Abstract

We have observed increases in assembled clathrin on the plasma membrane during "frustrated phagocytosis," the spreading of macrophages on immobilized immune complexes. Resident macrophages freshly harvested from the peritoneal cavity of mice and attached to bovine serum albumin (BSA)-anti-BSA-coated surfaces at 4 degrees C had almost no clathrin basketworks on their adherent plasma membrane (less than 0.01 coated patch/micron 2), as observed by immunofluorescence, immunoperoxidase, and platinum-carbon replica techniques, although abundant assembled clathrin was observed in the perinuclear Golgi region. When the cells were warmed to 37 degrees C they started to spread by 4 min and reached their maximum extent by 20 min. Spreading preceded clathrin assembly at the plasma membrane. Clathrin-coated patches were first observed on the adherent plasma membrane at 6 min. Between 12 and 20 min assembled clathrin coats appeared on both adherent and nonadherent plasma membranes with a concomitant decrease in identifiable clathrin in the perinuclear region. A new steady state emerged by 2 h, as perinuclear clathrin began to reappear. At 20 min at 37 degrees C the adherent plasma membranes of macrophages spreading on BSA alone had 0.9 coated patch/micron 2, whereas in cells spread on immune complex-coated surfaces, the clathrin patches increased, dependent on ligand concentration, to a maximum of 2.1 coated patches/micron 2. Because frustrated phagocytosis of immune complex-coated surfaces at 37 degrees C increased the area of adherent plasma membrane, the total area coated by clathrin basket-works increased 5-fold (28 micron 2/cell) as compared with cells plated on BSA alone (5.6 micron 2/cell) and 200-fold as compared with cells adhering to immune complexes at 4 degrees C. We then determined that macrophages cultured on BSA-coated coverslips for 24 h already have abundant surface clathrin. When immune complexes were formed by the addition of anti-BSA IgG to already spread macrophages cultured on BSA-coated coverslips for 24 h, clathrin assembled at the sites of ligand-receptor interaction even at 4 degrees C, before spreading, and a 2.6-fold increase in assembled clathrin was observed on the adherent plasma membrane of cells on immune complexes as compared with cells on BSA alone. Clathrin was reversibly redistributed to the Golgi region, returning to the steady state by 2 h.(ABSTRACT TRUNCATED AT 400 WORDS)

Highlights

  • We determined that macrophages cultured on bovine serum albumin (BSA)-coated coverslips for 24 h already have abundant surface clathrin

  • When immune complexes were formed by the addition of anti-BSA IgG to already spread macrophages cultured on BSA-coated coverslips for 24 h, clathrin assembled at the sites of ligandreceptor interaction even at 4°C, before spreading, and a 2.6-fold increase in assembled clathrin was observed on the adherent plasma membrane of cells on immune complexes as compared with cells on BSA alone

  • In this report we have described dynamic changes in the distribution of morphologically recognizable assembled clathfin during Fc receptor-mediated frustrated phagocytosis

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Summary

Introduction

We determined that macrophages cultured on BSA-coated coverslips for 24 h already have abundant surface clathrin. The spreading of macrophages on immune complex-coated surfaces is believed to represent an attempt to phagocytose these surfaces and has been used to analyze membrane receptor mobilization [21, 32, 60] This model system, called frustrated phagocytosis [24], is suitable for studying clathrin assembly because a large area of plasma membrane surface that interacts with the ligand is fixed on the ligand-coated surface and is available for examination. In the present study we have determined that clathrin assembly at the cell surface is induced by receptor-ligand interaction in this model system of Fc receptor-mediated phagocytosis and occurs with a concomitant decrease in clathrin-coated structures in the Golgi region

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