Related Experiment Videos
Internalization of monomeric lipopolysaccharide occurs after transfer out of cell surface CD14
T Vasselon1, E Hailman, R Thieringer
1Department of Endocrinology and Chemical Biology, Merck Research Laboratories, Rahway, New Jersey 07065, USA.
Abstract:
Lipopolysaccharide (LPS) fluorescently labeled with boron dipyrromethane (BODIPY) first binds to the plasma membrane of CD14-expressing cells and is subsequently internalized. Intracellular LPS appears in small vesicles near the cell surface and later in larger, punctate structures identified as the Golgi apparatus. To determine if membrane (m)CD14 directs the movement of LPS to the Golgi apparatus, an mCD14 chimera containing enhanced green fluorescent protein (mCD14-EGFP) was used to follow trafficking of mCD14 and BODIPY-LPS in stable transfectants. The chimera was expressed strongly on the cell surface and also in a Golgi complex-like structure. mCD14-EGFP was functional in mediating binding of and responses to LPS. BODIPY-LPS presented to the transfectants as complexes with soluble CD14 first colocalized with mCD14-EGFP on the cell surface. However, within 5-10 min, the BODIPY-LPS distributed to intracellular vesicles that did not contain mCD14-EGFP, indicating that mCD14 did not accompany LPS during endocytic movement. These results suggest that monomeric LPS is transferred out of mCD14 at the plasma membrane and traffics within the cell independently of mCD14. In contrast, aggregates of LPS were internalized in association with mCD14, suggesting that LPS clearance occurs via a pathway distinct from that which leads to signaling via monomeric LPS.
Insights
Lipopolysaccharide (LPS) initially binds to cell surfaces via CD14. Monomeric LPS then moves independently within cells, while LPS aggregates are cleared with CD14.
Area of Science:
- Immunology
- Cell Biology
- Molecular Biology
Background:
- Lipopolysaccharide (LPS) is a key component of Gram-negative bacteria.
- CD14 is a receptor involved in LPS recognition and cellular response.
- The intracellular trafficking pathway of LPS remains incompletely understood.
Purpose of the Study:
- To investigate the role of membrane-bound CD14 (mCD14) in the intracellular transport of LPS.
- To differentiate the trafficking pathways of monomeric versus aggregated LPS.
- To determine if mCD14 accompanies LPS during endocytosis and subsequent intracellular movement.
Main Methods:
- Utilized fluorescently labeled LPS (BODIPY-LPS).
- Employed a membrane-bound CD14-enhanced green fluorescent protein (mCD14-EGFP) chimera in stable transfectants.
- Tracked the colocalization and movement of mCD14-EGFP and BODIPY-LPS using live-cell imaging.
Main Results:
- mCD14-EGFP localized to the cell surface and Golgi apparatus.
- BODIPY-LPS initially colocalized with mCD14-EGFP on the cell surface.
- Within minutes, BODIPY-LPS trafficked to intracellular vesicles independently of mCD14, suggesting dissociation at the plasma membrane.
- LPS aggregates, however, were internalized in association with mCD14.
Conclusions:
- Membrane-bound CD14 facilitates LPS binding but does not accompany monomeric LPS during its endocytic trafficking.
- Monomeric LPS appears to be transferred from mCD14 at the plasma membrane and moves intracellularly independently.
- LPS aggregates utilize a distinct internalization pathway involving mCD14, separate from the signaling pathway of monomeric LPS.