Immunocytochemical localization of the α-subunit of G-proteins in macrophages andEscherichia coli interacting with each other

Abstract

The immunocytochemical localization of the c~-subunit of G-proteins is established in murine macrophage-like P388D1 cells, in E. coli, and in L-forms of E. coil. It is shown that the cytoplasmic concentration of G-proteins is increased in macrophages interacting with the bacteria. Key Words: G-proteins; phagocytosis; E. coli; immunocytochemistryHeterotrimeric proteins (G-proteins) capable of binding guanine nucleotides mediate signal transduction from an activated cell receptor to the effector (adenylate cyclase, ion channels, etc.) [2]. Lipopolysaccharides, the major component of the cell wall of Gram-negative bacteria, serve as primary messengers along with other physical and chemical factors. Lipopolysaccharides stimulate the production of inflammation mediators by macrophages (MPH) via activation of the c~-subunits of G-proteins and generation of second messengers [6,10]. The uptake of bacteria and endocytosis are associated with components of the cytoskeleton, primarily with the microtubules, which are controlled predominantly by G-proteins [8].G-proteins have been found in a wide variety of mammalian, plant, and microbial cells; however, their localization in bacteria is unknown and their role in the MATERIALS AND METHODSMurine P388D1 MPH were incubated for 96 h in modified Dulbecco's medium containing 10% fetal calf serum (Gibco). The suspended cells were washed three times with phosphate-buffered saline (PBS), the adherent cells were collected in PBS containing 0.02% EDTA, washed three times with PBS, and resuspended in PBS to a final concentration of 10 B cells/ml. Pooled cells were incubated with E. coil, L-forms of E. COil, or latex particles (diameter 0.5-1.0 ~) for 1 h at 25~After incubation with the bacteria or latex, a drop of the MPH suspension was transferred to a glass slide, air dried, fixed with absolute methanol or acetone for 5 min at room temperature, and washed in PBS-T