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Expression and subcellular localization of NRAMP1 in human neutrophil granules
François Canonne-Hergaux1, Jero Calafat, Etienne Richer
1Department of Biochemistry, Center for the Study of Host Resistance, McGill Cancer Center, McGill University, Montreal, QC, Canada.
Abstract:
Mutations at the Nramp1 gene cause susceptibility to infections with intracellular pathogens. In human blood, polymorphonuclear (PMN) leukocytes are the most abundant site of NRAMP1 messenger RNA (mRNA) expression, suggesting that NRAMP1 plays an important role in the activity of these cells. By Northern blot analysis, NRAMP1 mRNA was only detected in most mature neutrophils from bone marrow (band and segmented cells). A high-affinity polyclonal rabbit antihuman NRAMP1 antibody directed against the amino terminus of the protein was produced and used to study cellular and subcellular localization of the protein in primary human neutrophils. Subcellular fractionation of granule populations together with immunoblotting studies with granule-specific markers indicate that NRAMP1 expression is primarily in tertiary granules. These granules are positive for the matrix enzyme gelatinase and the membrane subunit of the vacuolar H(+)/ATPase and can be recruited for exocytosis by treatment of neutrophils with phorbol myristate acetate. Immunogold studies by cryoelectron microscopy with primary neutrophils confirm that a majority (75%) of NRAMP1-positive granules are also positive for gelatinase, but they also suggest further heterogeneity in this granule population. Presence of NRAMP1 in tertiary granules is in agreement with the late-stage appearance of NRAMP1 mRNA during neutrophil maturation in bone marrow. Finally, immunofluorescence studies of Candida albicans-containing phagosomes formed in neutrophils indicate that NRAMP1 is recruited from tertiary granules to the phagosomal membrane on phagocytosis, supporting a role for NRAMP1 in the antimicrobial defenses of human neutrophils.
Insights
Mutations in the NRAMP1 gene impact infection susceptibility. This study shows NRAMP1 protein localizes to tertiary granules in neutrophils and moves to phagosomes during infection, aiding antimicrobial defense.
Area of Science:
- Immunology
- Cell Biology
- Genetics
Background:
- The NRAMP1 gene is crucial for defense against intracellular pathogens.
- Polymorphonuclear (PMN) leukocytes, particularly neutrophils, show high NRAMP1 mRNA expression, indicating a key role in their function.
- NRAMP1 mRNA is primarily detected in mature neutrophils.
Purpose of the Study:
- To investigate the cellular and subcellular localization of the NRAMP1 protein in human neutrophils.
- To understand the role of NRAMP1 in neutrophil antimicrobial activity.
Main Methods:
- Northern blot analysis to detect NRAMP1 mRNA.
- Production of a polyclonal antibody against human NRAMP1.
- Subcellular fractionation and immunoblotting of neutrophil granules.
- Cryoelectron microscopy with immunogold labeling.
- Immunofluorescence studies of phagosomes.
Main Results:
- NRAMP1 protein is predominantly found in tertiary granules of neutrophils.
- These tertiary granules are positive for gelatinase and vacuolar H(+)/ATPase.
- NRAMP1 is recruited to phagosomal membranes upon phagocytosis of Candida albicans.
- A majority (75%) of NRAMP1-positive granules also contain gelatinase.
Conclusions:
- NRAMP1's presence in tertiary granules aligns with its late-stage expression during neutrophil maturation.
- NRAMP1's recruitment to phagosomes suggests a direct role in human neutrophil antimicrobial defense mechanisms.