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Structural heterogeneity of endocytic membranes in macrophages as revealed by the cholesterol probe, filipin
Abstract:
The polyene antibiotic, filipin, by specifically interacting with cholesterol, produces approximately 25-nm protuberances (filipin-sterol complexes) in freeze-fractured membranes, and the addition of filipin to aldehyde fixatives has been recently introduced as a cytochemical technique for the localization of cholesterol in cell membranes. In a previous study we showed that, in fibroblasts filipin-sterol complexes are absent from endocytic coated pits. To establish whether the absence of filipin-sterol complexes is a phenomenon restricted to coated pits or is correlated with endocytosis in general, we applied the filipin probe to cultured mouse peritoneal macrophages, in which different forms of endocytosis take place. The macrophages were incubated with bovine albumin or concanavalin A (Con A) to induce pinocytosis, and with heat-killed straphylococci or opsonized erythrocytes to induce phagocytosis, then fixed in glutaraldehyde/filipin and freeze-fractured. Filipin-sterol complexes were plentiful on the plasma membrane, on the smooth-membrane invaginations and vesicles induced by albumin, on the large endocytic vacuoles induced by Con A, and on the membrane of phagosomes but, in contrast, they were absent from coated pits and vesicles, as well as from coated segments of invagination or vesicles. These results indicate that the membranes involved in different types of endocytosis do not react in the same way with filipin and may, therefore, have a different cholesterol content. This could reflect different mechanisms of formation for the various types of endocytic vesicles.
Insights
Filipin-sterol complexes, used to detect cholesterol, are absent in coated endocytic structures but present in other endocytic membranes. This suggests varying cholesterol levels during different endocytosis types.
Area of Science:
- Cell Biology
- Membrane Biology
- Biochemistry
Background:
- Filipin interacts with cholesterol, forming complexes visible in freeze-fractured membranes.
- Filipin-sterol complexes are absent in fibroblast endocytic coated pits.
- This absence may relate to coated pits specifically or endocytosis generally.
Purpose of the Study:
- To investigate the distribution of filipin-sterol complexes in various endocytic pathways in macrophages.
- To determine if cholesterol localization differs between coated and non-coated endocytic structures.
Main Methods:
- Cultured mouse peritoneal macrophages were used.
- Cells were stimulated to undergo pinocytosis (albumin, Concanavalin A) or phagocytosis (staphylococci, erythrocytes).
- Cells were fixed with glutaraldehyde/filipin and analyzed by freeze-fracture electron microscopy.
Main Results:
- Filipin-sterol complexes were abundant on the plasma membrane and in smooth vesicles/invaginations during pinocytosis.
- Complexes were present on large endocytic vacuoles (Con A) and phagosome membranes.
- Notably, complexes were absent from coated pits, coated vesicles, and coated membrane segments.
Conclusions:
- Membranes involved in different endocytic processes exhibit differential reactivity to filipin, indicating varied cholesterol content.
- The distinct cholesterol distribution suggests different formation mechanisms for various endocytic vesicles.