Related Experiment Videos
Localisation of Nramp1 in macrophages: modulation with activation and infection
S Searle1, N A Bright, T I Roach
1Department of Medicine, University of Cambridge Clinical School, Level 5, Addenbrooke's Hospital, Hills Road, Cambridge CB2 2QQ, UK. SS10057@MEDSCHL.cam.ac.uk.
Abstract:
The murine natural resistance-associated macrophage protein, Nramp1, has multiple pleiotropic effects on macrophage activation and regulates survival of intracellular pathogens including Leishmania, Salmonella and Mycobacterium species. Nramp1 acts as an iron transporter, but precisely how this relates to macrophage activation and/or pathogen survival remains unclear. To gain insight into function, anti-Nramp1 monoclonal and polyclonal antibodies are used here to localise Nramp1 following activation and infection. Confocal microscope analysis in uninfected macrophages demonstrates that both the mutant (infection-susceptible) and wild-type (infection-resistant) forms of the protein localise to the membranes of intracellular vesicular compartments. Gold labelling and electron microscopy defines these compartments more precisely as electron-lucent late endosomal and electron-dense lysosomal compartments, with Nramp1 colocalizing with Lamp1 and cathepsins D and L in both compartments, with macrosialin in late endosomes, and with BSA-5 nm gold in pre-loaded lysosomes. Nramp1 is upregulated with interferon-(gamma) and lipopolysaccaride treatment, coinciding with an increase in labelling in lysosomes relative to late endosomes and apparent dispersion of Nramp1-positive vesicles from a perinuclear location towards the periphery of the cytoplasm along the microtubular network. In both control and activated macrophages, expression of the protein is 3- to 4-fold higher in wild-type compared to mutant macrophages. In Leishmania major-infected macrophages, Nramp1 is observed in the membrane of the pathogen-containing phagosomes, which retain a perinuclear localization in resting macrophages. In Mycobacterium avium-infected resting and activated macrophages, Nramp1-positive vesicles migrated to converge, but not always fuse, with pathogen-containing phagosomes. The Nramp1 protein is thus located where it can have a direct influence on phagosome fusion and the microenvironment of the pathogen, as well as in the more general regulation of endosomal/lysosomal function in macrophages.
Insights
The natural resistance-associated macrophage protein 1 (Nramp1) localizes to macrophage vesicles, influencing pathogen survival. Its location shifts upon activation, impacting phagosome fusion and the pathogen's microenvironment.
Area of Science:
- Immunology
- Cell Biology
- Microbiology
Background:
- The murine natural resistance-associated macrophage protein 1 (Nramp1) plays a role in macrophage activation and resistance to intracellular pathogens.
- Nramp1 functions as an iron transporter, but its precise role in macrophage activation and pathogen survival is not fully understood.
Purpose of the Study:
- To investigate the localization of Nramp1 in macrophages following activation and infection using specific antibodies.
- To elucidate the functional significance of Nramp1's localization in relation to macrophage defense mechanisms against intracellular pathogens.
Main Methods:
- Confocal microscopy and immunoelectron microscopy with gold labeling were used to visualize Nramp1 localization.
- Macrophages were treated with interferon-gamma and lipopolysaccharide for activation and infected with Leishmania major and Mycobacterium avium.
Main Results:
- Nramp1 localizes to the membranes of late endosomal and lysosomal compartments in uninfected macrophages, co-localizing with Lamp1 and cathepsins.
- Upon activation, Nramp1 labeling increases in lysosomes, and Nramp1-positive vesicles redistribute towards the cell periphery.
- In infected macrophages, Nramp1 is found on pathogen-containing phagosomes, suggesting a direct role in modulating the phagosomal environment.
Conclusions:
- Nramp1 is strategically positioned within macrophages to directly impact phagosome fusion and the intracellular pathogen environment.
- The localization and dynamics of Nramp1 are crucial for its function in regulating macrophage activation and controlling intracellular pathogen survival.