated by a distance from the cell margin. In cells treated with taxol, microtubules remained clustered near the cell center after spreading, yet the dynamics of pseudopodia at the cell margin were reduced and cells maintained a circular profile. Similarly, in cells treated with low concentrations of nocodazole, a much reduced microtubule cytoskeleton nonetheless suppressed pseudopod dynamics. We propose that microtubules act to stabilize cell shape at a distance from the cell edge via a biochemical intermediate that affects the structure or function of the microfilament system.