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Freeze-fracture study of phagocytosis in Dictyostelium discoideum
Abstract:
The plasma membrane and its derivative, the phagosome membrane, were studied during and after ingestion of yeast of latex beads in Dictyostelium discoideum. Freeze-fracture electron microscopy, which provides information on the internal architecture of the membranes, and observation of thin sections of cells treated by cytochemical methods were used in parallel. For visualization of membrane sterols in the replicas, the cells were fixed in the presence of digitonin or the antibiotic filipin. No lateral phase separation occurred during yeast engulfment: the intramembranous particles (IMPs), phospholipids and sterols remained distributed at random in the forming phagosome membrane. In contrast architectural modifications of the membrane were observed upon phagosome internalization. Compared to the plasma membrane, the phagosome membrane displayed 2-3 times more IMPs a shift in the IMP size distribution and a higher sterol content. These changes were completed soon after phagosome closure; they were not related either to the nature of the ingested particles (yeast, latex beads) or to the pH in the membrane environment. The membrane changes too place when the phagosomes began to fuse with pre-existing digestive or autophagic vacuoles and lysosomes. Some of the experimental evidence suggests that the restructuring of the membrane may be related to the presence of hydrolases.
Insights
The plasma membrane of Dictyostelium discoideum undergoes significant restructuring after phagosome internalization, increasing intramembranous particles and sterol content. These changes occur during fusion with digestive vacuoles and may involve hydrolases.
Area of Science:
- Cell biology
- Membrane biophysics
- Molecular biology
Background:
- The plasma membrane and phagosome membrane are crucial for cellular processes like nutrient uptake and waste removal.
- Understanding membrane dynamics during phagocytosis is key to deciphering cellular defense and degradation mechanisms.
Purpose of the Study:
- To investigate the structural and compositional changes in the plasma membrane and phagosome membrane during and after yeast and latex bead ingestion in Dictyostelium discoideum.
- To determine the role of membrane sterols and intramembranous particles (IMPs) in phagosome formation and maturation.
Main Methods:
- Freeze-fracture electron microscopy to visualize membrane architecture.
- Cytochemical staining with digitonin and filipin to identify membrane sterols.
- Thin section electron microscopy for cellular observation.
Main Results:
- No significant lateral phase separation was observed in the forming phagosome membrane during yeast engulfment.
- Internalization of the phagosome led to a 2-3 fold increase in IMPs and higher sterol content compared to the plasma membrane.
- These membrane modifications were independent of ingested particle type and pH, occurring during fusion with digestive vacuoles.
Conclusions:
- Phagosome membrane restructuring, characterized by increased IMPs and sterols, occurs post-internalization and precedes fusion with lysosomes.
- The observed membrane changes are likely linked to the fusion process and potentially to the action of hydrolases.
- Dictyostelium discoideum provides a model system for studying fundamental membrane dynamics during phagocytosis and organelle fusion.
