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Freeze-fracture cytochemical study of membrane systems in human epidermis using filipin as a probe for cholesterol
Abstract:
Filipin (a polyene antibiotic) interacts specifically with cholesterol in membranes, producing characteristic 25 nm-diameter deformation (pitlike lesions) within the membrane plane detectable by freeze-fracture electron microscopy. Utilizing this probe, the distribution of cholesterol molecules in membranes and in lamellar structures between horny cells was investigated in human skin. The plasma membranes of basal, spinous, and granular cells reacted extensively with filipin except for desmosomal membrane portions. However, the plasma membranes of horny cells were rarely labeled with filipin, while lamellar structures between horny cells were well labeled. These observations indicate the distinct difference in susceptibility to filipin among the plasma membranes of viable cells and horny cells, and the lipid lamellar structures. Whenever horny cell plasma membranes were affected with filipin, they revealed a low deformability showing shallow pits or low protrusions. This low deformability may be due to greater membrane rigidity rather than a lower content of cholesterol, although the possibility of a low amount of cholesterol cannot be excluded. Lamellar bodies in granular cells were well labeled in the limiting membranes but poorly labeled in the internal lamellar structures. The regions of gap junctions were absolutely unlabeled. Filipin-cholesterol complexes were produced very close to the junctional strands but did not appear to disrupt the junctional structure of tight junctions. Nuclear membranes were affected only in the outer membrane with filipin. These results suggest that keratinocytes undergo a distinctive reduction in membrane deformability or in free-cholesterol content at the transition from living to dead cells, and display a heterogeneity in cholesterol distribution in human epidermal cell membranes.
Insights
Filipin (a polyene antibiotic) reveals cholesterol distribution in human skin. Cholesterol content and membrane deformability decrease in dead skin cells, impacting membrane structure.
Area of Science:
- Dermatology
- Cell Biology
- Biochemistry
Background:
- Cholesterol is a vital component of cell membranes, influencing their structure and function.
- Filipin, a polyene antibiotic, specifically binds to cholesterol, causing membrane deformations observable via electron microscopy.
- Understanding cholesterol distribution in skin cells is crucial for elucidating epidermal barrier function.
Purpose of the Study:
- To investigate the distribution and accessibility of cholesterol in human epidermal cell membranes and intercellular structures.
- To assess changes in membrane cholesterol content and deformability during keratinocyte differentiation and cell death.
Main Methods:
- Utilized filipin as a probe for cholesterol detection.
- Employed freeze-fracture electron microscopy to visualize filipin-cholesterol complexes and membrane alterations.
- Examined various cell layers of human epidermis, including basal, spinous, granular, and horny cells, as well as intercellular structures.
Main Results:
- Filipin extensively labeled plasma membranes of viable keratinocytes (basal, spinous, granular) but not desmosomes.
- Horny cell plasma membranes showed minimal filipin labeling, indicating reduced cholesterol accessibility or altered membrane properties.
- Lamellar structures between horny cells were well labeled, suggesting cholesterol-rich lipid bilayers.
- Horny cell membranes exhibited low deformability when interacting with filipin, possibly due to increased rigidity or lower free cholesterol.
- Lamellar bodies in granular cells showed differential labeling, and gap junctions remained unlabeled.
Conclusions:
- Keratinocytes exhibit a significant reduction in membrane deformability or free cholesterol content upon transition from living to terminally differentiated (dead) cells.
- Cholesterol distribution is heterogeneous across different epidermal cell types and structures.
- Filipin labeling provides insights into the structural and compositional changes of cell membranes during epidermal differentiation.