Fig. 1. Cholesterol and Src family kinases control the assembly of FcRII caps and tyrosine phosphorylation of the receptor in U937 cells. (A-F) Localization of FcRII on the cell surface. (A) Cells exposed to IV.3 mouse anti-FcRII, fixed and postlabeled with anti-mouse FITC-IgG. (B) Cells treated at 0°C with IV.3 mouse anti-FcRII and anti-mouse FITC-IgG (crosslinking). (C) Capping of crosslinked FcRII after 10 minutes of cell warming at 20°C. (D) Cells pretreated with 4 mM CDX (1 hour, 37°C) followed by crosslinking of FcRII at 0°C and (E) cells after subsequent incubation for 10 minutes at 20°C. (F) Reconstitution of FcRII cap assembly after reincorporation of cholesterol (30 minutes, 37°C) into cells pretreated with 4 mM CDX. Bar, 10 µm. (G) Quantification of FcRII cap formation as a function of cholesterol content. Bars on the left side depict a dose-dependent inhibition of FcRII capping by CDX treatment (1 hour, 37°C). Bars on the right side show the assembly of FcRII caps in cholesterol-depleted cells after 30 minutes of cholesterol reincorporation (+) or without the reincorporation (-). (-•-) Cholesterol content. (H) Tyrosine phosphorylation (PY) of FcRII immunoprecipitated from whole lysates of U937 cells (upper panel). Cells were either untreated with antibodies or exposed at 0°C to IV.3 anti-FcRII alone (non-crosslinked) or incubated at 0°C with mouse anti-FcRII and goat anti-mouse IgG (crosslinking). Prior to FcRII crosslinking, cells were preincubated with 8 µM herbimycin A (Herb), 5 mM CDX and 1 mM HMA or without inhibitors (control, Ctrl). A subset of the control cells was warmed at 20°C for 10 and 20 minutes to induce the formation of FcRII caps (capping). 52 is a molecular mass standard in kDa. Arrowhead indicates tyrosine phosphorylated FcRII. A corresponding part of the membrane was reprobed with rabbit anti-FcRII to reveal amounts of the precipitated receptor (lower panel). (I) Influence of herbimycin A (Herb), PP1, HMA, BPA, piceatannol (PCT) and wortmannin (Wort) on FcRII cap assembly. Results are the mean±s.e.m. of three to five experiments. (J) In vivo treatment of cells with PP1 and piceatannol led to the inhibition of Lyn and Syk activity. Cells were treated with 15 µM PP1 and 25-100 µM piceatannol (PCT) before (30 minutes) and during FcRII crosslinking. Syk and Lyn kinases were immunoprecipitated from lysates of the cells and the kinase assay was performed on the obtained immunocomplexes. The samples were blotted with anti-PY to reveal autophosphorylation of Syk and Lyn (arrowheads) and later reprobed with rabbit anti-Syk and mouse anti-Lyn IgG to indicate the amounts of the precipitated kinases. Lanes `non-crosslinked' show the autophosphorylation of the kinases in cells exposed to IV.3 anti-FcRII only. The level of Lyn and Syk autophosphorylation was estimated densitometrically in relation to the kinase content and is shown over corresponding lanes (the mean from two experiments). Lane "-", control of immunoprecipitation - cell lysates supplemented with Pansorbin only.